Control of chromosome duplication: Difference between revisions

Revision as of 19:53, 28 June 2011

Revision as of 19:55, 28 June 2011

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{{lead missing|date=December 2010}}

{{orphan|date=December 2010}}

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-R786R778–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 |issue= 5}}</ref> This article explores the regulatory system that ensures that DNA replication occurs only once per [[cell cycle]].

==The Replication Origin==

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===Yeast===

Origins in [[budding yeast]] are defined by the [[autonomously replicating sequence]] (ARS), a short stretch of DNA (100-200 bp) that that can initiate replication when transferred to any sequence of DNA.<ref name="The Cell Cycle" /><ref name = "Mechali 2010" >{{cite journal |author= Mechali, M. |title=Eukaryotic DNA replication origins: many choices for appropriate answers|journal=Nature Rev. Mol. Cell. Biol.|volume=11|pages=728–738 |year=2010 |doi= 10.1038/nrm2976 |issue= 10}}</ref> 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.<ref name="The Cell Cycle" /><ref name = "Gilbert2001" >{{cite journal |author= Gilbert, D.M |title=Making sense of eukaryotic replication origins|journal=Science|volume=294|pages=96–100 |year=2001 | pmid=11588251 | doi=10.1126/science.1061724 |issue= 5540 |pmc= 1255916 }}</ref> The ORC, a component of the initiation complex, binds the ACS [[in vivo]] throughout the cell cycle, and [[in vitro]] in an [[Adenosine triphosphate|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.<ref name="The Cell Cycle" /> 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.<ref name="Mechali 2010" /> 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.<ref name="The Cell Cycle" />

===Animals===

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==The Pre-Replication Complex==

[[Image:pre-RC.png|thumb|350px|Pre-RC assembly involves the assembly of the ORC subunits, Cdc6 and Cdt1 and the Mcm2-7 complex]] <ref name = "Bell2002" >{{cite journal |author= Bell, S.P. and 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> 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]].<ref name="Bell2002" />

===Components of the Pre-Rc===

===The ORC===

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.<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===

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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 |author= T.J McGarry and M.W Kirschner |title=Geminin, an inhibitor of [[DNA replication]], is degraded during mitosis|journal=Cell |volume=93|pages=1043–1053 |year=1998 |doi=10.1016/S0092-8674(00)81209-X |issue= 6 |pmid= 9635433}}</ref> Studies have shown that Geminin prevents pre_RC assmebly by binding to cdt1 and preventing its association with the pre-RC.<ref name = "Dutta2000" >{{cite journal |author= J.A. Wohlschlegel, B.T. Dwyer, S.K. Dhar, C. Cvetic, J.C. Walter and A. Dutta|title= Inhibition of eukaryotic DNA replication by geminin binding to Cdt1|journal=Science |volume=290|pages=2309–2312 |year=2000 |doi=10.1126/science.290.5500.2309 |issue=5500 |pmid= 11125146}}</ref> Since geminin is degraded by the APC/C, pre-Rc assembly can proceed only when APC/C activity is high, which occurs in G1.<ref name ="Diffley2008" />

The importance of CDKs in preventing re-licensing in metazoan cells is still unclear. Some studies have showed that under some conditions, CDKs can also promote licensing. In G0 mammalian cells, APC mediated degradation of Cdc6 prevents licencing. However, when the cells transition into a proliferative state, CDK phosphorylates Cdc6 to stabilizes it and allow it to accumulate and bind to origins before licensing inhibitors such as geminin accumulate.<ref name = "Mailand2005" >{{cite journal |author= Mailand, N. and Diffley J.F |title=CDKs promote DNA replication origin liscensing in human cells by promoting Cdc6 from APC/C dependent proteolysis |journal=Cell |volume=122|pages=915–926 |year=2005 | doi=10.1016/j.cell.2005.08.013 |issue= 6 |pmid= 16153703}}</ref>

==Activation of Replication Origins==