Editing DNA replication (section)

=== Eukaryotes ===
Within eukaryotes, DNA replication is controlled within the context of the [[cell cycle]]. As the cell grows and divides, it progresses through stages in the cell cycle; DNA replication takes place during the S phase (synthesis phase). The progress of the eukaryotic cell through the cycle is controlled by [[cell cycle checkpoint]]s. Progression through checkpoints is controlled through complex interactions between various proteins, including [[cyclin]]s and [[cyclin-dependent kinase]]s.<ref>{{Cite book |title=Molecular Biology of the Cell |vauthors=Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P |publisher=Garland Science |year=2002 |isbn=0-8153-3218-1 |chapter=Intracellular Control of Cell-Cycle Events: S-Phase Cyclin-Cdk Complexes (S-Cdks) Initiate DNA Replication Once Per Cycle |chapter-url=https://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mboc4.section.3214#3215}}</ref> Unlike bacteria, eukaryotic DNA replicates in the confines of the nucleus.<ref>{{Cite book |title=Genomes |vauthors=Brown TA |publisher=Wiley-Liss |year=2002 |edition=2nd |location=Oxford |chapter=Chapter 13: Genome Replication |chapter-url=https://www.ncbi.nlm.nih.gov/books/NBK21113/}}</ref>

The G1/S checkpoint (restriction checkpoint) regulates whether eukaryotic cells enter the process of DNA replication and subsequent division. Cells that do not proceed through this checkpoint remain in the G0 stage and do not replicate their DNA.{{cn|date=November 2024}}

Once the DNA has gone through the "G1/S" test, it can only be copied once in every cell cycle. When the Mcm complex moves away from the origin, the pre-replication complex is dismantled. Because a new Mcm complex cannot be loaded at an origin until the pre-replication subunits are reactivated, one origin of replication can not be used twice in the same cell cycle.<ref name="Morgan-2007" />

Activation of S-Cdks in early S phase promotes the destruction or inhibition of individual pre-replication complex components, preventing immediate reassembly. S and M-Cdks continue to block pre-replication complex assembly even after S phase is complete, ensuring that assembly cannot occur again until all Cdk activity is reduced in late mitosis.<ref name="Morgan-2007" />

In budding yeast, inhibition of assembly is caused by Cdk-dependent phosphorylation of pre-replication complex components. At the onset of S phase, phosphorylation of Cdc6 by [[Cyclin-dependent kinase 1|Cdk1]] causes the binding of Cdc6 to the [[SCF complex|SCF]] [[Ubiquitin--protein ligase|ubiquitin protein ligase]], which causes proteolytic destruction of Cdc6. Cdk-dependent phosphorylation of Mcm proteins promotes their export out of the nucleus along with Cdt1 during S phase, preventing the loading of new Mcm complexes at origins during a single cell cycle. Cdk phosphorylation of the origin replication complex also inhibits pre-replication complex assembly. The individual presence of any of these three mechanisms is sufficient to inhibit pre-replication complex assembly. However, mutations of all three proteins in the same cell does trigger reinitiation at many origins of replication within one cell cycle.<ref name="Morgan-2007" /><ref>{{Cite journal |vauthors=Nguyen VQ, Co C, Li JJ |date=June 2001 |title=Cyclin-dependent kinases prevent DNA re-replication through multiple mechanisms |journal=Nature |volume=411 |issue=6841 |pages=1068–1073 |bibcode=2001Natur.411.1068N |doi=10.1038/35082600 |pmid=11429609 |s2cid=4393812}}</ref>

In animal cells, the protein [[geminin]] is a key inhibitor of pre-replication complex assembly. Geminin binds Cdt1, preventing its binding to the origin recognition complex. In G1, levels of geminin are kept low by the APC, which ubiquitinates geminin to target it for degradation. When geminin is destroyed, Cdt1 is released, allowing it to function in pre-replication complex assembly. At the end of G1, the APC is inactivated, allowing geminin to accumulate and bind Cdt1.<ref name="Morgan-2007" />

Replication of chloroplast and mitochondrial genomes occurs independently of the cell cycle, through the process of [[D-loop replication]].{{cn|date=November 2024}}<ref>{{Cite journal |last1=Kabeya |first1=Yukihiro |last2=Miyagishima |first2=Shin-ya |date=February 27, 2013 |title=Chloroplast DNA Replication Is Regulated by the Redox State Independently of Chloroplast Division in Chlamydomonas reinhardtii |journal=National Library of Medicine |volume=161 |issue=4 |pages=2102–2112 |doi=10.1104/pp.113.216291 |pmid=23447524 |pmc=3613479 }}</ref>

==== Replication focus ====
In vertebrate cells, replication sites concentrate into positions called '''replication foci'''.<ref name="in&out" /> Replication sites can be detected by immunostaining daughter strands and replication enzymes and monitoring GFP-tagged replication factors. By these methods it is found that replication foci of varying size and positions appear in S phase of cell division and their number per nucleus is far smaller than the number of genomic replication forks.

'''P. Heun et al.''',<ref name="in&out" />(2001) tracked GFP-tagged replication foci in budding yeast cells and revealed that replication origins move constantly in G1 and S phase and the [[Molecular dynamics|dynamics]] decreased significantly in S phase.<ref name="in&out" /> Traditionally, replication sites were fixed on spatial structure of chromosomes by [[nuclear matrix]] or [[lamin]]s. The Heun's results denied the traditional concepts, budding yeasts do not have lamins, and support that replication origins self-assemble and form replication foci.{{cn|date=November 2024}}

By firing of replication origins, controlled spatially and temporally, the formation of replication foci is regulated. D. A. Jackson et al.(1998) revealed that neighboring origins fire simultaneously in mammalian cells.<ref name="in&out" /> Spatial juxtaposition of replication sites brings '''clustering''' of replication forks. The clustering do '''rescue of stalled replication forks''' and favors normal progress of replication forks. Progress of replication forks is inhibited by many factors; collision with proteins or with complexes binding strongly on DNA, deficiency of dNTPs, nicks on template DNAs and so on. If replication forks get stuck and the rest of the sequences from the stuck forks are not copied, then the daughter strands get nick nick unreplicated sites. The un-replicated sites on one parent's strand hold the other strand together but not daughter strands. Therefore, the resulting sister chromatids cannot separate from each other and cannot divide into 2 daughter cells. When neighboring origins fire and a fork from one origin is stalled, fork from other origin access on an opposite direction of the stalled fork and duplicate the un-replicated sites. As other mechanism of the rescue there is application of '''dormant replication origins''' that excess origins do not fire in normal DNA replication.{{cn|date=November 2024}}