Mjh

helicases associate with each other

DNA primase makes leading strand primer synthesis

BOX 8-5 FIGURE 1 In the ¡eft panel, the two DNA heiicases function independently, in the right panel, the two DNA helicases remain associated with one another. Note that in the right panel, one DNA Pol III holoenzyme uses only the Watson strand as a template and the other uses only the Crick strand as a template. For simplicity, the DNA Pol ill is not shown associated with the ONA helicases.

Eukaryotic Chromosomes Are Replicated Exactly Once per Cell Cycle

As discussed in Chapter 7, the events required for eukaryotic cell division occur at distinct times during cull cycle. Cliroirtosoiiitil DNA replication occurs only during the S phase of the cell cycle. During this time, all the DNA in the cell must be duplicated exactly once. Incomplete replication of any part of a chromosome causes inappropriate links between daughter chromosomes. Segregation of linked chromosomes causes chromosome breakage or loss (Figure 8-27). Rereplication of DNA can also have severe consequences, increasing the number of copies of particular regions of the genome. Addition of even one or two more copies of critical regulatory genes can lead to catastrophic defects in gene expression, eel) division, or the response to environmental signals. Thus, it is critical that every base pair in each chromosome is replicated oncc and only once each time a eukaryotic cell divides.

The need to replicate the DNA once and only once is a particular challenge for eukaryotic chromosomes because they each have many origins of replication. First, enough origins must be activated to ensure that each chromosome is fully replicated during each S phase. Typically, not all potential origins need to be activated to complete replication hut, if too few are activated, regions of the genome will escape replication (see Figure 8-27). Second, although some potential origins may not be used in any given round of ceil division, no origin of replication can initiate after it has been replicated. Thus, whether an origin is activated to cause its own replication or replicated by a replication fork derived from an adjacent origin, it must be inactivated until the next round of cell division (Figure 8-28). If these conditions were not true, the DNA associated with an origin could be replicated twice in the same cell cycle.

Pre-Replicative Complex Formation Directs the Initiation of Replication in Eukaryotes

The initiation of replication in eukaryotic cells requires two steps to occur at distinct times in the cell cycle (see Chapter 7): replicator selection and origin activation. Replicator selection is the process of identifying sequences thai will direct the initiation of replication

FIGURE 8-27 Chromosome breakage as a result of incomplete DNA replication.

This illustration shows the consequences of incomplete replication follower! by chromosome segregation. The top of each illustration shows the entire chromosome. The bottom shows the details of the chromosome breakage at the DNA level (For the details of chromosome segregation, see Chapter 7.) As the chromosomes are pulled apart, stress « plated on the unreplicateci DMA, resulting in the breakage of the chromosome.

break!

chromosome segregation

break!

FIGURE 8-28 Replicators are inactivated by DNA replication. A chrcrmosomc with five replicators is shown. The replicators labded 3 and 5 are the first to be activated, leading to the formation of two pairs of bidirectional replication forks. Activation of the parental replicator results in the inactivation of the copies of each replicator on both daughter DNA molecules until the next cell cycle (indicated by a red X). Further extension of the resulting replication forks replicates the DNA overlapping with the number 2 and 4 replicators. When a replicator ts coped by a fork derived from an adjacent origin prior to initiation, it is said to have been passively replicated. Although these replicators have not initiated, they are nevertheless inactivated by the act of replicating their DNA In contrast, replicator 1 is not reached by an adjacent fork prior to initiation and is able to initiate normally The presence of more replicators than needed to complete DNA replication is a form of redundancy to ensure the complete replication o( each chromosome origin 3 and 5 initiate origin 3 and 5 initiate

I origin 1 initiates I origin 2 is passively replicated

I origin 1 initiates I origin 2 is passively replicated l origin A is passively replicated origin A is passively replicated and occurs in Gl (prior to S phase). This process leads to the assembly of a multiprotein complex at each replicator in the genome. Origin activation only occurs after cells enter S phase and triggers the replicator-associated protein complex to initiate DNA unwinding and DNA polymerase recruitment.

The separation of replicator selection and origin activation is different from the situation in prokaryolic celts, where the recognition of replicator DNA is intrinsically coupled to DNA unwinding and polymerase recruitment. As we will see below, the temporal separation of these two events in eukaryotic cells ensures that each chromosome is replicated only once during each cell cycle (bacterial cells solve this problem differently, see Box 8-4, E. coli DNA Replication Is Regulated by DnaA*ATP Levels and SeqA,

Replicator selection is mediated by the formation of pre-replicative complexes (pre-RCs) (Figure 8-29). The pre-RC is composed of four separate proteins that assemble in an ordered fashion at each replicator. The first step in the formation of the pre-RC is the recognition of the replicator by the eukaryotic initiator, ORC. Once ORC is bound, it recruits two helicasc loading proteins (Cdc6 and Cdtl). Together, ORC and the loading proteins recruit a protein that is thought to be the eukaryotic replication fork helicase (the Mem 2-7 complex). Formation of the pre-RC does not lead to the immediate unwinding of origin DNA or the recruitment of DNA polymerases. Instead the pre-RCs that are formed during Gl are only activated to initiate replication after cells pass from the Gl to the S phase of the cell cycle.

Pre-RCs are activated fo initiate replication by two protein kinases (Cdk and Ddk; Figure 8-30). Kinases are proteins that covalently attach phosphate groups to target proteins (see Chapter 5), Each of these kinases is inactive in Gl and is activated only when cells enter S phase. Once activated, these kinases target the pre-RC and other replication proteins. Phosphorylation of these proteins results in the assembly of additional replication proteins at the

FIGURE 8-29 The steps in the formation of the prereplicative complex {pre-RC). The assembly of the pre RC is an ordered process that is initiated by the association of the origin recognition complex with the replicator. Once bound to the replicator, ORC recruits at least two additional proteins, CdcG and Cdti. These three proteins function together to recruit the putative eukaryotic DNA helicase—the Mcrn2-7 complex to complete the formation of the pre-RC.

origin and the initiation of replication (see Figure 8-30). These new proteins include the three enkaryotic DNA polymerases and a number of other proteins required for their recruitment. Interestingly, the polymerases assemble at the origin in a particular order. DNA Pol 5 and e associate first, followed by DNA Pol et/primase. This order ensures that all three DNA polymerases are present at the origin prior to the synthesis of the first RNA primer (by DNA Pol ct/primase).

Only a subset of the proteins that assemble at the origin go on to function as part of the eukaryotic rep)isome. In addition to the three DNA polymerases, the Mem complex and many of the factors required fur DNA polymerase recruitment become part of the replication fork machinery. Similar to the E. coli DNA helicase loader (DnaC), the other factors (such as Cdc6 and Cdt1) are released or destroyed after their role is complete (see Figure 8-30).

Pre-RC Formation and Activation Is Regulated to Allow only a Single Round of Replication during Each Cell Cycle

How do eukaryotic cells control the activity of hundreds or even thousands of origins of replication such that not even one is activated more than once during a cell cycle? The answer lies in the tight regulation of the formation and activation of pre-RCs by cyclin-dependcmt kinases (Cdks).

Cdks play two seemingly contradictory roles in regulating pre-RC function (Figure 8-31). First, as we described above, they are required to activate. pre-RCs to initiate DNA replication. Second,. Cdk activity inhibits the fonnation of new pre-KCs,

FIGURE 8-30 Activation of the pre-RC leads to the assembly of the eukaryotic replication fork. As cells enter into the S phase of the cell cyde, Cdk and Ddk phosphorylate replication proteins to trigger the initiation of replication. The events that lead to DNA unwinding at the origin are poorly understood but are likely to require the activity of the Mem complex and resuit in the recruitment of a number of auxiliary replication factors and DNA Pol 8 and e. DNA Pol «.'primase is only recruited aftei DNA Pol & and e. Once present at the origin, DNA Pol a/pnmase synthesizes an RNA priner and briefly extends tt. the resulting primerrtcmplate junction is recognized by the eukaryotic sliding clamp loader (RF C), which assembles a sliding damp (PCNA) at these sites. Either DIMA Pol 6 or e recognises this primer and begins leading strand synthesis. After a period of DNA unwinding, DNA Pol u/primase synthesizes additional primers, which allow the initiation of lagging strand DNA synthesis by either DNA Pol & or e. Here we illustrate Pol B on the leading strand and Pol £ on the lagging strand.

start tagging strand synthesis

sliding clamp and damp loader (PCNA + RFC)

auxiliary factors and polymerases s

auxiliary factors and polymerases s o|EJ

polymerase«/

primase VtHpr^

sliding clamp and damp loader (PCNA + RFC)

start tagging strand synthesis

Cdk activity low f pre-RC formation allowed

pre-RC formation allowed no prc-RC activation

FIGURE 8-31 Effect of Cdk activity on pre-RC formation and activation. High Cdk activity is requited f« existing pre-RC complexes to initiate DNA replication. These same elevated levels of Cdk activity completely inhibit the formation of new pre-RC complexes. In contrast, low Cdk activity is conducive to new pre RC formation but is inadequate to trigger DMA replication initiation by the newly formed pre-RC complexes.

Cdk activity high

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