The actual synthesis of DNA occurs in the S phase of the cell cycle. This includes the initiation of DNA replication, when the first nucleotide of the new strand is laid down during the synthesis of the primer. The DNA replication preinitiation events begin in late M or early G1 phase.
View original pathway at Reactome.
Maga G, Villani G, Tillement V, Stucki M, Locatelli GA, Frouin I, Spadari S, Hübscher U.; ''Okazaki fragment processing: modulation of the strand displacement activity of DNA polymerase delta by the concerted action of replication protein A, proliferating cell nuclear antigen, and flap endonuclease-1.''; PubMedEurope PMCScholia
Podust VN, Podust LM, Müller F, Hübscher U.; ''DNA polymerase delta holoenzyme: action on single-stranded DNA and on double-stranded DNA in the presence of replicative DNA helicases.''; PubMedEurope PMCScholia
Chang LM, Rafter E, Augl C, Bollum FJ.; ''Purification of a DNA polymerase-DNA primase complex from calf thymus glands.''; PubMedEurope PMCScholia
Zheng N, Schulman BA, Song L, Miller JJ, Jeffrey PD, Wang P, Chu C, Koepp DM, Elledge SJ, Pagano M, Conaway RC, Conaway JW, Harper JW, Pavletich NP.; ''Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex.''; PubMedEurope PMCScholia
Mossi R, Hübscher U.; ''Clamping down on clamps and clamp loaders--the eukaryotic replication factor C.''; PubMedEurope PMCScholia
Tsurimoto T, Stillman B.; ''Replication factors required for SV40 DNA replication in vitro. II. Switching of DNA polymerase alpha and delta during initiation of leading and lagging strand synthesis.''; PubMedEurope PMCScholia
Pacek M, Tutter AV, Kubota Y, Takisawa H, Walter JC.; ''Localization of MCM2-7, Cdc45, and GINS to the site of DNA unwinding during eukaryotic DNA replication.''; PubMedEurope PMCScholia
Harrington JJ, Lieber MR.; ''The characterization of a mammalian DNA structure-specific endonuclease.''; PubMedEurope PMCScholia
Schaarschmidt D, Ladenburger EM, Keller C, Knippers R.; ''Human Mcm proteins at a replication origin during the G1 to S phase transition.''; PubMedEurope PMCScholia
Bae SH, Bae KH, Kim JA, Seo YS.; ''RPA governs endonuclease switching during processing of Okazaki fragments in eukaryotes.''; PubMedEurope PMCScholia
Jiang W, Wells NJ, Hunter T.; ''Multistep regulation of DNA replication by Cdk phosphorylation of HsCdc6.''; PubMedEurope PMCScholia
Wang TS, Hu SZ, Korn D.; ''DNA primase from KB cells. Characterization of a primase activity tightly associated with immunoaffinity-purified DNA polymerase-alpha.''; PubMedEurope PMCScholia
Podust VN, Tiwari N, Stephan S, Fanning E.; ''Replication factor C disengages from proliferating cell nuclear antigen (PCNA) upon sliding clamp formation, and PCNA itself tethers DNA polymerase delta to DNA.''; PubMedEurope PMCScholia
Maga G, Stucki M, Spadari S, Hübscher U.; ''DNA polymerase switching: I. Replication factor C displaces DNA polymerase alpha prior to PCNA loading.''; PubMedEurope PMCScholia
Nethanel T, Zlotkin T, Kaufmann G.; ''Assembly of simian virus 40 Okazaki pieces from DNA primers is reversibly arrested by ATP depletion.''; PubMedEurope PMCScholia
Li Y, Asahara H, Patel VS, Zhou S, Linn S.; ''Purification, cDNA cloning, and gene mapping of the small subunit of human DNA polymerase epsilon.''; PubMedEurope PMCScholia
Mossi R, Keller RC, Ferrari E, Hübscher U.; ''DNA polymerase switching: II. Replication factor C abrogates primer synthesis by DNA polymerase alpha at a critical length.''; PubMedEurope PMCScholia
Zhang Y, Baranovskiy AG, Tahirov TH, Pavlov YI.; ''The C-terminal domain of the DNA polymerase catalytic subunit regulates the primase and polymerase activities of the human DNA polymerase α-primase complex.''; PubMedEurope PMCScholia
Burgers PM.; ''Saccharomyces cerevisiae replication factor C. II. Formation and activity of complexes with the proliferating cell nuclear antigen and with DNA polymerases delta and epsilon.''; PubMedEurope PMCScholia
Voges D, Zwickl P, Baumeister W.; ''The 26S proteasome: a molecular machine designed for controlled proteolysis.''; PubMedEurope PMCScholia
Lee SH, Hurwitz J.; ''Mechanism of elongation of primed DNA by DNA polymerase delta, proliferating cell nuclear antigen, and activator 1.''; PubMedEurope PMCScholia
Bambara RA, Murante RS, Henricksen LA.; ''Enzymes and reactions at the eukaryotic DNA replication fork.''; PubMedEurope PMCScholia
Lee MY, Tan CK, So AG, Downey KM.; ''Purification of deoxyribonucleic acid polymerase delta from calf thymus: partial characterization of physical properties.''; PubMedEurope PMCScholia
Sato M, Gotow T, You Z, Komamura-Kohno Y, Uchiyama Y, Yabuta N, Nojima H, Ishimi Y.; ''Electron microscopic observation and single-stranded DNA binding activity of the Mcm4,6,7 complex.''; PubMedEurope PMCScholia
Tsurimoto T, Melendy T, Stillman B.; ''Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin.''; PubMedEurope PMCScholia
Petersen BO, Wagener C, Marinoni F, Kramer ER, Melixetian M, Lazzerini Denchi E, Gieffers C, Matteucci C, Peters JM, Helin K.; ''Cell cycle- and cell growth-regulated proteolysis of mammalian CDC6 is dependent on APC-CDH1.''; PubMedEurope PMCScholia
Kamada K, Kubota Y, Arata T, Shindo Y, Hanaoka F.; ''Structure of the human GINS complex and its assembly and functional interface in replication initiation.''; PubMedEurope PMCScholia
Zhang SJ, Zeng XR, Zhang P, Toomey NL, Chuang RY, Chang LS, Lee MY.; ''A conserved region in the amino terminus of DNA polymerase delta is involved in proliferating cell nuclear antigen binding.''; PubMedEurope PMCScholia
Wei SJ, Williams JG, Dang H, Darden TA, Betz BL, Humble MM, Chang FM, Trempus CS, Johnson K, Cannon RE, Tennant RW.; ''Identification of a specific motif of the DSS1 protein required for proteasome interaction and p53 protein degradation.''; PubMedEurope PMCScholia
Li CJ, DePamphilis ML.; ''Mammalian Orc1 protein is selectively released from chromatin and ubiquitinated during the S-to-M transition in the cell division cycle.''; PubMedEurope PMCScholia
Liu L, Mo J, Rodriguez-Belmonte EM, Lee MY.; ''Identification of a fourth subunit of mammalian DNA polymerase delta.''; PubMedEurope PMCScholia
Plafker SM, Plafker KS, Weissman AM, Macara IG.; ''Ubiquitin charging of human class III ubiquitin-conjugating enzymes triggers their nuclear import.''; PubMedEurope PMCScholia
Li Y, Pursell ZF, Linn S.; ''Identification and cloning of two histone fold motif-containing subunits of HeLa DNA polymerase epsilon.''; PubMedEurope PMCScholia
Waga S, Bauer G, Stillman B.; ''Reconstitution of complete SV40 DNA replication with purified replication factors.''; PubMedEurope PMCScholia
The MCM2-7 related protein, MCM8, is required to replicate chromosomal DNA in Xenopus egg extracts. MCM8 binds chromatin upon initiation of DNA synthesis. It may function as an helicase in the elongation step.
In budding yeast, all MCM proteins have been proved to be essential for elongation. The active form of this protein complex may be a heterohexamer. A subcomplex of MCM proteins consisting fo MCM4,6, and -7 has a weak helicase activity that may contribute to DNA unwinding.
By applying the chromatin immunoprecipitation technique to paused forks, certain proteins like DNA pol alpha, DNA pol delta, DNA pol epsilon, MCM2-7, CDC45, GINS and MCM10 were identified. By uncoupling a helicase at the site using a polymerase inhibitor, MCM2-7, GINS complex and CDC45 alone were found to be enriched at the paused fork suggesting these proteins may form a part of an "unwindosome" at the replicating fork.
At the beginning of this reaction, 1 molecule of 'PSF3p', 1 molecule of 'SLD5P', 1 molecule of 'PSF2p', and 1 molecule of 'PSF1p' are present. At the end of this reaction, 1 molecule of 'GINS complex' is present.
At the beginning of this reaction, 1 molecule of 'DNA polymerase alpha:primase:DNA polymerase alpha:origin complex', and 1 molecule of 'NTP' are present. At the end of this reaction, 1 molecule of 'DNA polymerase epsilon', and 1 molecule of 'RNA primer:origin duplex:DNA polymerase alpha:primase complex' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'DNA-directed RNA polymerase activity' of 'DNA polymerase alpha:primase'.
At the beginning of this reaction, 1 molecule of 'ATP', and 1 molecule of 'pre-replicative complex' are present. At the end of this reaction, 1 molecule of 'phosphorylated Orc1', 1 molecule of 'pre-replicative complex (Orc1-minus)', and 1 molecule of 'ADP' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'kinase activity' of 'Cyclin A:Cdk2 complex'.
ORC1 is ubiquitinated by the SKP2-containing ubiquitin ligase complex and targeted for proteasome-mediated degradation, which may play an important role in the maintenace of ploidy. While the ORC1 region phosphorylated by the CCNA:CDK1 complex is involved in the interaction with SKP2, phosphorylation may not be a pre-requisite for ORC1 ubiquitination (Mendez et al. 2002).
At the beginning of this reaction, 1 molecule of 'dTTP', 1 molecule of 'dGTP', 1 molecule of 'dATP', 1 molecule of 'RNA primer:origin duplex:DNA polymerase alpha:primase complex', and 1 molecule of 'dCTP' are present. At the end of this reaction, 1 molecule of 'RNA primer-DNA primer:origin duplex' is present.
This reaction takes place in the 'nucleus' and is mediated by the 'DNA-directed DNA polymerase activity' of 'DNA polymerase alpha:primase'.
At the beginning of this reaction, 1 molecule of 'CDC6', and 1 molecule of 'ATP' are present. At the end of this reaction, 1 molecule of 'ADP', and 1 molecule of 'phosphorylated Cdc6' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'kinase activity' of 'CDK'.
At the beginning of this reaction, 1 molecule of 'phosphorylated Cdc6', 1 molecule of 'ubiquitin', and 1 molecule of 'ATP' are present. At the end of this reaction, 1 molecule of 'ubiquitinated Cdc6' is present.
This reaction takes place in the 'cytosol' and is mediated by the 'endopeptidase activity' of 'anaphase-promoting complex (APC)'.
At the start of the elongation phase of DNA replication, the Mcm2-7 complex may re-arrange to function as the replicative helicase associated with the replication fork. In general, a replicative helicase is associated with the replication fork and unwinds DNA ahead of the polymerase. In yeast, the Mcm proteins associate with origin DNA in G1 phase and then exit the origin upon replication initiation, consistent with moving out of the origin with the replication fork. The Mcm2-7 complex is a ring-shaped hexamer. Complexes of Mcm4, Mcm6 and Mcm7 proteins from humans or S. pombe display a modest ATP-dependent helicase activity in vitro. Consistent with the hypothesis that eukaryotic Mcm complexes function as helicases, an archaeal Mcm homolog is a ring-shaped double hexamer that has a processive DNA unwinding activity. Mcm proteins may have additional functions during elongation, as uninterrupted function of all six is required for replication fork progression in budding yeast. Mcm4,6,7 helicase activity may be negatively regulated in two ways. Mcm2, Mcm4, Mcm6, and Mcm7 also form a stable complex which, however, has no helicase activity, suggesting that Mcm2 inhibits DNA unwinding by Mcm4,6,7. In addition, phosphorylation of human Mcm4,6,7 complex by CDK inhibits its helicase activity.
Once the RNA-DNA primer is synthesized, replication factor C (RFC) initiates a reaction called "polymerase switching"; pol delta, the processive enzyme, replaces pol alpha, the priming enzyme. RFC binds to the 3'-end of the RNA-DNA primer on the Primosome, to displace the pol alpha primase complex. The binding of RFC triggers the binding of the primer recognition complex (Tsurimoto and Stillman 1991, Maga et al. 2000, Mossi et al. 2000). RFC is recruited to telomeres via interaction with 5'-phosphate ends of a telomere repeat sequence (Uchiumi et al. 1996, Uchiumi et al. 1999). In budding yeast, the alternative evolutionarily conserved RFC complex in which the RFC1 subunit is substituted with the CTF18 complex (composed of CHTF18, CHTF8 and DSCC1) plays a critical role in telomere maintenance (Hiraga et al. 2006, Gao et al. 2014). The CTF18-RFC complex is also implicated in telomere maintenance in fission yeast (Khair et al. 2010). It was shown that the human CTF18-RFC complex has a redundant function with the RFC pentamer in PCNA loading and DNA replication (Bermudez et al. 2003), but its role in human telomere maintenance has not been studied. Mouse CFT18 complex is necessary for proper development of germ cells (Berkowitz et al. 2012).
The binding of the primer recognition complex involves the loading of the proliferating cell nuclear antigen (PCNA). Replication Factor C (RFC) transiently opens the PCNA toroid in an ATP-dependent reaction, and then allows PCNA to re-close around the double helix adjacent to the primer terminus. This leads to the formation of the "sliding clamp" (Tsurimoto et al. 1990, Mossi and Hubscher 1998). In a human telomere replication model, RFC-mediated PCNA loading increases the processivity of telomeric C-strand synthesis, but does not eliminate polymerase delta stalling on the G-rich template (Lormand et al. 2013). Interaction of RTEL1 with PCNA is needed for telomere replication and maintenance of telomere integrity (Vannier et al. 2013).
It is assumed that, as shown for generic DNA replication (Podust et al. 1998), the RFC complex dissociates from PCNA following sliding clamp formation at the telomere, and the DNA toroid alone tethers pol delta to the DNA.
The loading of proliferating cell nuclear antigen (PCNA) leads to recruitment of pol delta, the process of polymerase switching. Human PCNA is a homotrimer of 36 kDa subunits that form a toroidal structure. The loading of PCNA by RFC is a key event in the transition from the priming mode to the extension mode of DNA synthesis. The processive complex is composed of the pol delta holoenzyme and PCNA (Murakami et al.2010). Both PCNA and the DNA polymerase delta are needed for telomeric C-strand synthesis in a human telomere replication model (Lormand et al. 2013).
After RFC initiates the assembly of the primer recognition complex, the complex of pol delta and PCNA is responsible for incorporating the additional nucleotides prior to the position of the next downstream initiator RNA primer. On the lagging strand, short discontinuous segments of DNA, called Okazaki fragments, are synthesized on RNA primers. The average length of the Okazaki fragments is 100 nucleotides. Polymerase switching is a key event that allows the processive synthesis of DNA by the pol delta and PCNA complex (Lee and Hurwitz 1990, Tsurimoto and Stillman 1991, Nethanel et al. 1992, Brown and Campbell 1993, Waga et al.1994, Bambara et al. 1997).
When the polymerase delta:PCNA complex reaches a downstream Okazaki fragment, strand displacement synthesis occurs. The primer containing 5'-terminus of the downstream Okazaki fragment is folded into a single-stranded flap.
The first step in the removal of the flap intermediate is the binding of Replication Protein A (RPA) to the long flap structure. RPA is a eukaryotic single-stranded DNA binding protein.
After RPA binds the long flap, it recruits the Dna2 endonuclease. Dna2 endonuclease removes most of the flap, but the job of complete removal of the flap is then completed by FEN-1.
The Dna2 endonuclease removes the initiator RNA along with several downstream deoxyribonucleotides. The cleavage of the single-stranded RNA substrate results in the disassembly of RPA and Dna2. The current data for the role of the Dna2 endonuclease has been derived from studies with yeast and Xenopus Dna2.
The remaining flap, which is too short to support RPA binding, is then processed by FEN-1. There is evidence that binding of RPA to the displaced end of the RNA-containing Okazaki fragment prevents FEN-1 from accessing the substrate. FEN-1 is a structure-specific endonuclease that cleaves near the base of the flap at a position one nucleotide into the annealed region. Biochemical studies have shown that the preferred substrate for FEN-1 consists of a one-nucleotide 3'-tail on the upstream primer in addition to the 5'-flap of the downstream primer.
Removal of the flap by FEN-1 leads to the generation of a nick between the 3'-end of the upstream Okazaki fragment and the 5'-end of the downstream Okazaki fragment. DNA ligase I then seals the nicks between adjacent processed Okazaki fragments to generate intact double-stranded DNA.
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DataNodes
alpha:primase:DNA polymerase alpha:origin
complexcomplex:Okazaki
fragment complexcomplex:Okazaki fragment:Flap:RPA
heterotrimer:dna2complex:Okazaki fragment:Flap:RPA
heterotrimercomplex:Okazaki
fragment:Flapcomplex:Okazaki fragments:Remaining
Flapcomplex:nicked DNA from adjacent
Okazaki fragmentsHeteropentamer:RNA primer-DNA primer:origin duplex:PCNA
homotrimerHeteropentamer:RNA primer-DNA primer:origin
duplexprimer:origin
duplex:PCNAprimer:origin
duplexduplex:DNA polymerase alpha:primase
complexcomplex
(Orc1-minus)Annotated Interactions
alpha:primase:DNA polymerase alpha:origin
complexalpha:primase:DNA polymerase alpha:origin
complexcomplex:Okazaki
fragment complexcomplex:Okazaki
fragment complexcomplex:Okazaki fragment:Flap:RPA
heterotrimer:dna2complex:Okazaki fragment:Flap:RPA
heterotrimer:dna2complex:Okazaki fragment:Flap:RPA
heterotrimercomplex:Okazaki fragment:Flap:RPA
heterotrimercomplex:Okazaki
fragment:Flapcomplex:Okazaki
fragment:Flapcomplex:Okazaki fragments:Remaining
Flapcomplex:Okazaki fragments:Remaining
Flapcomplex:nicked DNA from adjacent
Okazaki fragmentsThis reaction takes place in the 'nucleus'.
This reaction takes place in the 'nucleus' and is mediated by the 'DNA-directed RNA polymerase activity' of 'DNA polymerase alpha:primase'.
This reaction takes place in the 'nucleus' and is mediated by the 'kinase activity' of 'Cyclin A:Cdk2 complex'.
This reaction takes place in the 'nucleus' and is mediated by the 'DNA-directed DNA polymerase activity' of 'DNA polymerase alpha:primase'.
This reaction takes place in the 'nucleus' and is mediated by the 'kinase activity' of 'CDK'.
This movement of the molecule occurs through the 'nuclear pore'.
This reaction takes place in the 'cytosol' and is mediated by the 'endopeptidase activity' of 'anaphase-promoting complex (APC)'.
This reaction takes place in the 'cytosol' and is mediated by the 'endopeptidase activity' of '26S proteasome'.
Interaction of RTEL1 with PCNA is needed for telomere replication and maintenance of telomere integrity (Vannier et al. 2013).
Heteropentamer:RNA primer-DNA primer:origin duplex:PCNA
homotrimerHeteropentamer:RNA primer-DNA primer:origin duplex:PCNA
homotrimerHeteropentamer:RNA primer-DNA primer:origin
duplexHeteropentamer:RNA primer-DNA primer:origin
duplexprimer:origin
duplex:PCNAprimer:origin
duplex:PCNAprimer:origin
duplexprimer:origin
duplexduplex:DNA polymerase alpha:primase
complexduplex:DNA polymerase alpha:primase
complexcomplex
(Orc1-minus)