Alternation problem

The ability to alternate S phase w ith M phase is one of the key problem s a cell has to solve. CDKs are required for the onset of both of these events and the regulation of this activity is essential in m aintaining the correct genom e ploidy. In fact disruption of norm al CDK activity is sufficient to alter the dependency of S phase on the completion of mitosis. This was first discovered in a screen for diploidizing m utants, ie. cells that undergo an additional ro u n d of DNA replication in the absence of an intervening mitosis. Two previously identified cdc2 alleles w ere re­ isolated, cdc2-33 and cdc2-M26. Loss of cdc2p from a G2 cell is therefore able to change cdc2p from the mitotic form to the S phase form (Broek et al., 1991). The

Chapter 1 Introduction

significance of the level of kinase activity in determ ining the cell cycle stage was further dem onstrated by two discoveries: first that depletion of the mitotic cyclin, cdcISp, could induce multiple rounds of DNA replication in the absence of mitosis (Hayles et al., 1994) and secondly that the CDK inhibitor, ru m l, could also induce re-replication w hen overexpressed (Correa-Bordes and N urse, 1995; Moreno and N urse, 1994).

Deletion of the B-type cyclins cigl and cigl dem onstrated that the mitotic cyclin, cdcl3, alone was able to prom ote both DNA replication, albeit w ith a slight delay and m itosis indicating that the level of cdc2p associated kinase activity is the determ ining factor for w hether cells undergo S phase or M phase (Fisher and N urse, 1996). This w ork led to the proposal of the quan titativ e m odel which suggested th at a low level of kinase activity is required to p rep are for DNA replication, an increase from a low to m oderate level of kinase activity prom otes DNA replication, maintenance of this level of activity prevents a subsequent round of replication and the increase to a high level brings about the onset of mitosis (Fig. 1.3) (reviewed in Stern and N urse, 1996). The m echanism s used to control the cdc2p associated kinase level in a norm al cell cycle include availability of a cyclin p artn er (controlled by transcription and proteolysis), phosphorylation and the presence of CDK inhibitors, all of which have been described previously (Fig. 1.2). Putative cdc2p substrates involved in both the initiation of replication and mitosis have previously been m entioned, however we have yet to address the substrates phosphorylated by cdc2p after S phase initiation to ensure that DNA replication occurs only once per cell cycle.

It is critical to tightly regulate cdclS protein levels as overexpression of cdclS leads to re-replication. CdclS is controlled both transcriptionally in a cdclOp dependent m anner (discussed previously) and post-translationally. CdcISp contains six cdc2p consensus phosphorylation sites (Kelly et al., 1993) an d has been show n to be phosphorylated by cdc2p, probably in association w ith cig2p (Baum et al., 1998; Jallepalli et al., 1997; Lopez-Girona et al., 1998). Phosphorylation prom otes binding of cdcISp to p o p lp and p o p 2 p /s u d lp , the F-box like com ponents of the SCF complex. These act as recognition factors for phosphorylated cdcISp and, along w ith the other m em bers of the complex, target cdcISp for degradation via the

Chapter 1 Introduction

proteasom e (Fig. 1.4 E) (Jallepalli et al., 1998; Kominami et al., 1998a; Kominami and Toda, 1997; Wolf et al., 1999b).

R u m lp is also ta rg e te d for p ro teo ly tic d e g ra d a tio n via the SCF after phosphorylation of two threonine residues, T58 and T62, by cdc2p (Benito et al., 1998). W hen these threonine residues are m utated to alanine, ru m lp is stabilized and cells occasionally re-initiate replication to become diploids (Benito et al., 1998). H ow ever, this d iploidization is m ore efficient if bo th r u m lp and cd cl8 p are stabilized, as in a popl or pop2/sudl m utant (Jallepalli et al., 1998; Kominami et al., 1998a; Kominami and Toda, 1997; Wolf et al., 1999b). Proteolysis following cdc2p phosphorylation is therefore a crucial m echanism by w hich DNA replication is restricted to once per cell cycle.

A nother im portant target of cdc2p phosphorylation is the MCM complex. Cdc21p (mcm4p) contains several cdc2p consensus phosphorylation sites and has been show n to exist in m ultiple phosphorylation states in fission yeast as well as hum ans and Xenopus (Coue et al., 1996; H endrickson et al., 1996; M usahl et al., 1995; N ishitani et al., 2000). Evidence initially suggested th at phosphorylation could p lay an in h ib ito ry role in the chrom atin b in d in g of cdc21p as the phosphorylated hum an Cdc21p appeared less tightly b ound to nuclear structures (M usahl et al., 1995), and X enopus Cdc21p becam e ph o sp h o ry lated by Cdc2p du rin g S phase, greatly decreasing its affinity for chrom atin (Coue et al., 1996; H endrickson et al., 1996). More recent data obtained from a m am m alian in vitro system suggests that the phosphorylation of Mcm4p by cyclin A /C d k 2 inhibits the helicase activity of the Mcm4p, 6p and 7p complex, thus providing a mechanism for the inhibitory effect of CDKs (Ishimi et al., 2000). The situation in b u d d in g yeast is som ew hat different as MCM localization is cell cycle regulated (as discussed earlier), being nuclear in G l and cytoplasmic follow ing S phase. This nuclear exclusion d uring 0 2 and mitosis is dependent on Cdc28p associated w ith either the G l cyclins (CLNs) or the B-type cyclins (CLBs) (Labib et al., 1999; N guyen et al., 2000). Thus the precise mechanism used by an organism to prevent re-association of MCMs and hence re-formation of pre-replicative complexes in G2 appears to vary bu t all involve phosphorylation of Mcm4 by CDKs.

Chapter 1 Introduction

A num ber of m echanisms have therefore evolved in fission yeast to ensure that M p hase alw ays succeeds S phase. These include the periodic transcription and d e g ra d atio n of key proteins such as cdcISp an d the inhibition of others by p hosphorylation such as cdc21p. Some of these m echanism s are redundant, for exam ple constitutive expression of cdcl8 does n o t affect cell cycle progression (Kelly et al., 1993). A m ulti-layered regulation system exists to preserve genome integrity by ensuring that DNA replication occurs only once in every cell cycle .