The replication of the genome is one of the most important events in the life of a cell. Eukaryotic cells replicate large amounts of genomic DNA distributed on multiple chromosomes. To accomplish this, replication initiates throughout S phase from multiple origins along each chromosome. Initiation events must be coordinated so that no region of the genome is left unreplicated, but no region is replicated more than once. Mistakes in DNA replication can result in loss or duplication of genetic information, which can play an important role in the genesis of cancer cells.
A two-step mechanism regulates the initiation of DNA replication, ensuring that the entire genome is precisely duplicated in each cell cycle. In the first step, Cdc6 and Cdt1 work with the origin recognition complex (ORC) to load the replicative helicase (Mcm2-7) into prereplicative complexes (pre-RCs) at replication origins. In the second step, two protein kinases, Cdc7 and the S phase cyclin dependent kinase (CDK), convert pre-RCs into bi-directional replisomes at each origin. DNA replication is limited to once per cell cycle because these two steps are temporally segregated: pre-RC assembly can only occur from late mitosis through G1 phase and, once cells enter S phase, pre-RCs can no longer assemble. Thus, origins that have fired once cannot be re-licensed until they pass through the following mitosis.
Evolution of licensing regulation
Despite its fundamental importance in maintaining an intact genome, the mechanisms by which pre-RC components are regulated differ widely across Eukarya. For example, the Cdc6 protein is targeted for SCF-dependent proteolysis by CDK phosphorylation during S phase in budding yeast, whilst in mammalian cells, Cdc6 is stable during S phase, but Cdt1 and Orc1 are SCF targets. We investigated this by studying the evolution of licensing control in Saccharomyces species. We found that even within the genus Saccharomyces, Cdc6 is regulated differently in different species. For example, the SCF binding site responsible for proteolysis in G2/M phases in Saccharomyces cerevisiae is absent from more distantly related Saccharomyces species. We propose that two factors contribute to the rapid evolution of licensing regulation. The first is redundancy: work from our lab and others has shown that eliminating any single pre-RC regulatory mechanism has very little affect on viability. The second is interchangeability: we showed that regulatory mechanisms can be swapped between pre-RC components without compromising the block to re-replication. Our experiments provide a framework for understanding the diversity of licensing regulation in eukaryotes and provide new tools for manipulating the chromosome replication cycle.
Effect of DNA replication on gene expression
DNA replication and transcription occur on a common template, and there are many ways in which these activities may influence each other. Firstly, the passage of DNA replication forks offers an opportunity to change gene expression patterns. For example, transcription of capsid genes generally occurs late in most viral infections and is often dependent upon prior replication of the viral genome. In bacteriophage T4, there is a direct coupling of replication and transcription because the sliding clamp processivity factor gp45 acts as a mobile transcriptional enhancer. Secondly, juxtaposed genes and replication origins can influence each other's activity. The origin of SV40 replication overlaps promoter and enhancer elements for both early and late gene expression and there are many examples of transcription factors influencing replication origin function. Moreover, induced transcription into a yeast replication origin can inactivate it. Finally, clashes between replication and transcription machinery are potentially important causes of genome instability.
We analyzed global mRNA expression in synchronized yeast cultures under two conditions that prevent the initiation of DNA replication without preventing cell cycle progression: the first prevents replication origin licensing by depleting Cdc6, the essential licensing factor; the second prevents origin firing at a step after licensing by inactivating Cdc45, the essential initiation factor. We found that the expression of at least 88% of the genome is unaffected by DNA replication. However, DNA replication did affect a small subset of genes. We found that histone gene expression was greatly diminished in the absence of DNA replication, independent of DNA damage checkpoint responses. We also found that the expression of genes with replication origins located near their 3' ends was inhibited by the presence of the pre-RC at origins, indicating that downstream origins can regulate the expression of upstream genes. Our work provides the first global view of how gene expression is affected by DNA replication.
Reconstitution of Pre-RC assembly with purified proteins
It has been 15 years since the discovery of the pre-RC and we have put considerable effort into its reconstitution over the years. We showed this year, using purified proteins that the loading of Mcm2-7 onto DNA requires ORC, Cdc6 and hydrolysable ATP, consistent with requirements in vivo. ORC and Cdc6 load the Mcm2-7 proteins from single Cdt1×Mcm2-7 heptamers into pre- RCs as head-to-head double hexamers. The loading of the double hexamer appears to be highly cooperative: double Cdt1×Mcm2-7 heptamers were never seen prior to loading and single Mcm2-7 hexamers were never detected on DNA after loading. These results indicate that the two hexamers are loaded together in a concerted reaction. Three-dimensional reconstruction from negatively stained EM images revealed the presence of a long central channel running through the double hexamer. We showed that double stranded DNA passes through this central channel and, once loaded, the double hexamer is mobile, capable of passive one-dimensional diffusion or 'sliding' along DNA (Figure 1). We proposed that the ability of the double hexamer to slide on double stranded DNA may provide a mechanism by which supernumerary Mcm2-7 double hexamers may be pushed ahead of the replisome during normal elongation. These excess double hexamers could play a role in restoring an active helicase at stalled forks. This might provide an important mechanism for replication restart that does not require the reloading of soluble Mcm2-7 and, therefore, does not compromise mechanisms designed to ensure once per cell cycle replication.
Figure 1. A: Images of the Mcm2-7 double hexamer loaded onto double stranded DNA visualised by electron microscopy after rotary shadowing. B:Time course of Mcm2-7 disassociation from linear and circular DNA. C. Quantification shows rapid release from linear DNA (squares) and slow release from circular DNA (triangles). See Remus et al., 2009; Cell, 139: 719-730 for details.
For a list of refereed research papers, see Publications (in navigation on left).
