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[[File:Overview of chromosome duplication in the cell cycle.svg|thumb|Overview of chromosome duplication in the 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]].<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]].▼
▲A key feature of the
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" />▼
== 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|
===Yeast===
Origins in [[budding yeast]] are defined by the [[autonomously replicating sequence]] (ARS), a short stretch of DNA (100-200 bp)
===Animals===
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.<ref name="The Cell Cycle" /> 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.<ref name="Mechali 2010" />
==The
[[Image:pre-RC.png|thumb|350px|Pre-RC assembly involves the assembly of the ORC subunits, Cdc6 and Cdt1 and the Mcm2-7 complex]]
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 phase|G1]]. Assembly of these pre-replicative complexes (pre-RCs) is regulated in a manner that coordinates [[DNA replication]] with the [[cell cycle]].<ref name = "Bell2002" >{{cite journal |author1=Bell, S.P. |author2=Dutta, A. |title=DNA replication in eukaryotic cells|journal=Annu. Rev. Biochem. |volume=71|pages=333–374 |year=2002 |doi=10.1146/annurev.biochem.71.110601.135425 |pmid= 12045100}}</ref>
===Components of the
====The ORC====
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 |
==== 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" />
==Regulation of pre-RC
[[Image:control.png|thumb|400px|pre-RC Assembly limited to late M and early G1]]
A two-step mechanism ensures that DNA is replicated only once per cycle. Assembly of the pre-RC complex (licensing) is limited to late mitosis and early G1 because it can occur only when [[Cyclin-dependent kinase|CDK]] activity is low, and [[Activated protein C|APC]] activity is high. Origin firing occurs only in S phase, when the APC is inactivated, and CDKs are activated.<ref name = "Diffley2008" />
===Yeast===
In budding yeast, CDK is the key regulator of pre-RC assembly.<ref name = "The Cell Cycle" /> Evidence for this is that inactivation of CDKs in cells arrested in G2/M or in S phase drives reassembly of pre-RCs.<ref name = "Diffley2008" /> CDK acts by inhibiting the individual components of the pre-RC. CDK phosphorylates Cdc6 to mark it for degradation by the SCF in late G1 and early S phase.<ref name = "Diffley2008" /> CDK also induces export of Mcm complexes and Cdt1 from the nucleus.<ref name = "Diffley2008" /> Evidence that CDKs regulate the localization of Mcm2-7
CDK also phosphorylates ORC proteins. It has been suggested that phosphorylation affects the ability of the ORC to bind other components of the pre-RC.<ref name="The Cell Cycle" />
To get substantial re-replication of DNA, regulation of all three components, Cdc6, Mcm2-7 and the ORC has to be prevented. Having multiple mechanisms to prevent re-replication is beneficial because it the regulatory network continues to function even if one of the components fails.<ref name="The Cell Cycle" />
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===Animals===
[[Geminin]] is an important inhibitor of pre-Rc assembly is metazoan cells.<ref name="The Cell Cycle" />
Geminin was identified in a screen for APC/C substrates in ''Xenopus''.<ref name = "Kirschner1998" >{{cite journal |
The importance of CDKs in preventing re-licensing in metazoan cells is still unclear. Some studies have
==Activation of
[[Image:Cdc7.png|thumb|300px|Regulation of Cdc7 activity throughout the cell cycle]]
While pre-RC complexes mark potential sites for origin activation, further proteins and complexes must assemble at these sites to activate replication (origin firing). The following events must occur in order to activate the origin: the DNA helix has to open, the helicase must be activated, and DNA polymerases and the rest of the replicative machinery have to load onto the DNA.<ref name="The Cell Cycle" /> These events depend on the assembly of
When the cell commits to a new cell cycle, after passing through the Start checkpoint, G1 and G1/S cyclin CDK complexes are activated. These activate the expression of the replicative machinery and of S-Cdk cylin complexes. S-Cdks and G1/S Cdks act to activate replication origins.<ref name="Bell2002" /> At the same time, S-Cdks suppress formation of new pre-RCs during S phase, G2 and early M, when S cyclin
===Role of CdK===
===Role of Cdc7===
▲'''Role of CdK:''' In ''S. cerevisiae'', the S cyclins Clb5 and Clb6 play and important role in inititating replication. In frog embryos, cyclin E-Cdk2 is primarily responsible for activating origins. Removal of cyclin E with antibodies blocks replication. Cyclin E-CDk2 is also important in ''Drosophila''. Levels of cyclin E rise during S phase and activate Cdk2.<ref name="Bell2002" />
==References==
[[Category:DNA replication]]
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