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Bacterial DNA polymerases, Chemistry of
Richard T. Pomerantz and Mike O'Donnell, Rockefeller University, New York, New York
All DNA polymerases share a common two-metal ion catalyzed chemistry of nucleotide incorporation. Structure analysis, however, suggests that DNA polymerases share one of two different ancestors, which converged to employ the same mechanism. Escherichia coli, the prototypical bacterium, encodes five different DNA polymerases. The chromosomal replicase functions closely with clamps, clamp-loaders, and other proteins. Oxidative damage to DNA during normal cell growth requires interplay among the several distinct DNA polymerases, which enable the replicase to circumvent these obstacles and complete chromosomal replication. Additional processes involving DNA polymerases are brought into action during heightened levels of DNA damage. We review here DNA polymerase structure, catalytic mechanism, and several pathways in which various bacterial DNA polymerases act.
E. coli DNA polymerase (Pol) I is so named because it was the first DNA polymerase to be isolated and characterized (1). Pol I remains the most intensively studied DNA polymerase, and its general structure and properties generalize to other DNA polymerases. Sequence comparisons of DNA polymerases derived from many organisms indicate the presence of at least five different classes of DNA polymerase (Table 1) (2). For simplicity, we will focus in this review on the DNA polymerases of Escherichia coli. E. coli contains five different DNA polymerases that assort into four polymerase families (Table 1). Pol I is a member of the A-family; it is ubiquitous among bacteria and plays a major role in DNA repair. Pol II is a B-family DNA polymerase that seems to be involved in repair. Interestingly, eukaryotic and archael chromosomal replicases are all members of the B-family. The C-family of DNA polymerase is specific to bacteria and functions as the chromosomal replicase. E. coli contains one C-family polymerase, which is referred to as Pol III. Some bacteria contain two C-family DNA polymerases, which are referred to as Pol C (the replicase) and DnaE polymerase (after the gene encoding it) (3). E. coli contains two Y-family DNA polymerases, Pol IV and Pol V. Y-family DNA polymerases have relatively low fidelity and thus differ from typical DNA polymerases, which usually have very high fidelity in DNA synthesis (2, 4). For example, DNA polymerases I, II, and III make mistakes only once every 104-106 nucleotide additions, and fidelity is assisted in these enzymes by the presence of a 3'-5' exonuclease, which is referred to as a proofreader that removes most of the mistakes made by the polymerase (i.e., misincorporated nucleotides) (2, 4). In contrast, Y-family polymerases have intrinsically high error rates (10-1-10-3) and are the only E. coli DNA polymerases that lack 3'-5' exonuclease activity (5, 6). These unique properties allow Pol IV and Pol V to pass over DNA lesions and thus enable chromosome duplication in the face of DNA damage, but they do so at the expense of creating mutations (5, 7). Pols II, IV, and V are induced by DNA damage and are proposed to function collectively and to enable replication over DNA lesions (4, 8-10)
In this review we summarize the general architecture of DNA polymerases and the chemistry of the DNA polymerase and 3'-5' exonuclease activities (see also DNA replication). The ultimate function of DNA polymerases is the duplication of genetic material, and therefore, we also describe how Pol III functions at a replication fork. Lastly, we present a brief overview of the different repair reactions in which the remaining DNA polymerases act.
Table 1. DNA polymerase families
Bacterial Pol I, phage T5, T7
B Family
Bacterial Pol II, Eukaryotic Pol α, Pol δ, Pol ε
C Family
Bacterial Replicases, Pol III, Pol C, DnaE
Y Family
E. coli Pol IV, Pol V
Eukaryotic Pol β, Nucleotidyl Transferases
Chemistry of DNA synthesis and 3'-5' exonucleolytic proofreading
The substrates for DNA synthesis are a 3' primed site and deoxyribonucleoside triphosphate (dNTP) (see Fig. 1). DNA polymerase catalyzes a phosphoryl transfer reaction that adds a dNMP moiety to a 3' terminus of an existing DNA strand, releasing pyrophosphate (Fig. 1a). The reaction is catalyzed exclusively by two metal ions (e.g., Mg++) (11, 12). Metal ion A extracts a proton from the DNA primer 3' terminal hydroxyl group to produce a oxyanion nucleophile, which attacks the α-phosphate of the incoming dNTP that is base-paired to the template. Metals A and B stabilize the resulting penta-coordinate transition state, and metal B also stabilizes the pyrophosphate leaving group.
The two metal ions are held in place by three conserved aspartic acid residues. The fact that catalysis is mediated exclusively by metal ions with no direct participation of amino acid side chains suggests that nucleotide polymerization may have originated before the evolution of proteins. Specifically, the two metals may have been chelated by RNA in the primordial "RNA World" that is thought to have operated before the evolution of proteins.
The 3'-5' proofreading exonuclease is called into action when an incorrect dNMP is added to the 3' terminus. The 3'-5' exonuclease is located in a separate domain with a distinct active site. The chemical reaction of the exonuclease proceeds by hydrolysis (see Fig. 1b), but it is remarkably similar to the polymerase reaction. Specifically, two metal ions catalyze the reaction; metal ion A activates water to form a hydroxyanion nucleophile that attacks the phosphodiester bond of the 3' terminal mismatched nucleotide. Metal B stabilizes the developing charge on the dNMP leaving group.
Figure 1. Chemical mechanism of DNA polymerase and 3'-5' exonuclease. (a) DNA polymerase reaction. The enzyme chelates two metal ions using three aspartic acid residues (only two are shown). Metal ion A abstracts the 3' hydroxyl proton of the primer terminus to generate a nucleophile that attacks the a-phosphate of an incoming dNTP substrate. The phosphoryl transfer results in production of a pyrophosphate leaving group, which is stabilized by metal ion B. (b) The 3'-5' exonuclease proofreading activity is located in a site that is distinct from the polymerase site; yet it uses two-metal-ion chemistry similar to DNA synthesis. The reaction type is hydrolysis in which metal ion A activates water to form the hydroxy anion nucleophile. Nucleophile attack on the phosphate of the mismatched nucleotide releases it as dNMP (dGMP in the case shown).
Structure of DNA polymerases
The crystal structures of multiple representatives of each class of DNA polymerase have been solved and compared (12). In all cases, the overall shape is that of a right hand, and it contains a minimum of three subdomains, which are referred to as the palm, fingers, and thumb. These subdomains are indicated for the structure of Pol I shown in Fig. 2a. Although the chain folding patterns of the fingers and thumb are different among the different polymerase families, the core architecture of the palm domain contains the catalytic site for DNA polymerization and is structurally conserved among the A, B, and Y families. The chain topology of the palm, and the relative location of the three Asp residues that hold the catalytic metal ions in polymerases of the A, B, and Y families, are shown in Fig. 2b. This conservation of structure in the palm suggests that the members of the A, B, and Y family share a common evolutionary ancestor.
Interestingly, C-family DNA polymerases (bacterial replicases) have a different chain topology in the palm, and the location of the Asp residues are also unique (13, 14). The palm architecture is shared by the X-family of DNA polymerases that include eukaryotic Pol P and certain nucleotidal transferases (Table 1). Presumably X-family polymerases share a common ancestor with DNA polymerases of the C-family.
In all DNA polymerases, the DNA lies on the palm of the polymerase and also interacts with the thumb (see Fig. 2c). The finger domain binds the incoming dNTP for incorporation into the DNA chain. The A-T and G-C base pairs have very similar geometry, and during binding of a dNTP, the fingers domain closes over the palm to match the dNTP to the template strand forming a tight enclosure into which only the geometry of a correct base pair can fit (15). Incorrect base pairs do not have the correct geometry to fit into this tight enclosure and are usually released instead of incorporated. In the rare instance of incorrect nucleotide incorporation, the primed site usually enters the 3'-5' exonuclease site for excision of the incorrect terminal nucleotide (see Fig. 2c).
The 3'-5' exonuclease intrinsic to DNA polymerase I, and most DNA polymerases, contributes to high fidelity by the kinetic scheme shown in Fig. 3. Most often, the DNA polymerase selects and incorporates the correct dNTP (pathway 1 in Fig. 3). Correct incorporation events are rapid. Incorporation of an incorrect dNTP (pathway 2 in Fig. 3) is slow, several orders of magnitude slower than incorporation of a correct dNTP (16, 17). Misincorporation is slow because the incorrect dNTP does not form a normal base pair with the template strand, and thus, the geometry of the incorrect base pair does not induce the correct fit in the enzyme active site needed to bring the two active site metal atoms into the proper juxtaposition for the chemical step. Furthermore, if the DNA polymerase incorporates the wrong nucleotide, the resulting mismatched 3' terminus presents yet another slow kinetic barrier to the DNA polymerase. The polymerase is very slow to extend the mismatched product by another nucleotide, because the correct geometry of the active site metals depends on the DNA substrate being correctly base paired. Therefore, the slow kinetics of two steps, misincorporation and extension of a mispaired 3' terminus, result in very few mistakes relative to the rapid incorporation of correct dNTP substrates. The kinetic pauses associated with incorporation of an incorrect dNTP give time for a mismatched primed template to melt and reposition into the separate active site of the 3'-5' exonuclease (Fig. 2c and Fig. 3). Once in the 3'-5' exonuclease active site, the proofreading 3'-5' exonuclease rapidly excises the incorrect 3' terminal nucleotide and thereby restores the substrate to one that is fully base paired (dashed arrow in the scheme of Fig. 3). Thus, the slow steps involved in misincorporation, combined with the rapid proofreading exonuclease removal of mismatched nucleotides, provide the DNA polymerase with a second try at chain extension. Most retrials have a positive outcome, which results in the incorporation of a correct nucleotide base.
Figure 2. Structure of DNA polymerases. (a) The structure of Pol I represents the general right-hand shape of all DNA polymerases (PDB ID 1QSY). Subdomains and the 3'-5' exonuclease domain are indicated. (b) The chain topology in the palm domain of DNA polymerases indicates their evolutionary heritage. Left: The A, B, and Y family have four antiparallel sheets supported by two a helices. The three aspartic residues that chelate the metal ions are indicated by dots. Right: The C-family and X-family DNA polymerases contain a set of parallel strands on which the acidic residues that chelate metal are oriented differently from the A-, B-, and Y-family DNA polymerases. (c) During misincorporation of an incorrect nucleotide, the DNA must leave the polymerase active site (left) and enter the 3'-5' exonuclease active site (right). This action requires the melting of three to four base pairs.
Figure 3. Kinetic steps during DNA synthesis favor incorporation of correct dNTPs. Most often the DNA polymerase selects the correct dNTP that forms a correct Watson-Crick base pair with the template strand (pathway 1, left). The chemistry of correct dNTP incorporation is rapid, and it allows the polymerase to proceed rapidly to incorporate subsequent dNTPs. The chemistry of incorporating an incorrect dNTP is slow (pathway 2, right), and subsequent elongation of the mispaired 3' terminus is also slow. These two kinetic barriers provide time for the primed template to switch into the proofreading 3'-5' exonuclease active site, where removal of the mispaired 3' terminus is rapid. The excised and fully base paired primed site then switches back to the DNA polymerase active site (dashed arrow).
DNA polymerase III holoenzyme
High-fidelity chromosomal replication in E. coli is executed by a multicomponent complex referred to as DNA polymerase III holoenzyme (see Fig. 4a) (18-21). Pol III holoenzyme consists of three main subcomponents: Pol III core, β-clamp, and γ-complex clamp-loader. Pol III core is the replicative DNA polymerase that consists of three subunits (α, ε, θ): a exhibits DNA polymerase activity, e performs 3'-5' exonuclease activity necessary for proofreading, and the function of θ is currently unclear.
Pol III core requires direct association with the β-clamp to perform processive DNA synthesis. β tethers the polymerase to its respective template by binding the α subunit of Pol III core while encircling DNA immediately behind the polymerase (see Fig. 4a). Coupling of Pol III core to β results in an increase in both the catalytic rate (~1kb s-1) and processivity (>50 kb).
The structure of the β-clamp resembles a symmetrical ring with sufficient space to accommodate double-stranded DNA (see Fig. 5a) (22). β is composed of a homodimer with each protomer consisting of three globular subdomains. The structure of the eukaryotic clamp, proliferating cell nuclear antigen (PCNA), is similar to β (23, 24). However, PCNA is composed of a homotrimer with each protomer consisting of two subdomains. The head-to-tail protomer organization of β results in two structurally distinct "faces"; the C-terminal face is involved in intermolecular interactions (i.e., binding Pol III core α subunit) (see Fig. 5a and 5b). β also binds Pol I, II, IV, and V and is therefore thought to play a role in polymerase trafficking during translesion synthesis (which is discussed in more detail below). Lastly, recent studies indicate that β interacts with DNA repair proteins MutS, MutL, and DNA ligase (25). The function of β in DNA repair is currently unclear.
The γ-complex clamp-loader is a heteropentameric ATPase responsible for assembling β onto DNA at primed sites (26-28). The clamp loading process is illustrated in Fig. 5b. The γ-complex binds and opens the ring-shaped β-clamp in the presence of ATP. The ATP-bound γ-complex selectively binds to a primed site that stimulates ATP hydrolysis and results in release of the clamp-loader and closure of β around DNA (20).
γ-complex is a member of the AAA+ (ATPases Associated with various cellular Activities) family of proteins that are involved in molecular remodeling activities (29). The "minimal" γ-complex includes only the subunits required for β-clamp assembly (γ3, δ, δ'). Importantly, the subunit composition of γ-complex exists in various states within the cell (reviewed in References 18 and 20) (which is discussed in more detail below). γ is the only subunit to exhibit ATPase activity and is therefore referred to as the "motor." δ is responsible for opening the P-clamp and is referred to as the "wrench." δ' is proposed to act as a "stator" (a part against which other parts of a machine move) because it is thought to be rigid relative to the other subunits.
The five clamp loading subunits (γ3, δ, δ') of the γ-complex are arranged in a spiral (see Fig. 5c) (26). A similar spiral-like subunit organization is found within the pentameric eukaryotic clamp-loader, RFC (30). The three ATP binding sites of γ-complex lie at the subunit interfaces and contain SRC (serine-arginine-cysteine) motifs common among all clamp-loaders, although mechanistic differences are proposed to exist in how ATP is coupled to the clamp loading process in bacterial and eukaryotic clamp loaders (31).
Pol III function at the replication fork
The proteins currently thought to act at the replication fork include two Pol III core molecules, two β-clamps, γ-complex, DnaB helicase, primase, and single-stranded DNA binding protein (SSB) (see Fig. 4a) (20, 32). Each Pol III core is dedicated to either the leading or the lagging strand and is tethered to its respective template via binding to the β-clamp. The unwound portion of the lagging strand is thought to form a "trombone" loop that enables codirectional movement of the lagging strand polymerase and the replisome (33).
The clamp loader acting at the replication fork is thought to be composed of subunits γ, Τ2, δ, δ', χ, ψ (32, 34, 35). In this configuration, two Y subunits are substituted by two τ subunits. γ (47kDa) and τ (71kDa) share identical N-terminal clamp loading sequences as they are encoded by the same gene, dnaX. However, γ is truncated because of an efficient translational frameshift that produces approximately equal amounts of γ and τ. The χ subunit connects to SSB and is held in the structure by ψ (36). The χ-to-SSB connection enhances processivity and enables efficient transfer of RNA primers from primase to the clamp loader (37). Each τ subunit binds to a single Pol III core thereby linking the leading and lagging strand polymerases together (see Fig. 4a) (14, 21, 38, 39).
DnaB is the replicative helicase that unwinds the parental duplex DNA ahead of the replication fork (see Fig. 4a) (40). DnaB encircles the lagging strand as a homohexamer and uses the energy of ATP hydrolysis to unwind DNA with 5'-3' polarity (see also DNA Helicases, Chemistry and Mechanisms of). DnaB is connected to Pol III holoenzyme via its interaction with the τ subunit of the clamp loader (41). This interaction greatly stimulates DnaB activity at the replication fork (42). DnaB also binds to and stimulates primase, which is a specialized RNA polymerase that synthesizes RNA primers approximately 12 nucleotides in length to initiate DNA synthesis (1, 43).
The mechanism of Pol III action at the replication fork is illustrated in Fig. 4. The lagging strand is synthesized as discontinuous sections of 1-3 kb called Okazaki fragments. An Okazaki fragment is initiated by primase action (see Fig. 4a). The clamp loader uses the energy of ATP hydrolysis to assemble P onto the newly synthesized RNA primer (see Fig. 4b). The Pol III core binds P and processively synthesizes the DNA portion of the Okazaki fragment, which creates a DNA loop (see Fig. 4c). The Okazaki fragment is completed when Pol III encounters the 5' end of the previous Okazaki fragment and dissociates from the DNA, which disassembles the loop. Pol I is required for Okazaki fragment maturation and is discussed below.
The leading strand polymerase performs DNA synthesis in a continuous fashion and therefore requires one initial priming event. However, DNA damage or other impediments along the leading strand may block progression of the replication fork and lead to additional priming events enabled by replication restart mechanisms (reviewed in Reference 44). Lagging strand blocks, such as damaged bases, are inconsequential to the progression of the replisome because the lagging strand polymerase readily cycles among numerous primed sites (45-47).
The repair of arrested replication forks involves two major pathways: recombinational repair and translesion synthesis. Recombinational repair uses RecA and a host of other recombination factors that facilitate strand exchange and non-mutagenic replication across damaged DNA (reviewed in References 48-50). Translesion synthesis employs low-fidelity DNA polymerases that often perform mutagenic replication opposite a damaged template (reviewed in References 5-7).
Figure 4. Pol III function at the replication fork. Leading and lagging strand synthesis are performed in a continuous and discontinuous fashion, respectively. The lagging strand is synthesized in contiguous sections called Okazaki fragments. (a) An Okazaki fragment is initiated as primase synthesizes a short RNA primer along the lagging strand. (b) The γ-complex clamp loader uses the energy of ATP hydrolysis to assemble p at the newly primed site. (c) The lagging strand polymerase cycles to a newly synthesized primed site after completing the previous Okazaki fragment. (d) The lagging strand polymerase synthesizes the DNA portion of the next Okazaki fragment. (Adapted with permission from Reference 20.)
Figure 5. Structure and function of β-clamp and γ-complex. (a) The structure of p resembles a ring with sufficient space to accommodate duplex DNA. β is a homodimer in which each protomer contains three globular subdomains. The protomers are arranged in a head-to-tail fashion resulting in two structurally distinct ''faces.'' The C-terminal face is involved in intermolecular interactions. Protomers are indicated in light and dark shades (PDB ID 2POL). (b) γ-complex uses the energy of ATP hydrolysis to assemble β onto DNA at primed sites. γ-complex binds to and opens β in the presence of ATP. The clamp loader selectively binds to primer-template junctions, which stimulates ATP hydrolysis and results in dissociation of γ-complex and closure of β around the DNA. DNA polymerase binds the C-terminal face of p and initiates DNA synthesis at the primed site. (c) Subunit organization of y-complex clamp loader. The γ-complex is composed of five subunits (γ3, δ', δ) that are arranged in a spiral-like fashion. γ-complex contains three ATP sites that lie at the subunit interfaces and include an ''arginine finger'' within a conserved SRC motif. The ATP site organization of the γ-complex is proposed to confer cooperativity between subunits. (Adapted with permission from Reference 20.)
Translesion synthesis by low-fidelity DNA polymerases
Chromosomal DNA is often damaged by exposure to exogenous (i.e., UV irradiation, chemical agents, etc.) and endogenous (i.e., oxidative damage) insults (see also DNA Damage, Chemical Biology of Diseases Related to (51). Spontaneous mutagenesis of nucleotide bases by cytosine deamination to yield uracil also occurs with high frequency in the cell. DNA repair mechanisms have evolved to excise and repair various types of DNA damage (discussed below) (see also DNA Damage Repair, Chemistry of). However, the replication fork sometimes encounters DNA damage that has evaded the repair machinery. Pol III and other high-fidelity DNA polymerases cannot replicate damaged DNA. Thus, the replication fork becomes arrested at the site of DNA damage. Low-fidelity lesion bypass DNA polymerases can extend DNA across various types of damaged DNA, but this often results in a mutation. Thus, switching between high-fidelity and low-fidelity DNA polymerases is thought to facilitate replication through sites of DNA damage (4, 8-10, 52).
Pol I and Pol III exhibit high-fidelity DNA synthesis and make mistakes on average of only 1 in 105-107 nucleotides (5, 16, 17). Y-family Pol IV and Pol V misincorporate nucleotides 10 to 1000 times more frequently and are error-prone lesion bypass polymerases that are associated with the mutagenic response of cells to DNA damage (5, 6, 16, 52). Structures of Y-family polymerases suggest that replication of past aberrant nucleotides is likely caused by a highly solvent exposed DNA binding cleft (53, 54). Thus, bulky lesions and nucleotide mismatches are more permissible among this class of enzyme.
Replication fork arrest by DNA damage leads to RecA mediated induction of the SOS response pathway. The SOS response involves upregulation of over 40 genes that facilitate increased viability during stress (i.e., chemical agents, UV light, and nutrient deficiency) (reviewed in References 5 and 6). The DNA polymerases that are upregulated during the SOS response include Pol II, Pol IV, and Pol V. Pol II is the first DNA polymerase to be induced (~1 minute). Interestingly, Pol II has been shown to perform high-fidelity DNA synthesis (error rate ~10-5 to 10-6 per base pair) on undamaged templates, but despite this, Pol II seems to be involved in error-prone DNA synthesis in vivo (4, 55). Pol II is somewhat of an enigmatic enzyme because it has been implicated in translesion synthesis, adaptive mutation, and recombinational repair (which is discussed below) (4-6, 56). Additional study is required to understand the relevant functions of Pol II in the cell.
Pol V is upregulated from <15 to ~200 copies per cell approximately 45 minutes after SOS induction. Pol V is a heterotrimer with a subunit composition of UmuD'2C (see Fig. 6a). UmuC contains the catalytic domain of Pol V. UmuD' is the product of RecA-mediated proteolytic cleavage of UmuD. Importantly, Pol V function requires RecA, and the mechanism by which RecA stimulates Pol V activity has been under investigation for several years (57). Recent data indicate that RecA nucleoprotein filaments act in trans to stimulate Pol V (see Fig. 6a) (57, 58). Interestingly, RecA filaments in cis (immediately 5' to Pol V on the DNA template) do not stimulate Pol V (59). Pol V and SSB may cooperate to displace RecA from DNA (57, 60). Alternatively, recent data indicate that RecA may be removed by UvrD helicase, as UvrD is also induced during the SOS response, but whether UvrD plays a direct role in translesion synthesis is currently an open question (61, 62).
Pol IV and Pol V use the β-clamp (63-66) and are capable of synthesizing DNA opposite various types of lesions (i.e., abasic site, thymine dimer, and benzo(a)pyrene) (see Table 2) (4, 8, 9, 67). Pol IV is expressed constitutively at ~250 copies per cell, suggesting a role during normal growth, and consistent with the finding that Pol IV is required for adaptive mutation under nonlethal conditions (6). Pol IV is increased 10-fold after SOS induction where it is presumably used to bypass lesions. In contrast, Pol V is undetectable during normal growth conditions and probably only plays a role during DNA damage.
All five E. coli DNA polymerases function with β, and therefore, it is perhaps not surprising that recent biochemical studies demonstrate that P plays a central role in different polymerases switching on DNA primed sites during translesion synthesis (7, 58, 68). A model for Pol IV/Pol III switching during translesion synthesis based on structural and biochemical studies is illustrated in Fig. 6b. The dimeric β-clamp can bind both Pol IV and Pol III simultaneously, and it is proposed to hold Pol IV away from the DNA template while Pol III actively synthesizes DNA (68, 69). However, during a Pol III encounter with a lesion, Pol IV is allowed to gain access to the template in place of Pol III. After the lesion is bypassed by Pol IV, the high-fidelity Pol III regains control of the primed site. These events limit the action of the low-fidelity Pol IV to the region in which it is required to bypass a lesion.
Figure 6. Mechanisms of translesion synthesis. (a) Activation mechanism of Pol V during translesion synthesis. Pol V is a heterotrimer composed of subunits UmuC, D'2: UmuC is the catalytic domain, and UmuD' is the product of RecA mediated proteolysis. Translesion synthesis by Pol V is activated by the presence of a RecA filament in trans. (b) Model of DNA polymerase switching during translesion synthesis. Pol III* and Pol IV each bind to a p protomer at a conserved hydrophobic protein binding pocket (QL[S/D]LF). 1. Pol III* is arrested at the site of DNA damage, whereas Pol IV is held in an inactive state away from the DNA. 2. Pol IV gains hold of the primer terminus from Pol III* at the stall site; Pol III* is now held away from the DNA. 3. Pol IV extends the DNA past the lesion. 4. Pol III* regains hold of the primer terminus from Pol IV.
DNA polymerase activity during repair
Oxidative damage that leads to replication fork arrest is common during bacterial growth and probably occurs at least once during each cell division even under the most favorable conditions (48, 51). Most lesions are not dealt with by mutagenic polymerases, but instead they are repaired by recombination, which is an error-free process. Recombinational repair can take many different paths and employs several different proteins, but ultimately, it involves high-fidelity DNA polymerases such as Pol I, Pol II, and/or Pol III (6, 49, 56). For additional information, we refer the reader to more thorough reviews of the various recombination repair pathways (44, 48-50, 70).
Even high-fidelity DNA polymerases sometimes incorporate an incorrect nucleotide. All cells contain a specialized mismatch repair system that catches these errors and corrects them. Mismatch repair involves excision of DNA past the mismatch, and eventually, it allows Pol III to try again (reviewed in References 71 and 72). The process of mismatch repair requires proteins MutS and MutL, among others. Mutations in the human homologues of these proteins lead to a predisposition to development of tumors (73).
Nucleotide excision repair (NER) is responsible for removing a wide array of DNA lesions that predominately occur as a result of exposure to oxygen or UV light (reviewed in Reference 74) (see also DNA Damage, Chemical Biology of Diseases Related to)). Unlike mismatch repair, NER employs Pol I to replace damaged DNA. NER also requires the action of Uvr (ultraviolet radiation) proteins that were originally discovered because of their ability to sustain cell growth after exposure to UV irradiation. Like MutS and MutL, mutations in the human homologues of the Uvr proteins predisposes individuals to cancer.
Base excision repair (BER), in which a single damaged base is excised and replaced, also uses Pol I to replace the modified nucleotide. BER is involved predominately in the repair of apurinic and apyrimidinic nucleotides [abasic (AP) sites) that frequently develop from spontaneous hydrolysis (reviewed in Reference 75). Importantly, BER is also responsible for removing uracil from DNA, which is the product of cytidine deamination. Bases modified by oxidation and alkylation are also repaired; however, these modifications occur less frequently.
A recent study suggests that Pol IV and Pol V may also be involved in BER because of their intrinsic lyase activity (76). Shen et al. demonstrate relatively weak 5'-phosphodiester bond cleavage of an abasic site (AP/5'-dRP activity) by Pol IV and Pol V. Interestingly, several DNA polymerases from the X, A, and Y families exhibit lyase activity. Human Pol P, for example, exhibits strong AP/5'-dRP activity and is thought to be involved in the excision and incorporation of nucleotides during BER (77, 78). Furthermore, eukaryotic pol i, which is a Y-family homolog of Pol IV and Pol V, has also been shown to exhibit AP/5'-dRP activity (79). Additional studies are required to determine whether Pol IV and Pol V are involved in BER.
Lastly, Pol I is responsible for repairing DNA during Okazaki fragment maturation. Pol I replaces the RNA portion of Okazaki fragments using its 5'-3' exonuclease activity and synthesizes DNA in its place. The remaining nick between adjacent Okazaki fragments is repaired by DNA ligase.
Table 2. Properties of translesion DNA polymerases
DNA polymerase
Abasic site (2, 80)
Pol IV
-1 frameshift
Pol V
Preferential incorporation of dATP and dGTP
Benzo(a)pyrene (4, 81)
Pol IV, Pol V
N-2-acetylaminofluorene (4)
Pol IV, Pol II
-1,-2 frameshift
8-oxo-guanine (9)
6-4 Thymine-thymine photoproduct (2)
T → C
Adapted with permission from Reference 5.
Future perspectives
The work of many laboratories has provided significant insight into the detailed workings of Pol III function at the replication fork. The way in which the replisome interacts with other DNA processes (i.e., DNA compaction, transcription, and recombination, repair), however, is poorly understood. For example, how does Pol III holoenzyme replicate through high-affinity protein-nucleic acid complexes such as RNA polymerase or repressor proteins? What is the fate of the replication components after an encounter with a blocking lesion? Does the replisome directly recruit or interact with recombination factors that repair broken forks? p has been shown to bind mismatch repair proteins MutS and MutL. What is the significance of these interactions?
The presence of five DNA polymerases in E. coli suggests that access to primed sites might be regulated. Indeed, recent biochemical data indicate that p plays a major role in switching between high-fidelity and low-fidelity DNA polymerases during translesion synthesis. High-fidelity DNA polymerases Pol I and Pol II also bind to p and are expressed at relatively high levels relative to Pol III. Do high-fidelity DNA polymerases compete for primed sites during normal growth? Furthermore, we still do not understand the major role of Pol II in the cell.
Lastly, translesion synthesis by Pol V is stimulated by RecA filaments in trans. The presence of RecA filaments in cis (on the DNA template), however, inhibits the activity of Pol V. Additional work is required to elucidate the exact mechanism and organization of RecA filaments in the cell responsible for Pol V activation. Future studies will likely reveal the answers to many of the questions addressed above.
The authors are grateful to their source of funding, NI-HGM38839
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28. Pritchard AE, Dallmann HG, Glover BP, McHenry CS. A novel assembly mechanism for the DNA polymerase III holoenzyme DnaX complex: association of deltadelta' with DnaX(4) forms DnaX(3)deltadelta'. Embo. J. 2000; 19:6536-6545.
29. Neuwald AF, Aravind L, Spouge JL, Koonin EV. AAA+: A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genome Res. 1999; 9:27-43.
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31. Neuwald AF. Hypothesis: bacterial clamp loader ATPase activation through DNA-dependent repositioning of the catalytic base and of a trans-acting catalytic threonine. Nucleic Acids Res. 2006; 34:5280-5290.
32. McHenry CS. Chromosomal replicases as asymmetric dimers: studies of subunit arrangement and functional consequences. Mol. Microbiol. 2003; 49:1157-1165.
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34. O'Donnell M, Jeruzalmi D, Kuriyan J. Clamp loader structure predicts the architecture of DNA polymerase III holoenzyme and RFC. Curr. Biol. 2001; 11:935-946.
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44. Heller RC, Marians KJ. Replisome assembly and the direct restart of stalled replication forks. Nat. Rev. Mol. Cell Biol. 2006; 7:932-943.
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46. Pages V, Fuchs RP. Uncoupling of leading- and lagging-strand DNA replication during lesion bypass in vivo. Science 2003; 300:1300-1303.
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48. Lusetti SL, Cox MM. The bacterial RecA protein and the recombinational DNA repair of stalled replication forks. Annu. Rev. Biochem. 2002; 71:71-100.
49. Cox MM. Recombinational DNA repair of damaged replication forks in Escherichia coli: questions. Annu. Rev. Genet. 2001; 35:53-82.
50. Kreuzer KN. Interplay between DNA replication and recombination in prokaryotes. Annu. Rev. Microbiol. 2005; 59:43-67.
51. Cox MM, Goodman MF, Kreuzer KN, Sherratt DJ, Sandler SJ, Marians KJ. The importance of repairing stalled replication forks. Nature 2000; 404:37-41.
52. Kuban W, Jonczyk P, Gawel D, Malanowska K, Schaaper RM, Fijalkowska IJ. Role of Escherichia coli DNA polymerase IV in in vivo replication fidelity. J. Bacteriol. 2004; 186:4802-4807.
53. Ling H, Boudsocq F, Woodgate R, Yang W. Crystal structure of a Y-family DNA polymerase in action: a mechanism for error-prone and lesion-bypass replication. Cell 2001; 107:91-102.
54. Zhou BL, Pata JD, Steitz TA. Crystal structure of a DinB lesion bypass DNA polymerase catalytic fragment reveals a classic polymerase catalytic domain. Mol. Cell 2001; 8:427-437.
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57. Schlacher K, Goodman MF. Lessons from 50 years of SOS DNA-damage-induced mutagenesis. Nat. Rev. Mol. Cell Biol. 2007; 8:587-594.
58. Schlacher K, Cox MM, Woodgate R, Goodman MF. RecA acts in trans to allow replication of damaged DNA by DNA polymerase V. Nature 2006; 442:883-887.
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60. Pham P, Bertram JG, O'Donnell M, Woodgate R, Goodman MF. A model for SOS-lesion-targeted mutations in Escherichia coli. Nature 2001; 409:366-370.
61. Centore RC, Sandler SJ. UvrD limits the number and intensities of RecA-green fluorescent protein structures in Escherichia coli K-12. J. Bacteriol. 2007; 189:2915-2920.
62. Veaute X, Delmas S, Selva M, Jeusset J, Le Cam E, Matic I, Fabre F, Petit MA. UvrD helicase, unlike Rep helicase, dismantles RecA nucleoprotein filaments in Escherichia coli. Embo. J. 2005; 24:180-189.
63. Dalrymple BP, Kongsuwan K, Wijffels G, Dixon NE, Jennings PA. A universal protein-protein interaction motif in the eubacterial DNA replication and repair systems. Proc. Natl. Acad. Sci. 2001; 98:11627-11632.
64. Wijffels G, Dalrymple BP, Prosselkov P, Kongsuwan K, Epa VC, Lilley PE, Jergic S, Buchardt J, Brown SE, Alewood PF, Jennings PA, Dixon NE. Inhibition of protein interactions with the beta 2 sliding clamp of Escherichia coli DNA polymerase III by peptides from beta 2-binding proteins. Biochemistry 2004; 43:5661-5671.
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67. Fuchs RP, Koffel-Schwartz N, Pelet S, Janel-Bintz R, Napolitano R, Becherel OJ, Broschard TH, Burnouf DY, Wagner J. DNA polymerases II and V mediate respectively mutagenic (-2 frameshift) and error-free bypass of a single N-2- acetylaminofluorene adduct. Biochem. Soc. Trans. 2001; 29:191-195.
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Scrap yards and landfill sites investigations
Gas monitoring
Gas and groundwater monitoring will require the insertion of boreholes to BS10175 specification and then a programme of monitoring that is usually agreed with the local authority contaminated land officer. This typically requires a minimum of three return visits to the site and then sampling for water analysis to pre agreed specifications or gas (typically: methane, carbon dioxide and oxygen) monitoring at different atmospheric pressures.
Leachate assessment and monitoring
Site contamination assessment for planning
Risk assessment work can range from a contamination desk-top survey at a site through to detailed Phase 3 intrusive investigations involving drilling to depth. In all circumstances, the primary goal is to determine all the risks from contamination at a site but with a phased and economical approach. This work is undertaken for private home owners, developers and large organisations.
Our aim is to ensure your site is risk-assessed to the exact requirements of the local planning authority and the Environment Agency as quickly as possible. Our assessments are not off-the-shelf contaminated land surveys that can leave more questions than answers. We engage in dialogue immediately with the regulatory authorities and manage all the land contamination issues needed to gain your planning permission or assess any liabilities.
A typical assessment incorporates and evaluates the findings from a desk top investigation and a site survey. The desk top investigation includes information detailing the history of the site together with any previous reports or correspondence. Following this, a site visit is undertaken to inspect ground conditions, talk with appropriate persons and check on surrounding land uses.
The risk assessment is designed within the source/pathway/receptor framework for contaminated site investigation as outlined in CLR 11, Model Procedures for the Management of Land Contamination and carried out on-site with reference to BS10175, Investigation of Potentially Contaminated Sites – Code of Practice. This framework, in its broadest sense, outlines a dynamic methodology for assessing risk which focuses on a Conceptual Site Model (CSM), the ultimate aim being to pronounce a site suitable for use and if not, the numerical and/or qualitative value of any risk.
A conceptual site model (CSM) is developed with a view to examining the potential risks given the intended operations on, and future use of, the site. The CSM describes the significant parameters, characteristics and processes at the site which have relevance to the likely risk of contamination. An effective CSM requires good quality and relevant information which can then be used within the Contaminated Land Exposure Assessment model (CLEA) and/or for consideration with information from the Total Petroleum Hydrocarbon (TPH) working group recommendations or other approved risk assessment procedures and models such as RISC4.
Reporting will be in a format following the guidance documents recommendations, which in most cases will require separate sections on:
conceptual site model
Please contact us if you require this type of investigation.

redpajama_set_name: RedPajamaCommonCrawl


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Ingrown Hairs & Acne
What Causes Chin Acne?
Dermabrasion or Laser Resurfacing for Acne Scars
Deep Acne Cysts
Causes of Acne & Facial Hair
Scars From Nodular Acne
Topical Treatment for Acne Nodules
By Kristin Leigh ; Updated July 18, 2017
The severity of acne varies from person to person. Most cases of acne consist of non-inflammatory blackheads and whiteheads as well as smaller inflammatory pimples. But some people suffer from more severe blemishes, called nodules. While these blemishes may not be as serious as acne cysts, having multiple acne nodules can become painful. If you suffer from acne nodules, various topical treatments may lessen pain and inflammation.
Acne nodules differ from smaller pimples in both depth and size. Acne nodules occur when the wall of a clogged follicle bursts deep in the skin. The bacteria and debris from inside the pore then spills out into the dermis, or the middle layer of skin. The infection then invades surrounding follicles. This process results in a large, red bump that may be tender to the touch. Unlike cysts, nodules are firm and can be felt deep in the skin.
Benzoyl peroxide is an ingredient in many over-the-counter acne treatments. Products containing benzoyl peroxide may come in the form of cleansers, gels or lotions. Benzoyl peroxide helps reduce inflammatory breakouts by fighting bacteria as well as reducing excess oil production. According to the American Academy of Dermatology's AcneNet, the most common side effect of benzoyl peroxide treatment is excessive drying of the skin. Benzoyl peroxide is primarily indicated for mild acne, but may be effective at treating or preventing an occasional acne nodule.
Topical Antimicrobials
For chronic acne nodules or multiple nodules, prescription treatment may be warranted. Topical antimicrobials focus primarily on fighting the Propionibacterium acnes bacteria. These bacteria live on the skin and contribute to inflammatory acne breakouts. Topical antimicrobials may be prescribed along with treatments that focus on factors other than bacteria. Clindamycin, erythromycin and azelaic acid are examples of antimicrobials prescribed for acne. Possible side effects include skin dryness and irritation.
Topical Retinoids
For moderately severe cases of acne nodules, topical retinoids may be prescribed. According to MayoClinic.com, tretinoin, a topical retinoid, effectively treats acne partly by keeping the pores clear. Topical retinoids also lighten the skin, which may reduce discoloration or red spots caused by acne breakouts. They also replace old skin with new skin and slow down the sloughing of dead skin cells, which prevents a buildup of dead skin cells from clogging pores. Possible side effects include increased sun sensitivity and severe dryness, redness, stinging or peeling of the skin.
Acne nodules are a severe form of pimples and should be evaluated by a dermatologist. People who suffer from mild acne may get the occasional acne nodule that can be treated with over-the-counter products. However, chronic breakouts of multiple acne nodules and cysts warrant dermatological care. Scarring can result from delaying treatment. Sufferers should never attempt to squeeze or pop acne nodules because they occur deep in the skin. Topical treatments may not be sufficient to treat more severe forms of acne, which may require oral medications for successful treatment.
AcneNet: Acne Treatment Available Without a Prescription
MayoClinic.com: Tretinoin (Topical Route)
Kristin Leigh has been writing professionally since 2007. Her work appears on various websites, focusing on topics such as health, beauty, medicine and personal finance. Leigh has worked as a certified medical transcriptionist in various specialties, including family medicine, dermatology and psychology. She is pursuing a bachelor's degree in graphic design from Herzing University Online.
Comedonal Acne
Does Your Face Break Out When You Become Pregnant?
Vaginal Acne
About Causes of Cystic Acne
What Is a Safe Acne Treatment?
Abscesses & Acne


Sun outage may affect your digital TV reception if you receive it via satellite.
It's a technical phenomenon which typically occurs during the months of March/April and September/October, and can last as long as 15 minutes a day and take place over a period of 15 days. On these days your reception may be affected. For example, you may see the "No Signal" message on your television for a short period of time, up to 10 minutes. Normal service will resume after this time.
It happens because the radio frequency noise from the sun is at its peak strength and can interfere with the satellite signal, resulting in a brief service interruption.
You may experience temporary noise or service interruptions. Different areas will be affected on different dates and times during this period.
The effects of a sun outage vary from minimal to total outage throughout the 15 day period. Once it reaches its peak, the interference will gradually decrease becoming less noticeable each day after.
If you are experiencing the outage for longer periods of time, it's likely a symptom that your antenna is not operating in optimum condition. The dish may be affected by corrosion or rain fade, which amplifies the effects of these sun outages. This is especially likely if your satellite dish is more than a few years' old and hasn't had its LNB (Low Noise Blocker) replaced during that time.
We recommend contacting an installer who can address any problems on-site and prevent any future reception issues.
If you're using a UHF aerial and not a satellite dish, your aerial may not be correctly positioned or aligned, or it may be too close to high powered machinery that produce radio interference with your TV signal. It's best to contact your installer to check the aerial and realign or reposition it if necessary. If you don't have a trusted installer, you can find one near you in this installer directory.


A timeless baby handprint keepsake frame that will always be cherished. Our 8x10 wall or tabletop frame with space for 3x3.25 photo and baby's handprint, makes a wonderful new baby gift, baby shower gift or first birthday present for new or expectant parents. Grandparents to be would also treasure this gift to welcome a new grandchild.
Each frame comes with a child-safe stamp and instructions and is available in a white frame with gold stamp or black frame with black stamp. The classic look makes this a perfect addition to baby's nursery wall or a family living area, where it can be displayed for years to come.


Gracie Gold of U.S. competes in the Ladies Short Program event during the ISU figure skating Eric Bompard Trophy competition, at Bordeaux's skating arena, south western France, Friday, Nov. 13, 2015.
Consider the gossamer-thin dresses with tiny skirts, bright lights, judges, and dozens of cameras capturing every square inch of skin. It's no wonder figure skating can deliver a fierce blow to the body image.
Canadian stars Kaetlyn Osmond and Meagan Duhamel can understand how an eating disorder can force a skater to back away from the sport, like American star Gracie Gold. The Olympic bronze medallist confirmed recently she was being treated for an eating disorder, along with anxiety and depression, and is sitting out the Grand Prix season.
"I can't say it surprises me, but it saddens me," said Duhamel, a two-time world pairs champion with partner Eric Radford. "You don't want anybody to have to experience something like that."
According to Canada's National Eating Disorder Information Centre, female athletes in aesthetic sports — figure skating, dance, gymnastics — were found to be at the highest risk for eating disorders. Athletes competing in weight-class sports such as wrestling and endurance sports such as distance running were also at an elevated risk.
"Skating is really hard, especially women's skating where we're judged in little tiny dresses," said Osmond, who captured her second Skate Canada International title this past weekend. "Definitely the way you see yourself makes a big difference, and it's really hard if you are a little bit heavier, the jumps are harder because you're putting more weight up into the air."
Russia's Yulia Lipnitskaya, who captured gold at the Sochi Olympics at just 15, wowing the crowd with her "Schindler's List" program, opened up lately about her battles with anorexia. She said the disease dogged her for several years, and checked into a clinic last January. In her final competition, a Grand Prix last fall, the big jumps proved too much and she stopped mid-program, tears welling up. The judges allowed her to eventually finish her program, but she finished last.
"Ultimately, for me, weight is something I have to deal with every year, but I do it with food, because I love food," Osmond said. "But it's a struggle, it's something that I think about."
Duhamel, who with Radford captured the Skate Canada pairs title over the weekend, is all strength and power in her four-foot-nine frame, but it took her some time to accept her muscular body.
"I don't have a feminine lean body with these long lines, and I tried so hard to be able to get rid of some of the muscle and develop these lines, but you can't change genetics, you can't change your body in a healthy way to a certain extent," said the 31-year-old.
"Sometimes I would go home from competitions feeling so insecure, thinking 'Oh those girls in the changing room, they're so tiny.' I'm tiny, I just have a different body type, I'm more stocky, with a muscular body-type that's not seen as much in pairs skating. As I just got older, I accepted myself for what I have, and I learned I wouldn't be able to do the things I can do on the ice if I didn't have the body I have."
Duhamel said once started studying holistic nutrition — she's currently doing a certification program in sports and fitness nutrition — and became a vegan several years ago, she gained a greater understanding that "you can be in really great shape and enjoy food."
"You need to fuel your body and your mind. you can't focus for an entire day of training if you haven't eaten properly, if you haven't fuelled yourself properly. It goes beyond your body image, it goes to your focus, to your energy, to your sleep. All these things that are so important in your life are fuelled from nutrition."
It's unclear whether Gold will compete at the U.S. national championships for a spot on the Pyeongchang Olympic team. She was fourth at the Sochi Games, and part of the U.S. squad that won bronze in the team competition.
Duhamel and Osmond praised Gold for putting her health first.
"We don't want the sport to negatively affect somebody, and the fact that maybe being involved in a sport that is about performance and wearing these little dresses on the ice, to think that might have affected their life in such a negative way, it's sad and it's disappointing," Duhamel said. "Everybody wishes well for anybody who's going through that situation, and wants them to be able to come out the other side with an even better perspective."
Added Osmond: "The people who are dealing with it now, I have the utmost respect for them because this sport is really hard, it's hard on the body."


Family owned and operated with more than 30 years of combined experience in the turf and landscape industry, it is our goal to offer top quality products and services to assist you in all aspects of your landscape. Contact us today to learn more about our services and request a free estimate.
Absolute Precision Landscape & Supply is YOUR full-service landscaping company specializing in both residential and commercial properties. Take a look at some of our recent work!
At Absolute Precision Landscape & Supply, we take great pride in our work and feel there are several traits that set us apart from our competitors. One is a management style built on communication, prioritizing and the ability to set and achieve goals. We are self-motivated individuals who feel the key to a successful team lies in individual work ethic and leadership. We feel these experiences have instilled the commitment to provide exceptional work on a daily basis through an acute attention to detail. Overall, our team's professionalism, inherent drive and strong leadership skills will enable Absolute Precision Landscape and Supply to fulfill the responsibilities for your landscape needs.
Company owners and operators are graduates of The Ohio State University with degrees in Turfgrass Management, Landscape Architecture, Landscape Horticulture and Construction. All Technicians are certified commercial pesticide applicators.


Your patients and clients are searching for your services online, can they find you?
so that those searching for your services can find you.
Content marketing, in-bound and earned media marketing solutions for health and senior care industries.
Almost 90% of web traffic comes from unpaid content on search engines, social media, the blogosphere, online media and digital communities.
Work with our professionals to develop educational articles, ask-the-expert pieces, Q&A, events, and other relevent articles to engage your target audience.
Photography and videography services are available.
All content is presented in an accurate, fun and engaging way to drive trac to your website.
The goal is to maximize online media interest, social community discussion, search engine marketing and conversion.
All content is distributed automatically to numerous digital channels through our proprietary platform to drive trac to your business.


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< Back to Blog Listing
Exploring Trail Ridge Road
A couple of weeks ago, I ventured up Trail Ridge Road which goes above the tree line to 3600 metres above sea level(!!!).
For those of you in Melbourne, take two Mount Buller's (and a little bit more), put them on top of each other, and you get the same height above sea level on Trail Ridge Road. Averaging 4% for 35km and getting steeper towards the top, it took me just under two hours of climbing to reach the summit. The ride started in green paddocks and favourable conditions in the low 20's. However, by the time I reached the top I was riding alongside snow walls at least a couple of metres high, temperatures had dropped to barely above zero, and black clouds rolled in. Well, it doesn't rain at 3600m, it hails, and hail hurts! …so I didn't hang around for long and started descending ASAP.
ONLY two days earlier, conditions were like this and the road was closed. I'd have been blown of the road, and likely turned into a white walker…
This was one of the most memorable training rides I've done to date, (up there with the first time I decided to climb up Mt Hotham and bonked)… I'm sure riding something like the Stelvio in Italy lined with snow would trump this, but I've never had to opportunity to ride it. There's an information visitors centre at the top, which I'm guessing serves a great hot chocolate, but unfortunately I couldn't hang around for long. If you're after a ~300km epic, you can also descend down the other side and loop around via Grand Lake.
The descent was long and cold, and I had to slow down pretty quickly to avoid a few herds of Elk. (Are they called herds?). After climbing for pretty much for 4 hours and over 70km, the hard work was done and it was mostly all downhill back home, for a round trip of just under 7 hours and 190km. Oh, and a stop at the Bakery of course. Photo's don't do it justice, but here's a few extra photo's from the day!
And if you want to check it out, here's a link to the strava: https://www.strava.com/activities/592501528/overview
Thanks for reading, BC
DCIM100GOPROG0105925.
Alex McCallum
AWESOME – (adjective
"causing or inducing awe; inspiring an overwhelming feeling of reverence, admiration, or fear: an awesome sight)".
That's all I can about your Trail Ridge ride besides Congrats. Hopefully you get to race Passo di Stelvio soon enough. :))
bman11476@gmail.com
Thanks for sharing Alex! (I'll work on the share button so it has a relevant image on facbeook rather than a sponsor logo!) …Yes, can hope to race the Stelvio one day, or at the very least, go training up there!
Andrew Stalder
Very nice! Out there solo or with a crew at all? Nice to see no arm or leg warmers and meters of snow on the side of the roads……hopefully means you were working. 😉
Solo km's! …couldn't keep out of the wind 😉
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RT @drapac_cycling: "My team mates were really good today being active in a very aggressive race. It allowed me to sit back and save myself…
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RT @Ride_Argyle: We're in it to win it with this #PinkArgyle squad for #TOU18. Our seven-man roster includes 2015 winner @JoeDombro and 201…
RT @drapac_cycling: After taking out the Time Trial on Wednesday, @MagennisLiam continued his run of good form and rounded out the podium i…
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Deep Silver has released a new F1 2018 gameplay trailer, showcasing the new features and refinements Codemasters has added, to the game this year. F1 2018 launches on August 24 for PlayStation 4, Xbox One and PC. Check out the trailer and press release below.
Codemasters® has released the first full gameplay video for F1® 2018, the official videogame of the 2018 FIA Formula One World Championship™, which reveals the incredible depth and feature set of this year's game. Launching onto PlayStation®4 computer entertainment system, the Xbox One family of devices including the Xbox One X and Windows PC on August 24, 2018, F1® 2018 lets you follow the path and career of an F1® driver like never before.
"This is the most complete F1® experience that Codemasters has ever created," said Game Director Lee Mather. "The Career mode is the central point of the game and has been greatly expanded with the inclusion of pressurized press interviews that will directly affect your path through the sport. There is also far more flexibility for the player, including the ability to select your own rival and negotiate team moves during the season. The Career mode also expands the player's research and development options, with each F1® team now having its own unique technology tree with differing strengths and weaknesses. What's more, F1® 2018's Career will also feature significant rule changes between seasons during the Career for the first time.
When playing online, the game's new Super License system is designed to minimize the effects of poor driving in multiplayer races. Players are rated on their skill levels and driver etiquette and then matched with other gamers of a similar ability and driving style.
Looking and sounding great. Nice new additions to the game. Can't wait to jump into this one. I just hope it's a bit easier to earn resource points this year. Having to do all those tests during practice can get pretty time consuming if you're trying to earn max points. I love it, because it really helps create that feeling you're working with a real F1 team during a typical race weekend, it makes practice sessions mean something, but it sure can be time consuming if you're just looking for a qualify and race experience.
The game looks stunning! I can't wait to get my hands on it.
On the verge of buying my first F! video game. This one just might be it.
I wish Codemasters had the rights to NASCAR. I'd kill to get my hands on a NASCAR game that drives like these F1 games.


Oh Paris, you are heaven! You have the best patisseries. Your fashion week's a dream. You gave us Carine Roitfeld. And because her latest CR Fashion Book Issue 2 is just about to go on sale, she joined up with Mercedes-Benz and threw the biggest party of Paris Fashion Week at the Shangri-La Hotel Paris. Kate Moss sauntered in with Jamie Hince, Jessica Alba and Jessica Chastain sneaked off to catch up with Julia Restoin Roitfeld. Caroline Sieber did a turn on the dancefloor, Alber Elbaz chatted to Alexander Wang and everyone sipped champagne while Marie-Agnès Gillot performed. And Carine? Well, she spent the night giggling with Karl Lagerfeld and Kanye West. Obviously.


text:
Q: Is it OK to overload ShowDialog() so that a child form returns information as an out parameter? In an earlier question about how to return a string from a dialog window, yapiskan suggested overloading the child form's ShowDialog() method to include an out parameter.
My question is whether or not this is a good approach in C#.
Here is some example code, based on yapiskan's suggestion.  In the child form (in this example, it's a form with a textbox on it), you just need to add the ShowDialog overload and assign UI values to the out parameter:
public DialogResult ShowDialog(out string s)
{
    DialogResult result = this.ShowDialog();
    s = this.textBox1.Text;
    return result;
}

And to show the form and retrieve the entered text, you do this:
using (CustomDialog frm = new CustomDialog())
{
    string s;
    if (frm.ShowDialog(out s) == DialogResult.OK)
    {
        // do something with s
    }
}

One advantage I can think of is that this approach forces the user of the CustomDialog form to get the information it contains through the form's ShowDialog method (rather than from a who-knows-what-it's-called method like GetMyData() or something).

A: Better to have a Public property/method and get the information.
What would you do if you would need 3..4..5 informations, having 5 parameters out? More clean to have accessors to get your information from the Dialog.

A: It should not be OK since .net framework does not use this design. In the case of OpenFileDialog class, it has a parameterless ShowDialog() method returning a DialogResult. Once this method called, user is supposed to get the selected files by using the FileName, FileNames, SafeFileName and SafeFileNames methods.
Let's assume that this implented in the "out parameter" way. I would have to write code like this just to get the SafeFileName:
string dummyFileName;
string[] dummyFileNames;
string safeFileName;
string[] dummySafeFileNames;

myDialog.ShowDialog(out dummyFileName, out dummyFileNames, out safeFileName, out dummySafeFileNames);

A: Personally I try to avoid out parameters wherever possible, although I understand that like GoTo they are sometimes a necessary evil. I would say that it would be much better to use properties or methods to return the information.

A: In my experience, a custom modal dialog that collects only one piece of information is a pretty extreme outlier.  Much more common are zero and many.
And a dialog that collects many pieces of data is almost certain to be modified at some point to collect just one more.  I'd much rather fix only the code that uses that one new piece of data than every single piece of code that uses the modified dialog.
Also, think about how a developer uses IntelliSense to use your class.  He's going to type this:
MyDialog d = new MyDialog();
d.ShowDialog(

...and at that last keystroke, IntelliSense will pop up telling him that he now has to declare three new string variables to hold the out parameters.  So he moves the cursor up, and starts typing:
string foo;
string

...and, what was the name of the second parameter again?  So it's back down to the open paren, hit CTRL+SPACE, oh yeah, it's bar, back up to the previous line, etc.
The problem with using properties on a custom dialog is that the Form class already has a million properties, and the three or four special ones that you're creating are going to get lost in the mix.  To fix this, create a class for the dialog parameters and a Parameters property of that type on the custom dialog.  That makes code like this easy to write:
MyDialog d = new MyDialog();
d.Parameters.Foo = "foo";
d.Parameters.Bar = "bar";
d.Parameters.Baz = "baz";

because the parameter names pop up in IntelliSense, and you don't need to declare any variables to hold their values.

A: My approach is typically to write a method that internally calls ShowDialog, then formats the output data appropriately. For (contrived) example:
public string GetFolderName(){
    if(this.ShowDialog() == DialogResult.OK) {
        return this.FolderName.Text;
    }
    return String.Empty;
}

In most cases I make this method static, and instantiate the dialog itself from within the body of the method - that way the caller doesn't have to deal with form references, or the notion of having to chose which 'show' method to call.
In the non-edge cases of having multiple output values, I typically construct a struct that holds these values, then have my 'Get' function return that struct.
public struct FolderData {
    public static FolderData Empty = new FolderData();

    public string FolderName {get; set;}
    public int FilesInFolder {get; set;}
}

public FolderData GetFolderData(){
    if(this.ShowDialog() == DialogResult.OK) {
        return new FolderData {
            FolderName = this.FolderName.Text;
            FilesInFolder = int.Parse(this.FilesInFolder.Text);
        }
    }
    return FolderData.Empty;
}

A: I prefer this one because I don't like the approach of getting result from a property or a method after you have done with the class. After dialog form was shown and closed I think the object should not be used any more because logically you have done with the dialog then why should I use its property or method to get the result?

A: @Musigenesis, you really don't want to force client code to break when you change your dialog, and using an out parameter that is only sometimes valid isn't a good design. As @Daok says, when you have more than 1 value returned this starts to get messy and ugly fast.
You also can't force client code to use the result any more than the .net framework ensures that you call properties on a file dialog. You're also not forcing the caller to do anything with the out parameter, all you've forced them to do is to accept a variable that they may not want to use.
If the dialog is very generic this may not be applicable, but instead of chucking all sorts of properties to the dialog itself, have a single method that you use consistently throughout your application, and have that return a specific class that holds the relevant data.
public sealed class MySaveDialogResult
{
    public static MySaveDialogResult NonOkResult(); // Null Object pattern
    public MySaveDialogResult( string filePath ) { ... }

    // encapsulate the dialog result
    public DialogResult DialogResult { get; private set; }
    // some property that was set in the dialog
    public string FilePath { get; private set; }
    // another property set in the dialog
    public bool AllowOVerwrite { get; private set; }
}

and your dialog is
public MySaveDialog ...
{
    public MySaveDialogResult GetDialogResult() { .... }
}

The essence is a small immutable utility class that also implements null object pattern. The null object is returned whenever the dialog result wasn't OK. Obviously the above is a shot in the dark for your needs, so alter it at will, make inheritance hierarchies, etc.
The main point is to have GetDialogResult(), a single method on the dialog, to returned a class that encapsulates all the relevant dialog data.

edit:
@yapiskan wonders why not just 'out' the MyDialogResult versus calling GetDialogResult().
IMO - The points are simply:

*

*That's not the convention

*A method call is trivially easy, and made easier when you follow the 'convention' argument as made above.

*out is awkward to use. GetDialogResult() is not forcing the caller to write awkward code, and it doesn't force the user to consume the dialog result at the point of invoking the dialog.

*Normally the dialog isn't re-instantiated or re-shown to get the result, it's already there. Show() and Hide() do just that.

The reality is you're trading a method call for an awkward ShowDialog() syntax. Method calls are cheap, and you can't guarantee the caller will use your out parameter any more than you can guarantee they will call GetDialogResult(). So why bother. Make the thing easy to use, or don't overload ShowDialog in the first place.
Maybe whatever you're sub-classing is funky and acts differently and it's not applicable to your situation, but general design is forms don't go away when you click OK, they go away when they are Disposed() of.

redpajama_set_name: RedPajamaStackExchange


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