Dna

replication

0 S phase

▲ FIGURE 21-24 Control of the G1 ^ S phase transition in S. cerevisiae by regulated proteolysis of the S-phase inhibitor Sic1. The S-phase cyclin-CDK complexes (Clb5-CDK and Clb6-CDK) begin to accumulate in G1, but are inhibited by Sic1. This inhibition prevents initiation of DNA replication until the cell is fully prepared. G1 cyclin-CDK complexes assembled in late G1

(Cln1-CDK and Cln2-CDK) phosphorylate Sic1 (step 1 ), marking it for polyubiquitination by the SCF ubiquitin ligase, and subsequent proteasomal degradation (step 2| ). The active S-phase cyclin-CDK complexes then trigger initiation of DNA synthesis (step 3 ) by phosphorylating substrates that remain to be identified. [Adapted from R. W. King et al., 1996, Science 274:1652.]

▲ FIGURE 21-25 Activity of S. cerevisiae cyclin-CDK complexes through the course of the cell cycle. The width of the colored bands is approximately proportional to the demonstrated or proposed protein kinase activity of the indicated cyclin-CDK complexes. S. cerevisiae produces a single cyclin-dependent kinase (CDK) whose activity is controlled by the various cyclins, which are expressed during different portions of the cell cycle.

cyclins in that the complexes they form with CDK trigger formation of mitotic spindles. The remaining two cerevisiae cyclins, Clb1 and Clb2, whose concentrations peak midway through mitosis, function exclusively as mitotic cyclins, forming complexes with CDK that trigger chromosome segregation and nuclear division (see Table 21-1).

Cdc14 Phosphatase Promotes Exit from Mitosis

Genetic studies with ,S. cerevisiae have provided insight into how B-type cyclin-CDK complexes are inactivated in late anaphase, permitting the various events constituting telophase and then cytokinesis to occur. These complexes are the sS. cerevisiae equivalent of the mitosis-promoting factor (MPF) first identified in Xenopus oocytes and early embryos. As mentioned previously, the specificity factor Cdh1, which targets the APC to polyubiquitinate B-type cyclins, is phos-phorylated by both late G1 and B-type cyclin-CDK complexes, thereby inhibiting Cdh1 activity during late G1, S, G2, and mitosis before late anaphase.

When daughter chromosomes have segregated properly in late anaphase, the Cdc14 phosphatase is activated and de-phosphorylates Cdh1, allowing it to bind to the APC. This interaction quickly leads to APC-mediated polyubiquitina-tion and proteasomal degradation of B-type cyclins, and hence MPF inactivation (see Figure 21-10). Since MPF is still active when Cdc14 is first activated in late anaphase, it po tentially could compete with Cdc14 by re-phosphorylating Cdh1. However, Cdc14 also induces expression of Sic1 by removing an inhibitory phosphate on a transcription factor that activates transcription of the ,SIC1 gene. Sic1 can bind to and inhibit the activity of all B-type cyclin-CDK complexes. Thus, starting late in mitosis, the inhibition of MPF by Sic1 allows the Cdc14 phosphatase to get the upper hand; the B-type cyclin APC specificity factor Cdh1 is dephosphorylated and directs the precipitous degradation of all the ,S. cerevisiae B-type cyclins. In Section 21.7, we will see how the activity of Cdc14 itself is controlled to assure that a cell exits mitosis only when its chromosomes have segregated properly.

As discussed already, Sic1 also inhibits the S-phase cyclin-CDKs as they are formed in mid-G1 (see Figure 21-24). This inhibitor of B-type cyclins thus serves a dual function in the cell cycle, contributing to the exit from mitosis and delaying entry into the S phase until the cell is ready.

Replication at Each Origin Is Initiated Only Once During the Cell Cycle

As discussed in Chapter 4, eukaryotic chromosomes are replicated from multiple origins. Some of these initiate DNA replication early in the S phase, some later, and still others toward the end. However, no eukaryotic origin initiates more than once per S phase. Moreover, the S phase continues until replication from all origins along the length of each chromosome results in replication of the chromosomal DNA in its entirety. These two factors ensure that the correct gene copy number is maintained as cells proliferate.

Yeast replication origins contain an 11-bp conserved core sequence to which is bound a hexameric protein, the origin-recognition complex (ORC), required for initiation of DNA synthesis. DNase I footprinting analysis (Figure 11-15) and immunoprecipitation of chromatin proteins cross-linked to specific DNA sequences (Figure 11-40) during the various phases of the cell cycle indicate that the ORC remains associated with origins during all phases of the cycle. Several replication initiation factors required for the initiation of DNA synthesis were initially identified in genetic studies in S. cerevisiae. These include Cdc6, Cdt1, Mcm10, and the MCM hexamer, a complex of six additional, closely related Mcm proteins; these proteins associate with origins during G1, but not during G2 or M. During G1 the various initiation factors assemble with the ORC into a pre-replication complex at each origin. The MCM hexamer is thought to act analogously to SV40 T-antigen hexamers, which function as a helicase to unwind the parental DNA strands at replication forks (see Figure 4-36). Cdc6, Cdt1, and Mcm10 are required to load opposing MCM hexamers on the origin.

The restriction of origin "firing" to once and only once per cell cycle in ,S. cerevisiae is enforced by the alternating cycle of B-type cyclin-CDK activities throughout the cell cycle: low in telophase through G1 and high in S, G2, and M through anaphase (see Figure 21-25). As we just discussed, S-phase cyclin-CDK complexes become active at the beginning of the S phase when their specific inhibitor, Sicl, is degraded. In the current model for cerevisiae replication, pre-replication complexes assemble early in G1 when B-type cyclin activity is low (Figure 21-26, step 1). Initiation of DNA replication requires an active S-phase cyclin-CDK complex and a second heterodimeric protein kinase, Dbf4-Cdc7, which is expressed in G1 (step 2). By analogy with cyclin-dependent kinases (CDKs), which must be bound by a partner cyclin to activate their protein kinase activity, the Dbf4-dependent Ainase Cdc7 is often called DDK. Although the complete set of proteins that must be phosphorylated to activate initiation of DNA synthesis has not yet been determined, there is evidence that phosphorylation of at least one subunit of the hexameric MCM helicase and of Cdc6 is required. Another consequence of S-phase cyclin-CDK acti vation is binding of the initiation factor Cdc45 to the pre-replication complex. Cdc45 is required for the subsequent binding of RPA, the heterotrimeric protein that binds single-stranded DNA generated when the MCM helicase unwinds the parental DNA duplex.

By stabilizing the unwound DNA, RPA promotes binding of the complex between primase and DNA polymerase a (Pol a) that initiates the synthesis of daughter strands (see Figure 21-26, step [3). Like the MCM helicase, Cdc45 remains associated with the replication forks as they extend in both directions from the origin. Presumably, it functions in the further cycles of RPA and primase-Pol a binding required to prime synthesis of the lagging daughter strand. Subsequent binding of DNA polymerase 8 and its accessory cofactors Rfc and PCNA is thought to occur as it does in

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