View source for DNA replication

{{short description|Biological process}}
[[File:DNA replication split.svg|thumb|200px|DNA replication: The [[double helix]] is 'unzipped' and unwound, then each separated strand (turquoise) acts as a template for replicating a new partner strand (green). [[Nucleotide]]s (bases) are matched to synthesize the new partner strands into two new double helices.]] 
'''DNA replication''' is the [[biological process|process]] by which a [[Cell (biology)|cell]] makes exact copies of its [[DNA]].<ref>{{Cite web |date=2022-03-01 |title=What Is DNA Replication And Its Steps? |url=https://www.funbiology.com/what-is-dna-replication/ |access-date=2023-08-04 |language=en-US |vauthors=Sabhadiya A}}</ref><ref>{{Cite journal |vauthors=O'Donnell M, Langston L, Stillman B |date=July 2013 |title=Principles and concepts of DNA replication in bacteria, archaea, and eukarya |journal=Cold Spring Harbor Perspectives in Biology |volume=5 |issue=7 |article-number=a010108 |doi=10.1101/cshperspect.a010108 |pmc=3685895 |pmid=23818497}}</ref><ref>{{Cite journal |vauthors=Prioleau MN, MacAlpine DM |date=August 2016 |title=DNA replication origins-where do we begin? |journal=Genes & Development |volume=30 |issue=15 |pages=1683–1697 |doi=10.1101/gad.285114.116 |pmc=5002974 |pmid=27542827}}</ref><ref>{{Cite journal |vauthors=Dewar JM, Walter JC |date=August 2017 |title=Mechanisms of DNA replication termination |journal=Nature Reviews. Molecular Cell Biology |volume=18 |issue=8 |pages=507–516 |doi=10.1038/nrm.2017.42 |pmc=6386472 |pmid=28537574}}</ref> This process occurs in all [[organism]]s and is essential to [[heredity|biological inheritance]], [[cell division]], and [[DNA repair|repair]] of damaged tissues. DNA replication ensures that each of the newly divided [[daughter cell]]s receives its own copy of each DNA molecule.<ref>{{Cite web |title=GENETICS / DNA REPLICATION (BASIC) – Pathwayz |url=https://www.pathwayz.org/Tree/Plain/DNA+REPLICATION+(BASIC)#:~:text=The%20purpose%20of%20DNA%20replication,own%20copy%20of%20the%20DNA. |access-date=2020-12-10 |website=pathwayz.org}}</ref>

DNA most commonly occurs in double-stranded form, made up of two [[Complementary DNA|complementary]] [[DNA strand|strands]] held together by [[base pairing]] of the [[nucleotide]]s comprising each strand. The two linear strands of a double-stranded DNA molecule typically twist together in the shape of a [[double helix]].<ref>{{Cite web |title=double helix |url=https://www.nature.com/scitable/definition/double-helix-277/ |access-date=2020-12-10 |website=Learn Science at Scitable |publisher=Nature Education}}</ref> During replication, the two strands are separated, and each strand of the original DNA molecule then serves as a template for the production of a complementary counterpart strand, a process referred to as [[semiconservative replication]]. As a result, each replicated DNA molecule is composed of one original DNA strand as well as one newly synthesized strand.<ref>{{Cite journal |vauthors=Pray LA |date=2008 |title=Semi-Conservative DNA Replication; Meselson and Stahl |url=https://www.nature.com/scitable/topicpage/semi-conservative-dna-replication-meselson-and-stahl-421/ |journal=Nature Education |volume=1 |issue=1 |page=98}}</ref> Cellular [[Proofreading (Biology)|proofreading]] and error-checking mechanisms ensure near-perfect [[fidelity]] for DNA replication.<ref name="Berg"> Imperfect DNA replication results in [[mutation]]s.{{Cite book |url=https://archive.org/details/biochemistrychap00jere |title=Biochemistry |vauthors=Berg JM, Tymoczko JL, Stryer L, Clarke ND |publisher=W.H. Freeman and Company |year=2002 |isbn=0-7167-3051-0 |chapter=Chapter 27: DNA Replication, Recombination, and Repair |access-date=2019-08-09 |chapter-url=https://www.ncbi.nlm.nih.gov/books/bv.fc,kgi?rid=stryer.chapter.3740 |archive-url=https://web.archive.org/web/20200326053135/https://archive.org/details/biochemistrychap00jere |archive-date=2020-03-26}}</ref><ref name="Alberts">{{Cite book |title=Molecular Cell Biology |vauthors=Lodish H, Berk A, Zipursky SL, etal |publisher=WH Freeman |year=2000 |isbn=0-7167-3136-3 |edition=4th |chapter=DNA Replication, Repair, and Recombination |chapter-url=https://www.ncbi.nlm.nih.gov/books/NBK21656/}}</ref>

DNA replication usually begins at specific locations known as [[origin of replication|origins of replication]]<ref name="Hu 352–372">{{Cite journal |vauthors=Hu Y, Stillman B |date=February 2023 |title=Origins of DNA replication in eukaryotes |journal=Molecular Cell |volume=83 |issue=3 |pages=352–372 |doi=10.1016/j.molcel.2022.12.024 |pmc=9898300 |pmid=36640769}}</ref> which are scattered across the [[genome]].<ref name="origins">{{Cite book |url=https://archive.org/details/biochemistrychap00jere |title=Biochemistry |vauthors=Berg JM, Tymoczko JL, Stryer L, Clarke ND |publisher=W.H. Freeman and Company |year=2002 |isbn=0-7167-3051-0 |chapter=Chapter 27, Section 4: DNA Replication of Both Strands Proceeds Rapidly from Specific Start Sites |access-date=2019-08-09 |chapter-url=https://www.ncbi.nlm.nih.gov/bofghoks/bv.fcvbngi?rid=stayer.section.3794 |archive-url=https://web.archive.org/web/20200326053135/https://archive.org/details/biochemistrychap00jere |archive-date=2020-03-26}}</ref> Unwinding of DNA at the origin is accommodated by [[enzyme]]s known as [[helicase]]s and results in [[replication fork]]s growing bi-directionally from the origin. Numerous [[protein]]s are associated with the replication fork to help in the initiation and continuation of [[DNA synthesis]]. Most prominently, [[DNA polymerase]] synthesizes the new strands by incorporating [[nucleotide]]s that complement the nucleotides of the template strand. DNA  replication occurs during the S (synthesis) stage of [[interphase]].<ref>{{Cite journal |last1=Chagin |first1=Vadim O. |last2=Stear |first2=Jeffrey H. |last3=Cardoso |first3=M. Cristina |date=April 2010 |title=Organization of DNA replication |journal=Cold Spring Harbor Perspectives in Biology |volume=2 |issue=4 |article-number=a000737 |doi=10.1101/cshperspect.a000737 |issn=1943-0264 |pmc=2845211 |pmid=20452942}}</ref>

DNA replication can also be performed ''[[in vitro]]'' (artificially, outside a cell).<ref name="Jarillo-2021">{{Cite journal |vauthors=Jarillo J, Ibarra B, Cao-García FJ |date=2021 |title=DNA replication: ''In vitro'' single-molecule manipulation data analysis and models |journal=Computational and Structural Biotechnology Journal |volume=19 |pages=3765–3778 |doi=10.1016/j.csbj.2021.06.032 |pmc=8267548 |pmid=34285777}}</ref> DNA polymerases isolated from cells and artificial DNA primers can be used to start DNA synthesis at known sequences in a template DNA molecule. [[Polymerase chain reaction]] (PCR), [[ligase chain reaction]] (LCR), and [[transcription-mediated amplification]] (TMA) are all common examples of this technique. In March 2021, researchers reported evidence suggesting that a preliminary form of [[transfer RNA]], a necessary component of [[Translation (biology)|translation]] (the biological synthesis of new [[protein]]s in accordance with the [[genetic code]]), could have been a replicator molecule itself in the early [[abiogenesis]] of primordial life.<ref name="EL-20210302">{{Cite journal |vauthors=Kühnlein A, Lanzmich SA, Braun D |date=March 2021 |title=tRNA sequences can assemble into a replicator |journal=eLife |volume=10 |article-number=e63431 |doi=10.7554/eLife.63431 |pmc=7924937 |pmid=33648631 |doi-access=free}}</ref><ref name="STD-20210403">{{Cite news |date=3 April 2021 |title=Solving the Chicken-and-the-Egg Problem – "A Step Closer to the Reconstruction of the Origin of Life" |url=https://scitechdaily.com/solving-the-chicken-and-the-egg-problem-a-step-closer-to-the-reconstruction-of-the-origin-of-life/ |access-date=3 April 2021 |work=[[SciTech (magazine)|SciTechDaily]] |vauthors=Maximilian L}}</ref>

== DNA structure ==
[[File:DNA Structure+Key+Labelled.pn NoBB.png|thumb|right|upright=1.55|The structure of the DNA [[double helix]] (type [[B-DNA]]). The [[atom]]s in the structure are color-coded by [[Chemical element|element]], and the detailed structures of two [[base pair]]s are shown in the bottom right.]]
DNA is a double-stranded structure, with both strands coiled together to form the characteristic [[double helix]]. Each single strand of DNA is a chain of four types of [[nucleotide]]s. Nucleotides in DNA contain a [[deoxyribose]] sugar, a [[phosphate]], and a [[nucleobase]]. The four types of [[nucleotide]] correspond to the four [[nucleobase]]s: [[adenine]], [[cytosine]], [[guanine]], and [[thymine]], commonly abbreviated as A, C, G, and T, respectively. Adenine and guanine are [[purine]]<ref>{{Cite journal |vauthors=Friedberg EC |date=January 2003 |title=DNA damage and repair |journal=Nature |volume=421 |issue=6921 |pages=436–440 |bibcode=2003Natur.421..436F |doi=10.1038/nature01408 |pmid=12540918 |doi-access=free}}</ref> nucleobases, while cytosine and thymine are [[pyrimidine]]s. These nucleotides form [[phosphodiester bonds]], creating phosphate-deoxyribose backbone of the DNA double helix with the nucleobases pointing inward (i.e., toward the opposing strand). Complementary nucleobases are matched between strands through [[hydrogen bonding|hydrogen bonds]] to form [[base pair]]s. Adenine pairs with thymine (two hydrogen bonds), and guanine pairs with cytosine (three [[hydrogen bonds]]).<ref>{{Cite web |date=2023-03-13 |title=Base Pair: Definition, Rules In DNA And RNA |url=https://www.funbiology.com/base-pair-definition-rules-in-dna-and-rna/ |access-date=2023-08-04 |language=en-US |vauthors=Sabhadiya A}}</ref>

[[Directionality (molecular biology)|DNA strands have a directionality]], and the different ends of a single strand are called the "3′ (three-prime) end" and the "5′ (five-prime) end". By convention, if the base sequence of a single strand of DNA is given, the left end of the sequence is the 5′ end, while the right end of the sequence is the 3′ end. The strands of the double helix are anti-parallel, with one being 5′ to 3′, and the opposite strand 3′ to 5′. These terms refer to the chemical convention of numbering the carbon atoms comprising the deoxyribose molecule and indicate the specific carbon atom to which the next phosphate in the chain attaches. Directionality has consequences in DNA synthesis, because DNA polymerase can synthesize DNA in only one direction by adding nucleotides to the 3′ end of a DNA strand.<ref>{{Cite book |last1=Alberts |first1=B. |title=Molecular Biology of the Cell |last2=Johnson |first2=A. |last3=Lewis |first3=J. |last4=Raff |first4=M. |last5=Roberts |first5=K. |last6=Walter |first6=P. |publisher=Garland Science. |year=2002 |edition=4th |location=New York |chapter=DNA Replication Mechanisms |display-authors=3 |chapter-url=https://www.ncbi.nlm.nih.gov/books/NBK26850/}}</ref>

The pairing of complementary bases in DNA (through [[hydrogen bonding]]) means that the information contained within each strand is redundant. Phosphodiester (intra-strand) bonds are stronger than hydrogen (inter-strand) bonds. The actual job of the phosphodiester bonds is to connect the 5' carbon atom of one nucleotide to the 3' carbon atom of another nucleotide, while the hydrogen bonds stabilize DNA double helices across the helix axis but not in the direction of the longitudinal axis.<ref>{{Cite web |title=DNA function & structure (with diagram) (article) |url=https://www.khanacademy.org/test-prep/mcat/biomolecules/dna/a/dna-structure-and-function |access-date=2020-12-10 |website=Khan Academy |language=en}}</ref> This makes it possible to separate the strands from one another. The nucleotides on a single strand can therefore be used to reconstruct nucleotides on a newly synthesized partner strand.<ref>{{Cite book |title=Molecular Biology of the Cell |vauthors=Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P |date=2002 |publisher=Garland Science |isbn=0-8153-3218-1 |edition=4th |pages=238–240}}</ref>

== DNA polymerase ==
{{Main|DNA polymerase}}
[[File:DNA polymerase.svg|thumb|250px|right| DNA polymerases adds nucleotides to the 3′ end of a strand of DNA.<ref>{{Cite book |title=Fundamental Molecular Biology |vauthors=Allison L |date=2007 |publisher=Blackwell Publishing |isbn=978-1-4051-0379-4 |page=112}}</ref> If a mismatch is accidentally incorporated, the polymerase is inhibited from further extension. Proofreading removes the mismatched nucleotide and extension continues.]]
[[DNA polymerase]]s are a family of [[enzyme]]s that carry out all forms of DNA replication.<ref>{{Cite book |url=https://archive.org/details/biochemistrychap00jere |title=Biochemistry |vauthors=Berg JM, Tymoczko JL, Stryer L, Clarke ND |publisher=W.H. Freeman and Company |year=2002 |isbn=0-7167-3051-0}} [https://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=stryer.section.3769 Chapter 27, Section 2: DNA Polymerases Require a Template and a Primer]{{dead link|date=July 2025|bot=medic}}{{cbignore|bot=medic}}</ref> DNA polymerases in general cannot initiate synthesis of new strands but can only extend an existing DNA or RNA strand paired with a template strand. To begin synthesis, a short fragment of RNA, called a [[primer (molecular biology)|primer]], must be created and paired with the template DNA strand.

DNA polymerase adds a new strand of DNA by extending the 3′ end of an existing nucleotide chain, adding new [[nucleotide]]s matched to the template strand, one at a time, via the creation of [[phosphodiester bond]]s. The energy for this process of DNA polymerization comes from hydrolysis of the [[high-energy phosphate]] (phosphoanhydride) bonds between the three phosphates attached to each unincorporated [[nucleotide|base]]. Free bases with an attached sugar molecule (deoxyribose in the case of DNA) are called [[nucleoside]]s, and nucleosides with one or more attached phosphate groups are called [[nucleotide]]s; in particular, nucleosides with three attached phosphate groups are called [[nucleoside triphosphate]]s. When a free nucleotide is being added to a growing DNA strand, the formation of a phosphodiester bond between the proximal phosphate of the free nucleotide and the deoxyribose of another nucleotide within the growing chain is accompanied by hydrolysis of a high-energy phosphate bond with release of the free nucleotide's two distal phosphate groups as a [[pyrophosphate]]. Enzymatic hydrolysis of the resulting [[pyrophosphate]] into inorganic phosphate consumes a second high-energy phosphate bond and renders the reaction effectively irreversible.<ref group="Note">The [[Bioenergetics|energetics]] of this process may also help explain the directionality of synthesis—if DNA were synthesized in the 3′ to 5′ direction, the energy for the process would come from the 5′ end of the growing strand, rather than from free nucleotides. The problem is that if the high-energy triphosphates were on the growing strand and not on the free nucleotides, proofreading by removing a mismatched terminal nucleotide would be problematic: once a nucleotide is added, the triphosphate is lost and a single phosphate remains on the backbone between the new nucleotide and the rest of the strand. If the added nucleotide were mismatched, removal would result in a DNA strand terminated by a monophosphate at the end of the "growing strand" rather than a high-energy triphosphate. The strand would consequently be stuck and would not be able to grow anymore. In actuality, the high-energy triphosphates hydrolyzed at each step originate from free nucleotides, not the polymerized strand, so this issue does not exist.</ref>

In general, DNA polymerases are highly accurate, with an intrinsic error rate of less than one mistake for every 10<sup>7</sup> nucleotides added.<ref name="pmid18166979">{{Cite journal |vauthors=McCulloch SD, Kunkel TA |date=January 2008 |title=The fidelity of DNA synthesis by eukaryotic replicative and translesion synthesis polymerases |journal=Cell Research |volume=18 |issue=1 |pages=148–161 |doi=10.1038/cr.2008.4 |pmc=3639319 |pmid=18166979}}</ref> Some DNA polymerases can also delete nucleotides from the end of a developing strand in order to fix mismatched bases. This is known as proofreading. Finally, post-replication mismatch repair mechanisms monitor the DNA for errors, being capable of distinguishing mismatches in the newly synthesized DNA strand from the original strand sequence. Together, these three discrimination steps enable replication fidelity of less than one mistake for every 10<sup>9</sup> nucleotides added.<ref name="pmid18166979" />

The rate of DNA replication in a living cell was first measured as the rate of phage T4 DNA elongation in phage-infected ''E. coli''.<ref>{{Cite journal |vauthors=McCarthy D, Minner C, Bernstein H, Bernstein C |date=October 1976 |title=DNA elongation rates and growing point distributions of wild-type phage T4 and a DNA-delay amber mutant |journal=Journal of Molecular Biology |volume=106 |issue=4 |pages=963–981 |doi=10.1016/0022-2836(76)90346-6 |pmid=789903}}<!--|access-date=7 April 2016--></ref> During the period of exponential DNA increase at 37&nbsp;°C, the rate was 749 nucleotides per second. The mutation rate per base pair per replication during phage T4 DNA synthesis is 1.7 per 10<sup>8</sup>.<ref>Drake JW (1970) ''The Molecular Basis of Mutation.'' Holden-Day, San Francisco {{ISBN|0-8162-2450-1}}  {{ISBN|978-0-8162-2450-0}}</ref>

== Replication process ==
{{Main|Prokaryotic DNA replication|Eukaryotic DNA replication}}

[[File:Asymmetry in the synthesis of leading and lagging strands.svg|thumb|left|Overview of the steps in DNA replication]]
[[File:Steps in DNA synthesis.svg|thumb|Steps in DNA synthesis]]

DNA replication, like all biological polymerization processes, proceeds in three enzymatically catalyzed and coordinated steps: initiation, elongation and termination.

=== Initiation ===
[[File:Figure Role of initiators for initiation of DNA replication.png|thumb|200px|right|Role of initiators for initiation of DNA replication]]
[[File:EukPreRC.jpg|thumb|200px|Formation of pre-replication complex]]
For a [[cell division|cell to divide]], it must first replicate its DNA.<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=Chapter 5: DNA Replication Mechanisms |chapter-url=https://www.ncbi.nlm.nih.gov/books/NBK26850/}}</ref> DNA replication is an all-or-none process; once replication begins, it proceeds to completion. Once replication is complete, it does not occur again in the same cell cycle. This is made possible by the division of initiation of the [https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/pre-replication-complex pre-replication complex].{{cn|date=November 2024}}

=== Pre-replication complex ===
{{Main|Pre-replication complex}}

In late [[mitosis]] and early [[G1 phase]], a large complex of initiator proteins assembles into the pre-replication complex at particular points in the DNA, known as "[[origin of replication|origins]]".<ref name="origins" /><ref name="Hu 352–372" /> In ''[[Escherichia coli|E. coli]]'' the primary initiator protein is [[DnaA|Dna A]]; in [[yeast]], this is the [[origin recognition complex]].<ref>{{Cite journal |vauthors=Weigel C, Schmidt A, Rückert B, Lurz R, Messer W |date=November 1997 |title=DnaA protein binding to individual DnaA boxes in the Escherichia coli replication origin, oriC |journal=The EMBO Journal |volume=16 |issue=21 |pages=6574–6583 |doi=10.1093/emboj/16.21.6574 |pmc=1170261 |pmid=9351837}}</ref>  Sequences used by initiator proteins tend to be "AT-rich" (rich in adenine and thymine bases), because A-T base pairs have two hydrogen bonds (rather than the three formed in a C-G pair) and thus are easier to strand-separate.<ref>{{Cite book |url=https://archive.org/details/molecularcellbio00lodi |title=Molecular Cell Biology |vauthors=Lodish H, Berk A, Zipursky LS, Matsudaira P, Baltimore D, Darnell J |publisher=W. H. Freeman and Company |year=2000 |isbn=0-7167-3136-3}}[https://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.section.3163#3179 12.1.  General Features of Chromosomal Replication: Three Common Features of Replication Origins]{{dead link|date=July 2025|bot=medic}}{{cbignore|bot=medic}}</ref>  In eukaryotes, the origin recognition complex (ORC) catalyzes the assembly of initiator proteins into the pre-replication complex. In addition, a recent report suggests that budding yeast ORC dimerizes in a cell cycle dependent manner to control licensing.<ref>{{Cite journal |vauthors=Lin YC, Prasanth SG |date=July 2021 |title=Replication initiation: Implications in genome integrity |journal=DNA Repair |volume=103 |article-number=103131 |doi=10.1016/j.dnarep.2021.103131 |pmc=8296962 |pmid=33992866 |doi-access=free}}</ref><ref>{{Cite journal |display-authors=6 |vauthors=Amin A, Wu R, Cheung MH, Scott JF, Wang Z, Zhou Z, Liu C, Zhu G, Wong CK, Yu Z, Liang C |date=March 2020 |title=An Essential and Cell-Cycle-Dependent ORC Dimerization Cycle Regulates Eukaryotic Chromosomal DNA Replication |journal=Cell Reports |volume=30 |issue=10 |pages=3323–3338.e6 |doi=10.1016/j.celrep.2020.02.046 |pmid=32160540 |doi-access=free}}</ref> {{Clarify|date=September 2025}} In turn, the process of ORC dimerization is mediated by a cell cycle-dependent Noc3p dimerization cycle in vivo, and this role of Noc3p is separable from its role in ribosome biogenesis. [https://www.life-science-alliance.org/content/6/3/e202201594.full An essential Noc3p dimerization cycle mediates ORC double-hexamer formation in replication licensing] and Noc3p are continuously bound to the chromatin throughout the cell cycle.<ref>{{Cite journal |vauthors=Zhang Y, Yu Z, Fu X, Liang C |date=June 2002 |title=Noc3p, a bHLH protein, plays an integral role in the initiation of DNA replication in budding yeast |journal=Cell |volume=109 |issue=7 |pages=849–860 |doi=10.1016/s0092-8674(02)00805-x |pmid=12110182 |doi-access=free}}</ref>  [[CDC6|Cdc6]] and [[DNA replication factor CDT1|Cdt1]] then associate with the bound origin recognition complex at the origin in order to form a larger complex necessary to load the [[Minichromosome maintenance|Mcm complex]] onto the DNA. In eukaryotes, the Mcm complex is the helicase that will split the DNA helix at the replication forks and origins. The Mcm complex is recruited at late G1 phase and loaded by the ORC-Cdc6-Cdt1 complex onto the DNA via ATP-dependent protein remodeling. The loading of the MCM complex onto the origin DNA marks the completion of pre-replication complex formation.<ref name="Morgan-2007">{{Cite book |title=The cell cycle: principles of control |vauthors=Morgan DO |date=2007 |publisher=New Science Press |isbn=978-0-19-920610-0 |location=London |pages=64–75 |oclc=70173205}}</ref>

If environmental conditions are right in late G1 phase, the G1 and G1/S [[cyclin]]-[[Cyclin-dependent kinase|Cdk]] complexes are activated, which stimulate expression of genes that encode components of the DNA synthetic machinery. G1/S-Cdk activation also promotes the expression and activation of S-Cdk complexes, which may play a role in activating replication origins depending on species and cell type. Control of these Cdks vary depending on cell type and stage of development. This regulation is best understood in [[budding yeast]], where the S cyclins [[Clb 5,6 (Cdk1)|Clb5]] and [[Clb 5,6 (Cdk1)|Clb6]] are primarily responsible for DNA replication.<ref>{{Cite journal |vauthors=Donaldson AD, Raghuraman MK, Friedman KL, Cross FR, Brewer BJ, Fangman WL |date=August 1998 |title=CLB5-dependent activation of late replication origins in S. cerevisiae |journal=Molecular Cell |volume=2 |issue=2 |pages=173–182 |doi=10.1016/s1097-2765(00)80127-6 |pmid=9734354 |doi-access=free}}</ref> Clb5,6-Cdk1 complexes directly trigger the activation of replication origins and are therefore required throughout S phase to directly activate each origin.<ref name="Morgan-2007" />

In a similar manner, [[Cell division cycle 7-related protein kinase|Cdc7]] is also required through [[S phase]] to activate replication origins. Cdc7 is not active throughout the cell cycle, and its activation is strictly timed to avoid premature initiation of DNA replication. In late G1, Cdc7 activity rises abruptly as a result of association with the regulatory subunit [[DBF4]], which binds Cdc7 directly and promotes its protein kinase activity. Cdc7 has been found to be a rate-limiting regulator of origin activity. Together, the G1/S-Cdks and/or S-Cdks and Cdc7 collaborate to directly activate the replication origins, leading to initiation of DNA synthesis.<ref name="Morgan-2007" />

=== Preinitiation complex ===

In early S phase, S-Cdk and Cdc7 activation lead to the assembly of the preinitiation complex, a massive protein complex formed at the origin. Formation of the preinitiation complex displaces Cdc6 and Cdt1 from the origin replication complex, inactivating and disassembling the pre-replication complex. Loading the preinitiation complex onto the origin activates the Mcm helicase, causing unwinding of the DNA helix. The preinitiation complex also loads [[DNA polymerase alpha|α-primase]] and other DNA polymerases onto the DNA.<ref name="Morgan-2007" />

After α-primase synthesizes the first primers, the primer-template junctions interact with the clamp loader, which loads the sliding clamp onto the DNA to begin DNA synthesis. The components of the preinitiation complex remain associated with replication forks as they move out from the origin.<ref name="Morgan-2007" />

=== Elongation ===
DNA polymerase has 5′–3′ activity.
All known DNA replication systems require a free 3′ [[hydroxyl]] group before synthesis can be initiated (note: the DNA template is read in 3′ to 5′ direction whereas a new strand is synthesized in the 5′ to 3′ direction—this is often confused). Four distinct mechanisms for DNA synthesis are recognized:{{cn|date=November 2024}}
# All cellular life forms and many DNA [[virus]]es, [[phage]]s and [[plasmid]]s use a [[primase]] to synthesize a short RNA primer with a free 3′ OH group which is subsequently elongated by a DNA polymerase.
# The retroelements (including [[retrovirus]]es) employ a transfer RNA that primes DNA replication by providing a free 3′ OH that is used for elongation by the [[reverse transcriptase]].
# In the [[adenovirus]]es and the φ29 family of [[bacteriophage]]s, the 3′ OH group is provided by the side chain of an amino acid of the genome attached protein (the terminal protein) to which nucleotides are added by the DNA polymerase to form a new strand.
# In the single stranded DNA viruses—a group that includes the [[circovirus]]es, the [[geminivirus]]es, the [[parvovirus]]es and others—and also the many phages and [[plasmid]]s that use the rolling circle replication (RCR) mechanism, the RCR endonuclease creates a nick in the genome strand (single stranded viruses) or one of the DNA strands (plasmids). The 5′ end of the nicked strand is transferred to a [[tyrosine]] residue on the nuclease and the free 3′ OH group is then used by the DNA polymerase to synthesize the new strand.

Cellular organisms use the first of these pathways leading to it being the most well-known. In this mechanism, once the two strands are separated, [[primase]] adds RNA primers to the template strands. The leading strand receives one RNA primer while the lagging strand receives several. The leading strand is continuously extended from the primer by a DNA polymerase with high [[processivity]], while the lagging strand is extended discontinuously from each primer forming [[Okazaki fragments]]. [[RNase]] removes the primer RNA fragments, and a low processivity DNA polymerase distinct from the replicative polymerase enters to fill the gaps. When this is complete, a single nick on the leading strand and several nicks on the lagging strand can be found. [[Ligase]] works to fill these nicks in, thus completing the newly replicated DNA molecule.{{cn|date=November 2024}}

The primase used in this process differs significantly between [[bacteria]] and [[archaea]]/[[eukaryote]]s. Bacteria use a primase belonging to the [[DnaG]] protein superfamily which contains a catalytic domain of the TOPRIM fold type.<ref>{{Cite journal |vauthors=Aravind L, Leipe DD, Koonin EV |date=September 1998 |title=Toprim--a conserved catalytic domain in type IA and II topoisomerases, DnaG-type primases, OLD family nucleases and RecR proteins |journal=Nucleic Acids Research |volume=26 |issue=18 |pages=4205–4213 |doi=10.1093/nar/26.18.4205 |pmc=147817 |pmid=9722641}}</ref> The TOPRIM fold contains an α/β core with four conserved strands in a [[Rossmann fold|Rossmann-like]] topology. This structure is also found in the catalytic domains of [[topoisomerase]] Ia, topoisomerase II, the OLD-family nucleases and DNA repair proteins related to the RecR protein.{{cn|date=November 2024}}

The primase used by archaea and eukaryotes, in contrast, contains a highly derived version of the [[RNA recognition motif]] (RRM). This primase is structurally similar to many viral RNA-dependent RNA polymerases, reverse transcriptases, cyclic nucleotide generating cyclases and DNA polymerases of the A/B/Y families that are involved in DNA replication and repair.  In eukaryotic replication, the primase forms a complex with Pol α.<ref>{{Cite journal |vauthors=Frick DN, Richardson CC |date=July 2001 |title=DNA primases |journal=Annual Review of Biochemistry |volume=70 |pages=39–80 |doi=10.1146/annurev.biochem.70.1.39 |pmid=11395402 |s2cid=33197061}}</ref>

Multiple DNA polymerases take on different roles in the DNA replication process. In ''[[Escherichia coli|E. coli]]'', [[Pol III|DNA Pol III]] is the polymerase enzyme primarily responsible for DNA replication. It assembles into a replication complex at the replication fork that exhibits extremely high processivity, remaining intact for the entire replication cycle. In contrast, [[Pol I|DNA Pol I]] is the enzyme responsible for replacing RNA primers with DNA. DNA Pol I has a 5′ to 3′ [[exonuclease]] activity in addition to its polymerase activity, and uses its exonuclease activity to degrade the RNA primers ahead of it as it extends the DNA strand behind it, in a process called [[nick translation]]. Pol I is much less processive than Pol III because its primary function in DNA replication is to create many short DNA regions rather than a few very long regions.{{cn|date=November 2024}}

In [[eukaryote]]s, the low-processivity enzyme, Pol α, helps to initiate replication because it forms a complex with primase.<ref>{{Cite journal |vauthors=Barry ER, Bell SD |date=December 2006 |title=DNA replication in the archaea |journal=Microbiology and Molecular Biology Reviews |volume=70 |issue=4 |pages=876–887 |doi=10.1128/MMBR.00029-06 |pmc=1698513 |pmid=17158702}}</ref>  In eukaryotes, leading strand synthesis is thought to be conducted by Pol ε; however, this view has recently been  challenged, suggesting a role for Pol δ.<ref>{{Cite journal |vauthors=Stillman B |date=July 2015 |title=Reconsidering DNA Polymerases at the Replication Fork in Eukaryotes |journal=Molecular Cell |volume=59 |issue=2 |pages=139–141 |doi=10.1016/j.molcel.2015.07.004 |pmc=4636199 |pmid=26186286}}</ref>  Primer removal is completed Pol δ<ref>{{Cite thesis |title=Distinguishing the pathways of primer removal during Eukaryotic Okazaki fragment maturation |date=February 2009 |degree=Ph.D. |publisher=School of Medicine and Dentistry, University of Rochester |vauthors=Rossi ML |hdl=1802/6537}}</ref> while repair of DNA during replication is completed by Pol ε.

As DNA synthesis continues, the original DNA strands continue to unwind on each side of the bubble, forming a [[replication fork]] with two prongs. In bacteria, which have a single origin of replication on their circular chromosome, this process creates a "[[theta structure]]" (resembling the Greek letter theta: θ). In contrast, eukaryotes have longer linear chromosomes and initiate replication at multiple origins within these.<ref>{{Cite journal |vauthors=Huberman JA, Riggs AD |date=March 1968 |title=On the mechanism of DNA replication in mammalian chromosomes |journal=Journal of Molecular Biology |volume=32 |issue=2 |pages=327–341 |doi=10.1016/0022-2836(68)90013-2 |pmid=5689363}}<!--|access-date=7 April 2016--></ref>

=== Replication fork ===
[[File:Replication fork.svg|right|thumb|Scheme of the replication fork.<br />a: template, b: leading strand, c: lagging strand, d: replication fork, e: primer, f: [[Okazaki fragments]]]]
[[File:Eukaryotic DNA replication.svg|thumb|437x437px|Many enzymes are involved in the DNA replication fork.]]
The replication fork is a structure that forms within the long helical DNA during DNA replication. It is produced by enzymes called helicases that break the hydrogen bonds that hold the DNA strands together in a helix. The resulting structure has two branching "prongs", each one made up of a single strand of DNA. These two strands serve as the template for the leading and lagging strands, which will be created as DNA polymerase matches complementary nucleotides to the templates; the templates may be properly referred to as the leading strand template and the lagging strand template.{{cn|date=November 2024}}

'''DNA is read by DNA polymerase in the 3′ to 5′ direction, meaning the new strand is synthesized in the 5' to 3' direction.''' Since the leading and lagging strand templates are oriented in opposite directions at the replication fork, a major issue is how to achieve synthesis of new lagging strand DNA, whose direction of synthesis is opposite to the direction of the growing replication fork.{{cn|date=November 2024}}

==== Leading strand ====
The leading strand is the strand of new DNA which is synthesized in the same direction as the growing replication fork. This sort of DNA replication is continuous.{{cn|date=November 2024}}

==== Lagging strand ====
The lagging strand is the strand of new DNA whose direction of synthesis is opposite to the direction of the growing replication fork. Because of its orientation, replication of the lagging strand is more complicated as compared to that of the leading strand.  As a consequence, the DNA polymerase on this  strand is seen to "lag behind" the other strand.{{cn|date=November 2024}}

The lagging strand is synthesized in short, separated segments. On the lagging strand ''template'', a [[primase]] "reads" the template DNA and initiates synthesis of a short complementary [[RNA]] primer. A DNA polymerase extends the primed segments, forming [[Okazaki fragment]]s. The RNA primers are then removed and replaced with DNA, and the fragments of DNA are joined by [[DNA ligase]].{{cn|date=November 2024}}

==== Dynamics at the replication fork ====
[[File:1axc tricolor.png|thumb|200px|The assembled human DNA clamp, a [[trimer (biochemistry)|trimer]] of the protein [[PCNA]]]]

In all cases the helicase is composed of six polypeptides that wrap around only one strand of the DNA being replicated. The two polymerases are bound to the helicase hexamer. In eukaryotes the helicase wraps around the leading strand, and in prokaryotes it wraps around the lagging strand.<ref name="replisome-in-Science">{{Cite journal |display-authors=6 |vauthors=Gao Y, Cui Y, Fox T, Lin S, Wang H, de Val N, Zhou ZH, Yang W |date=February 2019 |title=Structures and operating principles of the replisome |journal=Science |volume=363 |issue=6429 |page=835 |article-number=eaav7003 |doi=10.1126/science.aav7003 |pmc=6681829 |pmid=30679383}}</ref>

As helicase unwinds DNA at the replication fork, the DNA ahead is forced to rotate. This process results in a build-up of twists in the DNA ahead.<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=DNA Replication Mechanisms: DNA Topoisomerases Prevent DNA Tangling During Replication |chapter-url=https://www.ncbi.nlm.nih.gov/books/NBK26850/}}</ref> This build-up creates a torsional load that would eventually stop the replication fork. Topoisomerases are enzymes that temporarily break the strands of DNA, relieving the tension caused by unwinding the two strands of the DNA helix; topoisomerases (including [[DNA gyrase]]) achieve this by adding negative [[DNA supercoil|supercoils]] to the DNA helix.<ref>{{Cite journal |vauthors=Reece RJ, Maxwell A |date=26 September 2008 |title=DNA gyrase: structure and function |journal=Critical Reviews in Biochemistry and Molecular Biology |volume=26 |issue=3–4 |pages=335–375 |doi=10.3109/10409239109114072 |pmid=1657531}}<!--|access-date=7 April 2016--></ref>

Bare single-stranded DNA tends to fold back on itself forming [[Biomolecular structure#Secondary structure|secondary structures]]; these structures can interfere with the movement of DNA polymerase. To prevent this, [[single-strand binding protein]]s bind to the DNA until a second strand is synthesized, preventing secondary structure formation.<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=DNA Replication Mechanisms: Special Proteins Help to Open Up the DNA Double Helix in Front of the Replication Fork |chapter-url=https://www.ncbi.nlm.nih.gov/books/NBK26850/}}</ref>

Double-stranded DNA is coiled around [[histone]]s that  play an important role in regulating gene expression so the replicated DNA must be coiled around histones at the same places as the original DNA.<ref>{{Cite journal |last1=Koonin |first1=Eugene V. |last2=Krupovic |first2=Mart |last3=Ishino |first3=Sonoko |last4=Ishino |first4=Yoshizumi |date=2020-06-09 |title=The replication machinery of LUCA: common origin of DNA replication and transcription |journal=BMC Biology |volume=18 |issue=1 |page=61 |doi=10.1186/s12915-020-00800-9 |issn=1741-7007 |pmc=7281927 |pmid=32517760 |doi-access=free}}</ref> To ensure this, histone [[Chaperone (protein)|chaperones]] disassemble the [[chromatin]] before it is replicated and replace the histones in the correct place. Some steps in this reassembly are somewhat speculative.<ref>{{Cite journal |vauthors=Ransom M, Dennehey BK, Tyler JK |date=January 2010 |title=Chaperoning histones during DNA replication and repair |journal=Cell |volume=140 |issue=2 |pages=183–195 |doi=10.1016/j.cell.2010.01.004 |pmc=3433953 |pmid=20141833}}<!--|access-date=24 July 2020--></ref>

Clamp proteins act as a sliding clamp on DNA, allowing the DNA polymerase to bind to its template and aid in processivity. The inner face of the clamp enables DNA to be threaded through it. Once the polymerase reaches the end of the template or detects double-stranded DNA, the sliding clamp undergoes a conformational change that releases the DNA polymerase. Clamp-loading proteins are used to initially load the clamp, recognizing the junction between template and RNA primers.<ref name="Alberts" /><sup>:274-5</sup>

=== DNA replication proteins ===
At the replication fork, many replication enzymes assemble on the DNA into a complex molecular machine called the [[replisome]]. The following is a list of major DNA replication enzymes that participate in the replisome:<ref>{{Cite book |title=Introduction to Genetic Analysis |vauthors=Griffiths AJ, Wessler SR, Lewontin RC, Carroll SB |publisher=W. H. Freeman and Company |year=2008 |isbn=978-0-7167-6887-6}}[Chapter 7: DNA: Structure and Replication. pg 283–290]</ref>

{| class="wikitable"
|-
! Enzyme !! Function in DNA replication
|-
| [[DNA helicase]] || Also known as helix destabilizing enzyme. Helicase separates the two strands of DNA at the [[Replication Fork]] behind the topoisomerase.
|-
| [[DNA polymerase]] || The enzyme responsible for catalyzing the addition of nucleotide substrates to DNA in the 5′ to 3′ direction during DNA replication. Also performs proof-reading and error correction.  There exist many different types of DNA Polymerase, each of which perform different functions in different types of cells.
|-
| [[DNA clamp]] || A protein which prevents elongating DNA polymerases from dissociating from the DNA parent strand.
|-
| [[Single-strand DNA-binding protein]] || Bind to ssDNA and prevent the DNA double helix from re-annealing after DNA helicase unwinds it, thus maintaining the strand separation, and facilitating the synthesis of the new strand.
|-
| [[Topoisomerase]] || Relaxes the DNA from its super-coiled nature.
|-
| [[DNA gyrase]] || Relieves strain of unwinding by DNA helicase; this is a specific type of topoisomerase
|-
| [[DNA ligase]]|| Re-anneals the semi-conservative strands and joins [[Okazaki Fragments]] of the lagging strand.
|-
| [[Primase]] || Provides a starting point of RNA (or DNA) for DNA polymerase to begin synthesis of the new DNA strand.
|-
| [[Telomerase]] || Lengthens telomeric DNA by adding repetitive nucleotide sequences to the ends of '''[[Eukaryotic chromosome fine structure|eukaryotic chromosomes]]'''.  This allows germ cells and stem cells to avoid the [[Hayflick limit]] on cell division.<ref>{{Cite web |date=2009-05-11 |title=Will the Hayflick limit keep us from living forever? |url=http://science.howstuffworks.com/life/genetic/hayflick-limit.htm |access-date=January 20, 2015 |website=Howstuffworks |vauthors=Clark J}}</ref>
|}
''[[In vitro]]'' [[single-molecule experiment]]s (using [[optical tweezers]] and [[magnetic tweezers]]) have found synergetic interactions between the replisome enzymes ([[helicase]], [[polymerase]], and [[Single-strand DNA-binding protein]]) and with the DNA replication fork enhancing [[DNA unwinding element|DNA-unwinding]] and DNA-replication.<ref name="Jarillo-2021" /> These results lead to the development of kinetic models accounting for the synergetic interactions and their stability.<ref name="Jarillo-2021" />

=== Replication machinery ===

[[File:E. coli replisome.png|thumb|left|E. coli Replisome. Notably, the DNA on lagging strand forms a loop. The exact structure of replisome is not well understood.]]

'''Replication machineries''' consist of factors involved in DNA replication and appearing on template ssDNAs. Replication machineries include primosotors are replication enzymes; DNA polymerase, DNA helicases, DNA clamps and DNA topoisomerases, and replication proteins; e.g. single-stranded DNA binding proteins (SSB). In the replication machineries these components coordinate. In most of the bacteria, all of the factors involved in DNA replication are located on replication forks and the complexes stay on the forks during DNA replication. Replication machineries are also referred to as replisomes, or DNA replication systems. These terms are generic terms for proteins located on replication forks. In eukaryotic and some bacterial cells the replisomes are not formed.{{cn|date=November 2024}}

In an alternative figure, DNA factories are similar to projectors and DNAs are like as cinematic films passing constantly into the projectors. In the replication factory model, after both DNA helicases for leading strands and lagging strands are loaded on the template DNAs, the helicases run along the DNAs into each other. The helicases remain associated for the remainder of replication process. Peter Meister et al. observed directly replication sites in [[budding yeast]] by monitoring [[green fluorescent protein]] (GFP)-tagged DNA polymerases α. They detected DNA replication of pairs of the tagged loci spaced apart symmetrically from a replication origin and found that the distance between the pairs decreased markedly by time.<ref name="in&out">{{Cite journal |vauthors=Meister P, Taddei A, Gasser SM |date=June 2006 |title=In and out of the replication factory |journal=Cell |volume=125 |issue=7 |pages=1233–5 |doi=10.1016/j.cell.2006.06.014 |pmid=16814710 |s2cid=15397410 |doi-access=free}}</ref> This finding suggests that the mechanism of DNA replication goes with DNA factories. That is, couples of replication factories are loaded on replication origins and the factories associated with each other. Also, template DNAs move into the factories, which bring extrusion of the template ssDNAs and new DNAs. Meister's finding is the first direct evidence of replication factory model. Subsequent research has shown that DNA helicases form dimers in many eukaryotic cells and bacterial replication machineries stay in single intranuclear location during DNA synthesis.<ref name="watson237">{{Cite book |title=Molecular Biology of the Gene |vauthors=Watson JD, Baker TA, Bell SP, Gann A, Levine M, Losick R, Inglis CH |date=2008 |publisher=Pearson/Benjamin Cummings |isbn=978-0-8053-9592-1 |edition=6th |location=San Francisco |page=237}}</ref>

Replication Factories Disentangle Sister Chromatids. The disentanglement is essential for distributing the chromatids into daughter cells after DNA replication. Because sister chromatids after DNA replication hold each other by '''[[Cohesin]]''' rings, there is the only chance for the disentanglement in DNA replication. Fixing of replication machineries as replication factories can improve the success rate of DNA replication. If replication forks move freely in chromosomes, catenation of nuclei is aggravated and impedes mitotic segregation.<ref name="in&out" />

=== Termination ===
Eukaryotes initiate DNA replication at multiple points in the chromosome, so replication forks meet and terminate at many points in the chromosome.  Because eukaryotes have linear chromosomes, DNA replication is unable to reach the very end of the chromosomes.  Due to this problem, DNA is lost in each replication cycle from the end of the chromosome.  [[Telomeres]] are regions of repetitive DNA close to the ends and help prevent loss of genes due to this shortening. Shortening of the telomeres is a normal process in [[somatic cell]]s.  This shortens the telomeres of the daughter DNA chromosome.  As a result, cells can only divide a certain number of times before the DNA loss prevents further division. (This is known as the [[Hayflick limit]].)  Within the [[germ cell]] line, which passes DNA to the next generation, [[telomerase]] extends the repetitive sequences of the telomere region to prevent degradation. Telomerase can become mistakenly active in somatic cells, sometimes leading to [[cancer]] formation. Increased telomerase activity is one of the hallmarks of cancer.<ref>{{Cite journal |last=Shay |first=Jerry W. |date=2016-06-01 |title=Role of Telomeres and Telomerase in Aging and Cancer |journal=Cancer Discovery |language=en |volume=6 |issue=6 |pages=584–593 |doi=10.1158/2159-8290.CD-16-0062 |pmid=27029895 |pmc=4893918 |issn=2159-8274}}</ref>

Termination requires that the progress of the DNA replication fork must stop or be blocked. Termination at a specific locus, when it occurs, involves the interaction between two components: (1) a termination site sequence in the DNA, and (2) a protein which binds to this sequence to physically stop DNA replication. In various bacterial species, this is named the DNA replication terminus site-binding protein, or [[Ter protein]].<ref>{{Cite journal |last1=Neylon |first1=Cameron |last2=Kralicek |first2=Andrew V. |last3=Hill |first3=Thomas M. |last4=Dixon |first4=Nicholas E. |date=September 2005 |title=Replication Termination in Escherichia coli: Structure and Antihelicase Activity of the Tus- Ter Complex |journal=Microbiology and Molecular Biology Reviews |language=en |volume=69 |issue=3 |pages=501–526 |doi=10.1128/MMBR.69.3.501-526.2005 |pmid=16148308 |pmc=1197808 |issn=1092-2172}}</ref>

Because bacteria have circular chromosomes, termination of replication occurs when the two replication forks meet each other on the opposite end of the parental chromosome. ''E. coli'' regulates this process through the use of termination sequences that, when bound by the [[Tus protein]], enable only one direction of replication fork to pass through. As a result, the replication forks are constrained to always meet within the termination region of the chromosome.<ref>{{Cite book |title=Genomes |vauthors=Brown TA |publisher=[[BIOS Scientific Publishers]] |year=2002 |isbn=1-85996-228-9 |chapter=Chapter 13.2.3. Termination of replication |chapter-url=https://www.ncbi.nlm.nih.gov/books/NBK21113/}}</ref>

== Regulation ==
{{Main|Cell division|Cell cycle}}

[[File:Cell Cycle 2.svg|thumb|150px|The cell cycle of eukaryotic cells]]

=== 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/NBK26856/}}</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= Plant Physiology|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}}

=== Bacteria ===
[[File:Hemimethylation.svg|thumb|200px|Dam methylates adenine of GATC sites after replication]]
Most bacteria do not go through a well-defined cell cycle but instead continuously copy their DNA; during rapid growth, this can result in the concurrent occurrence of multiple rounds of replication.<ref>{{Cite journal |vauthors=Tobiason DM, Seifert HS |date=June 2006 |title=The obligate human pathogen, Neisseria gonorrhoeae, is polyploid |journal=PLOS Biology |volume=4 |issue=6 |article-number=e185 |doi=10.1371/journal.pbio.0040185 |pmc=1470461 |pmid=16719561 |doi-access=free}}</ref>  In ''E. coli'', the best-characterized bacteria, DNA replication is regulated through several mechanisms, including: the hemimethylation and sequestering of the origin sequence, the ratio of [[Adenosine triphosphate|adenosine triphosphate (ATP)]] to [[Adenosine diphosphate|adenosine diphosphate (ADP)]], and the levels of protein DnaA. All these control the binding of initiator proteins to the origin sequences.<ref>{{Cite journal |last1=O'Donnell |first1=Michael |last2=Langston |first2=Lance |last3=Stillman |first3=Bruce |date=2013-07-01 |title=Principles and concepts of DNA replication in bacteria, archaea, and eukarya |journal=Cold Spring Harbor Perspectives in Biology |volume=5 |issue=7 |article-number=a010108 |doi=10.1101/cshperspect.a010108 |issn=1943-0264 |pmc=3685895 |pmid=23818497}}</ref>

Because ''E. coli'' [[DNA methylation|methylates]] GATC DNA sequences, DNA synthesis results in hemimethylated sequences. This hemimethylated DNA is recognized by the protein [[SeqA protein domain|SeqA]], which binds and sequesters the origin sequence; in addition, DnaA (required for initiation of replication) binds less well to hemimethylated DNA. As a result, newly replicated origins are prevented from immediately initiating another round of DNA replication.<ref>{{Cite journal |vauthors=Slater S, Wold S, Lu M, Boye E, Skarstad K, Kleckner N |date=September 1995 |title=E. coli SeqA protein binds oriC in two different methyl-modulated reactions appropriate to its roles in DNA replication initiation and origin sequestration |journal=Cell |volume=82 |issue=6 |pages=927–936 |doi=10.1016/0092-8674(95)90272-4 |pmid=7553853 |s2cid=14652024 |doi-access=free}}<!--|access-date=7 April 2016--></ref>

ATP builds up when the cell is in a rich medium, triggering DNA replication once the cell has reached a specific size. ATP competes with ADP to bind to DnaA, and the DnaA-ATP complex is able to initiate replication. A certain number of DnaA proteins are also required for DNA replication — each time the origin is copied, the number of binding sites for DnaA doubles, requiring the synthesis of more DnaA to enable another initiation of replication.{{cn|date=November 2024}}

In fast-growing bacteria, such as ''E. coli'', chromosome replication takes more time than dividing the cell. The bacteria solve this by initiating a new round of replication before the previous one has been terminated.<ref>{{Cite journal |vauthors=Cooper S, Helmstetter CE |date=February 1968 |title=Chromosome replication and the division cycle of Escherichia coli B/r |journal=Journal of Molecular Biology |volume=31 |issue=3 |pages=519–540 |doi=10.1016/0022-2836(68)90425-7 |pmid=4866337}}</ref> The new round of replication will form the chromosome of the cell that is born two generations after the dividing cell. This mechanism creates overlapping replication cycles.

{{See also|FtsZ|Min System|Plasmid|Plasmid copy number|Plasmid partition system}}

== Problems with DNA replication ==
{{Main|DNA replication stress}}
{{Expand section|date=May 2020}}
[[File:Replication fork restarts by homologous recombination following replication stress.png|thumb|left|Replication fork restarts by homologous recombination following replication stress]]
[[File:Epigenetic consequences of nucleosome reassembly defects at stalled replication forks.png|thumb|Epigenetic consequences of nucleosome reassembly defects at stalled replication forks]]

There are many events that contribute to replication stress, including:<ref>{{Cite journal |vauthors=Zeman MK, Cimprich KA |date=January 2014 |title=Causes and consequences of replication stress |journal=Nature Cell Biology |volume=16 |issue=1 |pages=2–9 |doi=10.1038/ncb2897 |pmc=4354890 |pmid=24366029}}</ref>
* Misincorporation of ribonucleotides
* Unusual [[Nucleic acid secondary structure|DNA structures]]
* Conflicts between replication and transcription
* Insufficiency of essential replication factors
* [[Chromosomal fragile site|Common fragile sites]]
* Overexpression or constitutive activation of [[oncogene]]s
* [[Chromatin]] inaccessibility

== Polymerase chain reaction ==
{{Main|Polymerase chain reaction}}
Researchers commonly replicate DNA ''in vitro'' using the [[polymerase chain reaction]] (PCR). PCR uses a pair of [[primer (molecular biology)|primers]] to span a target region in template DNA, and then polymerizes partner strands in each direction from these primers using a [[thermostable DNA polymerase]]. Repeating this process through multiple cycles amplifies the targeted DNA region. At the start of each cycle, the mixture of template and primers is heated, separating the newly synthesized molecule and template. Then, as the mixture cools, both of these become templates for annealing of new primers, and the polymerase extends from these. As a result, the number of copies of the target region doubles each round, [[exponential growth|increasing exponentially]].<ref name="Saiki2">{{Cite journal |display-authors=6 |vauthors=Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA |date=January 1988 |title=Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase |journal=Science |volume=239 |issue=4839 |pages=487–491 |bibcode=1988Sci...239..487S |doi=10.1126/science.239.4839.487 |pmid=2448875}}</ref>

== See also ==
{{Commons category|DNA replication}}
{{wikiversity|DNA#DNA_Replication}}
* [[Autopoiesis]]
* [[Cell (biology)]]
* [[Cell division]]
* [[Chromosome segregation]]
* [[Data storage device]]
* [[Gene]]
* [[Gene expression]]
* [[Epigenetics]]
* [[Genome]]
* [[Hachimoji DNA]]
* [[Life]]
* [[Replication (computing)]]
* [[Self-replication]]

== Notes ==

{{Reflist|group=Note}}

== References ==
{{Reflist|30em}}

{{DNA replication|state=expanded}}
{{Self-replicating organic structures|state=collapsed}}

{{Portal bar|Biology|Astronomy}}

{{Authority control}}

[[Category:DNA replication]]
[[Category:Senescence]]
[[Category:Cellular processes]]
[[Category:Molecular biology]]
[[Category:Copying]]