Figure 5–11 The synthesis of one of many DNA fragments on the lagging strand. In eukaryotes, RNA primers are made at intervals spaced by about 200 nucleotides on the lagging strand, and each RNA primer is approximately 10 nucleotides long. This primer is erased by a special DNA repair enzyme (an RNAse H) that recognizes an RNA strand in an RNA/DNA helix and fragments it; this leaves gaps that are filled in by DNA polymerase and DNA ligase.
DNA polymerase adds to new RNA primer to start new Okazaki fragment sealing by DNA ligase joins new Okazaki fragment to the growing chain
Special Proteins Help to Open Up the DNA Double Helix in Front of the Replication Fork
For DNA synthesis to proceed, the DNA double helix must be opened up (“melted”) ahead of the replication fork so that the incoming deoxyribonucleoside triphosphates can form base pairs with the template strands. However, the DNA double helix is very stable under physiological conditions; the base pairs are locked in place so strongly that it requires temperatures approaching that of boiling water to separate the two strands in a test tube. For this reason, two additional types of replication proteins—DNA helicases and single-strand DNA-binding proteins—are needed to open the double helix and provide the appropriate single-strand DNA templates for the DNA polymerase to copy.
DNA helicases were first isolated as proteins that hydrolyze ATP when they are bound to single strands of DNA. As described in Chapter 3, the hydrolysis of ATP can change the shape of a protein molecule in a cyclical manner that allows the protein to perform mechanical work. DNA helicases use this principle to propel themselves rapidly along a DNA single strand. When they encounter a region of double helix, they continue to move along their strand, thereby prying apart the helix at rates of up to 1000 nucleotide pairs per second (Figure 5–13 and Figure 5–14).
The two strands of DNA have opposite polarities, and, in principle, a helicase
Figure 5–12 The reaction catalyzed by DNA ligase. This enzyme seals a broken phosphodiester bond. As shown, DNA ligase uses a molecule of ATP to activate the 5ʹ end at the nick (step 1) before forming the new bond (step 2). In this way, the energetically unfavorable nick-sealing reaction is driven by being coupled to the energetically favorable process of ATP hydrolysis.
could unwind the DNA double helix by moving in the 5ʹ-to-3ʹ direction along one 5˜ 3˜ strand or in the 3ʹ-to-5ʹ direction along the other. In fact, both types of DNA helicase exist. In the best-understood replication systems in bacteria, a helicase moving 5ʹ to 3ʹ along the lagging-strand template appears to have the predominant role, for reasons that will become clear shortly.
Single-strand DNA-binding (SSB) proteins, also called helix-destabilizing proteins, bind tightly and cooperatively to exposed single-strand DNA without covering the bases, which therefore remain available as templates. These proteins are unable to open a long DNA helix directly, but they aid helicases by stabilizing the unwound, single-strand conformation. In addition, through cooperative binding, they coat and straighten out the regions of single-strand DNA, which occur routinely in the lagging-strand template, thereby preventing the formation of the short hairpin helices that readily form in single-strand DNA (Figure 5–15 and Figure 5–16). If not removed, these hairpin helices can impede the DNA synthesis catalyzed by DNA polymerase.
A Sliding Ring Holds a Moving DNA Polymerase Onto the DNA
On their own, most DNA polymerase molecules will synthesize only a short string of nucleotides before falling off the DNA template. The tendency to dissociate quickly from a DNA molecule allows a DNA polymerase molecule that has just