Control of chromosome duplication

In cell biology, eukaryotes possess a regulatory system that ensures that DNA replication occurs only once per cell cycle. In cell biology, eukaryotes possess a regulatory system that ensures that DNA replication occurs only once per cell cycle. A key feature of the DNA replication mechanism in eukaryotes is that it is designed to replicate relatively large genomes rapidly and with high fidelity. Replication is initiated at multiple origins of replication on multiple chromosomes simultaneously so that the duration of S phase is not limited by the total amount of DNA. This flexibility in genome size comes at a cost: there has to be a high-fidelity control system that coordinates multiple replication origins so that they are activated only once during each S phase. If this were not the case, daughter cells might inherit an excessive amount of any DNA sequence, which could lead to many harmful effects. Replication in eukaryotes begins at replication origins, where complexes of initiator proteins bind and unwind the helix. In eukaryotes, it is still unclear what exact combinations of DNA sequence, chromatin structure, and other factors define these sites. The relative contribution of these factors varies between organisms. Yeast origins are defined primarily by DNA sequence motifs, while origin locations in other organisms seem to be defined by local chromatin structure. Origins in budding yeast are defined by the autonomously replicating sequence (ARS), a short stretch of DNA (100-200 bp) that can initiate replication when transferred to any sequence of DNA. The ARS contains several specific sequence elements. One of these is the A element (ACS), an 11 bp consensus sequence rich in adenines and thymines that is essential for initiation. Single base-pair mutations in the ACS can abolish initiation activity. The ORC, a component of the initiation complex, binds the ACS in vivo throughout the cell cycle, and in vitro in an ATP dependent manner. When a few of these sequences are deleted, DNA is still copied from other intact origins, but when many are deleted, chromosome replication slows down dramatically. Still, presence of an ACS sequence is not sufficient to identify an origin of replication. Only about 30% of ACS sequences present in the genome are the sites of initiation activity. Origins in fission yeast contain long stretches of DNA rich in thymines and adenines that are important for origin function, but do not exhibit strong sequence similarity. In animals, no highly conserved sequence elements have been found to direct origin activity, and it has proved difficult to identify common features of replication origins. At some loci, initiation occurs within small, relatively definable stretches of DNA, while at others, larger initiation zones of 10–50 kb seem to direct origin activity. At the sequence level, AT rich elements and CpG islands have been found at origins, but their importance or role is not yet clear. At the level of DNA structure, bent DNA and loop formation have been identified as origin features. Features identified at the chromatin level include nucleosome free regions, histone acetylation and DNAse sensitive sites. Before DNA replication can start, the pre-replicative complex assembles at origins to load helicase onto DNA. The complex assembles in late mitosis and early G1. Assembly of these pre-replicative complexes (pre-RCs) is regulated in a manner that coordinates DNA replication with the cell cycle. The ORC is a six subunit complex that binds DNA and provides a site on the chromosome where additional replication factors can assemble. It was identified in S. cerevisiae by its ability to bind the conserved A and B1 elements of yeast origins. It is a conserved feature of the replication system in Eukaryotes. Studies in Drosophila showed that recessive lethal mutations in multiple drosophila ORC subunits reduces the amount of BrdU (a marker of active replication), incorporated. Studies in Xenopus extracts show that immuno-depletion of ORC subunits inhibits DNA replication of Xenopus sperm nuclei. In some organisms, the ORC appears to associate with chromatin throughout the cell cycle, but in others it dissociates at specific stages of the cell cycle. Cdc6 and Cdt1 assemble on the ORC and recruit the Mcm proteins. Homologs for these two S. cerevisiae proteins have been found in all eukaryotes. Studies have shown that these proteins are necessary for DNA replication. Mutations in S. pombe cdt1 blocked DNA replication. Mcm 2-7 form a six-subunit complex and is thought to have helicase activity. Deletion of any single subunit of the complex has a lethal phenotype in yeast. Studies in Xenopus revealed the Mcm2-7 complex is a critical component of DNA replication machinery. Inactivation of temperature sensitive mutants of any of the Mcm proteins in 'S. cerevisiae' caused DNA replication to halt if inactivation occurred during S phase, and prevented initiation of replication if inactivation occurred earlier. Although biochemical data support the hypothesis that the Mcm complex is a helicase, helicase activity was not detected in all species, and some studies suggest that some of the mcm subunits act together as the helicase, while other subunits act as inhibitors of this activity. If this is true, activation of the Mcm complex probably involves rearrangement of the subunits.