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.
Brown WC, Campbell JL.; ''Interaction of proliferating cell nuclear antigen with yeast DNA polymerase delta.''; PubMedEurope PMCScholia
Harrington JJ, Lieber MR.; ''DNA structural elements required for FEN-1 binding.''; PubMedEurope PMCScholia
Méndez J, Stillman B.; ''Chromatin association of human origin recognition complex, cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of prereplication complexes in late mitosis.''; PubMedEurope PMCScholia
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
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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
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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
Méndez J, Zou-Yang XH, Kim SY, Hidaka M, Tansey WP, Stillman B.; ''Human origin recognition complex large subunit is degraded by ubiquitin-mediated proteolysis after initiation of DNA replication.''; PubMedEurope PMCScholia
Hindges R, Hübscher U.; ''DNA polymerase delta, an essential enzyme for DNA transactions.''; 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
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The loading of proliferating cell nuclear antigen (PCNA) leads to recruitment of pol delta. 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.
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.
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.
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 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.
Polymerase switching is a key event that allows the processive synthesis of DNA by the pol delta and PCNA complex. Polymerase delta possesses polymerization and proofreading activities, which increases the overall fidelity of DNA replication. The pol delta holoenzyme is a heterotetrameric complex that contains p125, p66, p50, and p12 subunits, in human cells.
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.
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 'ubiquitin', and 1 molecule of 'phosphorylated Orc1' are present. At the end of this reaction, 1 molecule of 'ubiquitinated Orc1' is present.
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)'.
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.
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.
The binding of the primer recognition complex involves the loading of proliferating cell nuclear antigen (PCNA). Replication Factor C 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".
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.
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.
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'.
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.
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.
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 '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'.
Original Pathway at Reactome: http://www.reactome.org/PathwayBrowser/#DB=gk_current&FOCUS_SPECIES_ID=48887&FOCUS_PATHWAY_ID=69239
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DataNodes
Cdk2
substrate complexprimase DNA polymerase alpha
origin complexOkazaki fragment Flap RPA heterotrimer
dna2Okazaki fragment Flap
RPA heterotrimerOkazaki fragment
FlapOkazaki fragments
Remaining FlapRNA primer-DNA primer origin duplex
PCNA homotrimerRNA primer-DNA primer
origin duplexorigin duplex DNA polymerase alpha
primase complexorigin duplex
PCNAAnnotated Interactions
Cdk2
substrate complexprimase DNA polymerase alpha
origin complexOkazaki fragment Flap
RPA heterotrimerOkazaki fragment
FlapOkazaki fragments
Remaining FlapThis reaction takes place in the 'cytosol' and is mediated by the 'endopeptidase activity' of '26S proteasome'.
This reaction takes place in the 'nucleus' and is mediated by the 'kinase activity' of 'CDK'.
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'.
This reaction takes place in the 'cytosol' and is mediated by the 'endopeptidase activity' of 'anaphase-promoting complex (APC)'.
This movement of the molecule occurs through the 'nuclear pore'.
This reaction takes place in the 'nucleus' and is mediated by the 'kinase activity' of 'Cyclin A:Cdk2 complex'.
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 '26S proteasome'.
This reaction takes place in the 'nucleus'.
This reaction takes place in the 'nucleus' and is mediated by the 'DNA-directed DNA polymerase activity' of 'DNA polymerase alpha:primase'.
RNA primer-DNA primer origin duplex
PCNA homotrimerRNA primer-DNA primer
origin duplexorigin duplex DNA polymerase alpha
primase complexorigin duplex DNA polymerase alpha
primase complexorigin duplex
PCNAorigin duplex
PCNAorigin duplex
PCNA