Cdc6 ORC (origin recognition complex) P P P P P P P P originCdt1 Mcm helicase + prereplicative complex (preRC) initiator proteins S-Cdk DDK INACTIVATION OF ORC, Cdc6, Cdt1 DNA HELICASE ACTIVATION COMPLETION OF DNA REPLICATION G1 S G2 DNA
Figure 17–18 Control of the initiation of DNA replication. The replication origin is bound by the ORC throughout the cell cycle. In early g1, Cdc6 associates with the ORC, and these proteins bind the dna helicase, which contains six closely related subunits called mcm proteins. The helicase also associates with a protein called Cdt1. Using energy provided by aTp hydrolysis, the ORC and Cdc6 proteins load two copies of the dna helicase, in an inactive form, around the dna next to the origin, thereby forming the prereplicative complex (preRC). at the onset of s phase, s-Cdk stimulates the assembly of several initiator proteins on each dna helicase, while another protein kinase, ddk, phosphorylates subunits of the dna helicase. as a result, the dna helicases are activated and unwind the dna. dna polymerase and other replication proteins are recruited to the origin, and dna replication begins. The ORC is displaced by the replication machinery and then rebinds. s-Cdk and other mechanisms also inactivate the preRC components ORC, Cdc6, and Cdt1, thereby preventing formation of new preRCs at the origins until the end of mitosis (see text).
it is crucial that chromatin structure, like the DNA within, is reproduced accurately during S phase. How chromatin structure is reproduced is not well understood, however. During DNA synthesis, histone-modifying enzymes and various non-histone proteins are probably deposited onto the two new DNA strands as they emerge from the replication fork, and these proteins are thought to reproduce the local chromatin structure of the parent chromosome (see Figure 4–45).
At the end of S phase, each replicated chromosome consists of a pair of identical sister chromatids glued together along their length. This sister-chromatid cohesion sets the stage for a successful mitosis because it greatly facilitates the attachment of the two sister chromatids to opposite poles of the mitotic spindle. Imagine how difficult it would be to achieve this bipolar attachment if sister chromatids were allowed to drift apart after S phase. Indeed, defects in sister-chromatid cohesion—in yeast mutants, for example—lead inevitably to major errors in chromosome segregation.
Sister-chromatid cohesion depends on a large protein complex called cohesin, which is deposited at many locations along the length of each sister chromatid as the DNA is replicated in S phase. Two of the subunits of cohesin are members of a large family of proteins called SMC proteins (for Structural Maintenance of Chromosomes). Cohesin forms giant ringlike structures, and it has been proposed that these surround the two sister chromatids (Figure 17–19).
Sister-chromatid cohesion also results, at least in part, from DNA catenation, the intertwining of sister DNA molecules that occurs when two replication forks meet during DNA synthesis. The enzyme topoisomerase II gradually disentangles the catenated sister DNAs between S phase and early mitosis by cutting one DNA molecule, passing the other through the break, and then resealing the cut DNA (see Figure 5–22). Once the catenation has been removed, sister-chromatid cohesion depends primarily on cohesin complexes. The sudden and synchronous loss of sister cohesion at the metaphase-to-anaphase transition therefore depends primarily on disruption of these complexes, as we describe later.