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TimBentley (talk | contribs) m Disambiguated: G1 → G1 phase, ORC → Origin Recognition Complex, APC → Activated protein C, nucleus → Cell nucleus; Help needed: NCS |
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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]].<ref name = "Diffley2008" >{{cite journal |author= Diffley, J.F |title= Regulation of Early Events in Chromosome Replication |journal=Curr. Biol. |volume=14 |pages=R778–R786 |year=2008 |pmid= 15380092 |issue= 18 |doi= 10.1016/j.cub.2004.09.019}}</ref> 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.<ref name = "Kearsey2003" >{{cite journal |author= Kearsey, S.E and Cotteril, S. |title= Engimatic variations: divergent modes of regulating eukaryotic [[DNA replication]]|journal=Mol. Cell |volume=12 |pages=1067–1075 |year=2003 |pmid= 14636567 | doi = 10.1016/S1097-2765(03)00441-6 |issue= 5}}</ref> This article explores the regulatory system that ensures that DNA replication occurs only once per [[cell cycle]].
== The Replication Origin ==
Replication in eukaryotes begins at replication origins, where complexes of initiator [[proteins]] bind and unwind the [[helix]].<ref name="The Cell Cycle">{{cite book| author= Morgan, David, O.| title=The Cell Cycle: Principles of Control| year=2007| publisher=New London Press 1st ed.}}</ref> 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.<ref name="The Cell Cycle" />
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The [[Origin Recognition Complex|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.<ref name = "Bell2002" /> Studies in ''[[Drosophila]]'' showed that recessive lethal mutations in multiple ''drosophila'' ORC subunits reduces the amount of [[BrdU]] (a marker of active replication), incorporated.<ref name = "Pflumm2001" >{{cite journal |author= Pflumm, M.F and Bochtan, M.R. |title=Orc mutants arrest in metaphase with abnormally condensed chromosomes|journal=Development |volume=128|pages=1697–1707 |year=2001 |pmid= 11290306 |issue= 9}}</ref> 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.<ref name = "Bell2002" />
=== Cdc6 and Cdt1 ===
[[Cdc6]] and [[Cdt1]] assemble on the ORC and recruit the Mcm proteins.<ref name="The Cell Cycle" /> Homologs for these two S. cerevisiae proteins have been found in all eukaryotes.<ref name="The Cell Cycle" /> Studies have shown that these proteins are necessary for [[DNA replication]]. Mutations in ''[[Schizosaccharomyces pombe|S. pombe]]'' cdt1 blocked DNA replication.<ref name = "Bell2002" />
=== The Mcm Complex ===
Mcm 2-7 form a six-subunit complex and is thought to have helicase activity.<ref name = "Kearsey2003" /> Deletion of any single subunit of the complex has a lethal phenotype in yeast.<ref name = "Bell2002" /> Studies in ''Xenopus'' revealed the Mcm2-7 complex is a critical component of [[DNA replication]] machinery.<ref name = "Bell2002" /> 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.<ref name = "Bell2002" /> 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.<ref name = "Bell2002" />
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