Control of chromosome duplication: Difference between revisions

Revision as of 21:07, 10 November 2011

Latest revision as of 10:21, 19 February 2025

(34 intermediate revisions by 31 users not shown)

Line 1:

[[File:Overview of chromosome duplication in the cell cycle.svg|thumb|Overview of chromosome duplication in the cell cycle]]

{{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–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]].

In [[cell biology]], [[eukaryotes]] possess a 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" />

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|doi-access= free }}</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 |authorauthor1= Kearsey, S.E. and |author2=Cotteril, S. |title= EngimaticEnigmatic 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|doi-access=free }}</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.Owen Morgan|title title= The Cell Cycle: Principles of Control| year=2007| publisher=New LondonScience Press 1st ed.|isbn=978-0-19-920610-0}}</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" />

===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.Reviews Mol.Molecular Cell. Biol.Biology |volume=11|pages=728–738 |year=2010 |doi= 10.1038/nrm2976 |issue= 10 |pmid=20861881}}</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===

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 Prepre-Replicationreplication Complexcomplex==

[[Image:pre-RC.png|thumb|350px|Pre-RC assembly involves the assembly of the ORC subunits, Cdc6 and Cdt1 and the Mcm2-7 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 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" />

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 Prepre-RcRC===

===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 |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=The and Cdt1ORC====

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 |authorauthor1= Pflumm, M.F. and |author2=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]] 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 ''[[S. pombe]]'' cdt1 blocked DNA replication.<ref name = "Bell2002" />

===The= McmCdc6 Complexand 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 Complexcomplex Assemblyassembly==

[[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 this is that inactivation of CDKs in nocodozole[[nocodazole]] arrested cells induced accumulation of Mcm2-7 in the nucleus.<ref name = "Diffley2008" /> Cdt1 is also exported because it binds to the Mcm complex. In Mcm depleted cells, cdt1Cdt1 did not accumulate in the nucleus. Conversely, when ana [[NCS]]{{dnNuclear localization sequence|date=Novembernuclear 2011}}localization tagsignal]] was attached to Mcm7, Mcm2-7 and Cdt1 were always found in the nucleus.<ref name = "Diffley2008" /> Export of Mcm from the [[Cell nucleus|nucleus]] prevents loading of new Mcm complexes but does not affect the complexes that have already been loaded onto the DNA.<ref name="The Cell Cycle" />

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" />

Line 36 ⟶ 40:

===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 |authorauthor1= T.J. McGarry and |author2=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|doi-access=free }}</ref> Studies have shown that Geminin prevents pre_RC assmeblyassembly by binding to cdt1 and preventing its association with the pre-RC.<ref name = "Dutta2000" >{{cite journal |authorauthor1= J.A. Wohlschlegel, |author2=B.T. Dwyer, |author3=S.K. Dhar, |author4=C. Cvetic, |author5=J.C. Walter and |author6=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 showedshown that under some conditions, CDKs can also promote licensing. In G0 mammalian cells, APC mediated degradation of Cdc6 prevents licencinglicensing. 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 |authorauthor1= Mailand, N. and |author2=Diffley J.F. |title=CDKs promote DNA replication origin liscensinglicensing 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|doi-access=free }}</ref>

==Activation of replication origins==

[[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 a several proteins to form the pre-initiation complex at the replication origins loaded with pre-replicative complexes.<ref name="The Cell Cycle" /> Assembly of the pre-initiation complex depends on the activities of S-Cdks and the protein kinase Cdc7. The pre-initiation complex activates the Mcm helicase and recruits DNA polymerase.<ref name="The Cell Cycle" />

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 leveslevels remains high. Cdc7 is activated in late G1 and is required thourghoutthroughout S phase for origin firing. Mutations in this protein in budding yeast, and in its homolog in fission yeast block initiation of replication. Cdc7 is highly conserved – related proteins have been identified in frogs and humans. [[DNA replication]] is inhibited when Cdc7 homologs are inhibited with antibodies in frog or human cells. It is not known whether CDKs and Cdc7 just regulate protein assembly at origins, or whether they directly activate components of the pre-initiation complex.<ref name="Bell2002" />

==Activation=Role of Replication OriginsCdK===

'''Role of CdK:''' In ''S. cerevisiae'', the S cyclins Clb5 and Clb6 play and important role in inititatinginitiating 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" />

[[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 a several proteins to form the pre-initiation complex at the replication origins loaded with pre-replicative complexes.<ref name="The Cell Cycle" /> Assembly of the pre-initiation complex depends on the activities of S-Cdks and the protein kinase Cdc7. The pre-initiation complex activates the Mcm helicase and recruits DNA polymerase.<ref name="The Cell Cycle" />

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 leves remains high. Cdc7 is activated in late G1 and is required thourghout S phase for origin firing. Mutations in this protein in budding yeast, and in its homolog in fission yeast block initiation of replication. Cdc7 is highly conserved – related proteins have been identified in frogs and humans. [[DNA replication]] is inhibited when Cdc7 homologs are inhibited with antibodies in frog or human cells. It is not known whether CDKs and Cdc7 just regulate protein assembly at origins, or whether they directly activate components of the pre-initiation complex.<ref name="Bell2002" />

===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" />

'''Role of Cdc7''': Cdc7 levels remain relatively constant throughout the cell cycle, but its activity varies. Its activity is low in G1, increases in late G1, and remains high till late mitosis. Dbf4 is the key regulator of Cdc7 activity – association Cdc7 with Dbf4 activates its kinase activity. In a similar manner to cyclin levels, dbf4 levels fluctuate throughout the cell cycle.<ref name="Bell2002" /> In vitro biochemical studies have shown that Cdc7-Dbf4 phsphorylatesphosphorylates individual components of the Mcm complex. It also seems to be involved in the recruitment of Cdc45 to chromatin at the time of initiation. In Xenopus eggs, Cdc45 has been shown to interact with DNA polymerase α, and in yeast, mutations in Cdc45 prevent assembly of DNA pol α at origins, suggesting that Cdc45 recruits DNA pol α to chromatin in a Cdc7/Dbf4 dependantdependent manner.<ref name="The Cell Cycle" /><ref name="Bell2002" />

==References==

{{Reflist}}

<references/>

[[Category:DNA replication]]