Aspectos No-mon´ otonos de la L´ ogica Posibilista

The MCM proteins are a family of related proteins required for initiation o f DNA

replication. The MCM proteins were first identified in S. cerevisiae in genetic

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maintenance proteins) or cell cycle progression (Maine et al., 1984; Moir et at.,

1982). MCM genes have been identified in a wide range of eukaryotes, including yeast, frogs and humans, and based on sequence homology are clustered into six

groups called MCM2-7 (Chong et a l , 1996). The 240-aminoacid region o f high

conservation between MCM proteins from the six groups include a modified match to

the Walker A motif of the nucleotide binding site (Koonin, 1993; Walker et al.,

1982). However, different members of MCM family are quite diverse outside this 240 amino acid region.

Studies of a variety of eukaryotes suggests that MCM proteins function together in a

large multi-protein complex. In S. cerevisiae, individual MCM proteins were shown

to interact with each other both genetically and biochemically (Hennessy et al., 1991;

Lei et a i , 1996). Interaction between individual MCM subunits was also

demonstrated in S. pombe. Drosophila, Xenopus and mouse (Chong et al., 1995;

Hendrickson et a l , 1996; Kimura et al., 1995; Kubota et al., 1995; Madine et al.,

1995a; Madine et al., 1995b; Okishio et al., 1996; Romanowski et al., 1996a; Su et

al., 1996).

The levels o f MCM proteins are relatively constant throughout the cell cycle.

However, the localization of MCM proteins in S. cerevisiae is significantly altered as

cells replicate their DNA. MCM proteins enter the nucleus at the end o f mitosis, persist there throughout G l and are rapidly exported into cytoplasm soon after

initiation o f DNA replication (Dalton and Whitbread, 1995; Hennessy et a i , 1990;

Yan et al., 1993). Once inside the nucleus, a fraction o f MCM proteins becomes

tightly associated with DNA (Yan et a i , 1993). Chromatin binding experiments

clearly demonstrated that less that 5% of MCM5 (Cdc46p) and MCM7 (Cdc47p) are associated with chromatin at the G2/M transition, while about half of the total cellular

MCM5 and MCM7 is associated with chromatin in G l (Donovan et al., 1997).

Similar results were obtained for the S. cerevisiae MCM3 protein, which was shown

to be bound to chromatin in G l, becoming displaced during S phase (Liang and Stillman, 1997).

In vertebrate and fission yeast cells the nuclear localisation of MCM proteins does not seem to be cell cycle regulated. However, MCM association with the insoluble chromatin is under cell cycle control. MCM proteins associate with sperm chromatin

added to X en o p u s egg extract and are gradually released from chromatin as

replication proceeds (Chong et at., 1995; Hendrickson et al., 1996; Kubota et al.,

1995; Madine et al., 1995a; Madine et al., 1995b; Romanowski et al., 1996a).

The timing o f the association of the MCM proteins with chromatin implies their

presence in pre-replicative complexes. Indeed, in vivo cross-linking studies in

S. cerevisiae showed MCM4 (Cdc54p) and MCM7 (Cdc47p) associated specifically

with origin DNA during G l, supporting the idea that they are a part o f pre-replicative

complexes (Aparicio et al., 1997; Tanaka et al., 1997). During S phase, MCM

proteins associate with non-origin DNA, suggesting that after initiation they move

with the replication forks (Aparicio et al., 1997). The participation of MCM proteins

in the pre-replicative complexes was further supported by experiments showing that MCM association with chromatin was prevented in cells which carried a mutation in

the Orel or Orc5 gene (Aparicio etal., 1997; Tanaka etal., 1997).

In addition to functional ORC, association of MCM proteins with chromatin requires

the presence o f Cdc6 protein. In the absence o f Cdc6 expression in S. cerevisiae,

MCM proteins are unable to bind chromatin (Donovan et a i , 1997). Similarly, in

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(Aparicio et a l , 1997; Tanaka et a l , 1997). Despite the requirement for functional

ORC and Cdc6 for loading of the MCM proteins onto chromatin, these proteins are not required for the maintenance of the MCM-chromatin interaction, as MCM proteins remain chromatin bound following salt extraction o f ORC and Cdc6 (Donovan et a l , 1997).

Similar results were obtained in the Xenopus system, where binding o f Cdc6 to

chromatin was shown to require ORC to bind first (Coleman et a l , 1996). Loading

of MCM3 required both ORC and Cdc6 proteins to be pre-bound (Coleman et a l ,

1996; Romanowski et a l , 1996b; Rowles et a l , 1996). Thus, the assembly of

replication competent chromatin in Xenopus involves sequential binding o f ORC,

Cdc6 and MCMs to DNA. Interestingly, salt extraction o f ORC and Cdc6 from chromatin, following the loading of the MCM proteins does not inhibit DNA replication, suggesting that loading of MCMs might be the essential function of ORC

and Cdc6 (Rowles et a l, 1999).

Despite their importance in the process of DNA replication, the precise function of MCM proteins remains unclear. MCM complexes isolated from HeLa cells had both ATPase and helicase activities, suggesting a role as a replicative helicase (Ishimi, 1997; Koonin, 1993). The finding that MCMs, although initially associated with

replication origin sequences in S. cerevisiae, associate with chromatin distal to

replication origins as replication proceeds supports the idea that MCM proteins are associated with replication forks (Aparicio e t a l , 1997).

Interestingly, MCM proteins were identified as a part of a licencing factor, which is required for initiation, but is inactivated or destroyed as DNA replication progresses

MCMs in distinguishing the replicated and unreplicated DNA and restricting DNA replication to once per cell cycle. In agreement with this model, in replicating HeLa nuclei, localisation of MCM proteins coincides with subnuclear sites o f unreplicated chromatin. MCM proteins are displaced from their chromatin sites at the time when these sites are replicated (Krude et ah, 1996).