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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An important not yet fully understood event in DNA replication is the DNA polymerase (pol) switch from pol alpha to pol delta. Indirect evidence suggested that the clamp loader replication factor C (RF-C) plays an important role, since a replication competent protein complex containing pol alpha, pol delta and RF-C could perform pol switching in the presence of proliferating cell nuclear antigen (PCNA). By using purified pol alpha/primase, pol delta, RF-C, PCNA and RP-A we show that: (i) RF-C can inhibit pol alpha in the presence of ATP prior to PCNA loading, (ii) RF-C decreases the affinity of pol alpha for the 3'OH primer ends, (iii) the inhibition of pol alpha by RF-C is released upon PCNA loading, (iv) ATP hydrolysis is required for PCNA loading and subsequent release of inhibition of pol alpha, (v) under these conditions a switching from pol alpha/primase to pol delta is evident. Thus, RF-C appears to be critical for the pol alpha to pol delta switching. Based on these results, a model is proposed in which RF-C induces the pol switching by sequestering the 3'-OH end from pol alpha and subsequently recruiting PCNA to DNA.
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PMID:DNA polymerase switching: I. Replication factor C displaces DNA polymerase alpha prior to PCNA loading. 1065 91

A crucial event in DNA replication is the polymerase switch from the synthesis of a short RNA/DNA primer by DNA polymerase alpha/primase to the pro?cessive elongation by DNA polymerase delta. In order to shed light on the role of replication factor C (RF-C) in this process, the effects of RF-C on DNA polymerase alpha were investigated. We show that RF-C stalls DNA polymerase alpha after synthesis of approximately 30 nucleotides, while not inhibiting the polymerase activity per se. This suggested that RF-C and the length of the primer may be two important factors contributing to the polymerase switch. Furthermore the DNA binding properties of RF-C were tested. Band shift experiments indicated that RF-C has a preference for 5' recessed ends and double-stranded DNA over 3' ends. Finally PCNA can be loaded onto a DNA template carrying a RNA primer, suggesting that a DNA moiety is not necessarily required for the loading of the clamp. Thus we propose a model where RF-C, upon binding to the RNA/DNA primer, influences primer synthesis and sets the conditions for a polymerase switch after recruiting PCNA to DNA.
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PMID:DNA polymerase switching: II. Replication factor C abrogates primer synthesis by DNA polymerase alpha at a critical length. 1065 92

We examined cDNAs of the catalytic subunit of DNA polymerase alpha (185 kDa), the 70 kDa subunit of replication protein A (single-stranded DNA-binding protein) and the 140 kDa subunit of replication factor C for mutations. Surgical specimens from 12 patients with sporadic colon cancer and normal mucosae from the same patients were investigated. In addition, we analyzed 3 human colon cancer cell lines that exhibited defects in mismatch repair (DLD-1, HCT116, SW48) and 3 colon cancer cell lines without such a defect (HT29, SW480 and SW620). For detection of mutations, we used reverse transcription of mRNA, amplification of cDNAs by PCR, analysis of single-strand conformation polymorphism and DNA sequencing. Eleven colon cancers and 6 colon cancer cell lines were analyzed for DNA polymerase alpha. Only 2 silent point mutations were detected, in 1 colon carcinoma and in cell line HCT116. Two sequence alterations of the 70 kDa subunit of replication factor A were identified in 15 specimens (9 colon carcinomas and 6 cell lines). Colon carcinomas from 2 patients (CC5MA and CC25HN) exhibited an ACA-->GCA transition in codon 351, which caused a Thr-->Ala exchange. In carcinomas CC5MA and CC8MA, a TCC-->TCT (Ser-->Ser) transition in codon 352 was observed. The deviations in codons 351 and 352 occurred in both cancer tissues and normal mucosae, suggesting a genetic polymorphism. No mutation was found in the 140 kDa subunit of replication factor C from 16 specimens (10 tumors and 6 cell lines). Point mutations were identified in the p53 tumor-suppressor gene in 4 of the 6 colon cancer cell lines and 3 of the 8 carcinoma specimens. We did not find tumor-associated DNA sequence alterations that resulted in amino acid changes in the DNA replication genes analyzed. We infer that the scarcity of mutations found is due to stringent selection, eliminating functionally impaired replication proteins.
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PMID:Mutation analysis of replicative genes encoding the large subunits of DNA polymerase alpha and replication factors A and C in human sporadic colorectal cancers. 1076 Aug 17

Here we report the isolation and characterization of a clamp-loader complex from the thermoacidophilic archaeon Sulfolobus solfataricus (SsoRFC). SsoRFC is a hetero-pentamer composed of polypeptides of 37 kDa (small subunit) and 46 kDa (large subunit), which possess primary structure similarity with human replication factor C p40 and p140 subunits, respectively. The two SsoRFC polypeptides were co-expressed in Escherichia coli and purified as a complex (SsoRFC-complex) that was demonstrated to possess a native M(r) of about 200 kDa and a 4:1 (small to large) subunit stoichiometric ratio. The small subunit was individually expressed in E. coli, purified, and found to form a homo-tetramer (SsoRFC-small; native M(r) 156 kDa), which was also characterized. The SsoRFC-complex, but not SsoRFC-small, highly stimulated the synthetic activity of S. solfataricus B1-type DNA polymerase in reactions containing primed M13mp18 DNA, ATP, and either of the two poliferating cell nuclear antigen-like processivity factors of S. solfataricus (039p and 048p). Both SsoRFC-small and -complex were able to hydrolyze ATP, but only the ATPase activity of the holo-enzymatic assembly was activated by primed DNA templates, such as poly(dA)-oligo(dT). As measured by nitrocellulose filter binding assays, SsoRFC-complex bound poly(dA)-oligo(dT), but not the unprimed homopolymer, whereas SsoRFC-small was devoid of any DNA-binding activity. The peculiar properties of this archaeal clamp-loader complex and their significance for the understanding of the DNA replication process in Archaea are discussed.
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PMID:Biochemical characterization of a clamp-loader complex homologous to eukaryotic replication factor C from the hyperthermophilic archaeon Sulfolobus solfataricus. 1092 93

Proliferating cell nuclear antigen is best known as a DNA polymerase accessory protein but has more recently also been shown to have different functions in important cellular processes such as DNA replication, DNA repair, and cell cycle control. PCNA has been found in quaternary complexes with the cyclin kinase inhibitor p21 and several pairs of cyclin-dependent protein kinases and their regulatory partner, the cyclins. Here we show a direct interaction between PCNA and Cdk2. This interaction involves the regions of the PCNA trimer close to the C termini. We found that PCNA and Cdk2 form a complex together with cyclin A. This ternary PCNA-Cdk2-cyclin A complex was able to phosphorylate the PCNA binding region of the large subunit of replication factor C as well as DNA ligase I. Furthermore, PCNA appears to be a connector between Cdk2 and DNA ligase I and to stimulate phosphorylation of DNA ligase I. Based on our results, we propose the model that PCNA brings Cdk2 to proteins involved in DNA replication and possibly might act as an "adaptor" for Cdk2-cyclin A to PCNA-binding DNA replication proteins.
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PMID:A direct interaction between proliferating cell nuclear antigen (PCNA) and Cdk2 targets PCNA-interacting proteins for phosphorylation. 1093 Apr 25

The eukaryotic DNA sliding clamp that keeps DNA polymerase engaged at a replication fork, called proliferating cell nuclear antigen (PCNA), is loaded onto the 3' ends of primer DNA through its interaction with a heteropentameric protein complex called replication factor C (RFC). The ATPase activity of RFC is necessary for formation of a functional PCNA clamp. In the present study, the sensitivity of RFC to partial proteolysis is used to show that addition of ATP, ATPgammaS, or ADP induces different structural changes in RFC. Direct observation by electron microscopy reveals that RFC has a closed two-finger structure called the U form in the absence of ATP. This is converted into a more open C form on addition of ATP. In contrast, the structural changes induced by ATPgammaS or ADP are limited. These results suggest that RFC adapts on opened configuration intermediately after ATP hydrolysis. We further observe that PCNA is held between the two fingers of RFC and propose that the RFC structure change we observe during ATP hydrolysis causes the attached PCNA to form its active ring-like clamp on DNA.
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PMID:ATP-dependent structural change of the eukaryotic clamp-loader protein, replication factor C. 1112 Oct 20

An archaeal clamp loader, replication factor C (RFC), consists of two proteins, the small subunit (RFCS) and large subunit (RFCL), whose sequences are both highly homologous to those of the eukaryotic RFC components. We have investigated the oligomeric structure of RFCS from Pyrococcus furiosus by electron microscopy using single-particle analysis. RFCS forms mostly ring-shaped hexamers at pH 9.0, although it tends to form C-shaped tetramers or pentamers at a lower pH (pH 5.5). The three-dimensional (3D) structure of the RFCS hexamer was obtained by random conical tilt reconstruction at 24.0-A resolution. RFCS forms a hexameric ring with outer and inner diameters of 117 and 27 A, respectively, and with a height of about 55 A. The six subunits are arranged in a twisted manner with a sixfold symmetry around the channel. The 3D map revealed that the six subunits are arranged in a head-to-tail configuration. Although the RFC complex consists of RFCS and RFCL in vivo, RFCS alone, together with PCNA, substantially enhanced the DNA synthesizing activity of P. furiosus DNA polymerase I in vitro. The 3D reconstruction of RFCS with catalytic activity provides important insights into the organization mechanism and the functional state of the RFC complex.
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PMID:Three-dimensional electron microscopy of the clamp loader small subunit from Pyrococcus furiosus. 1146 75

In higher eukaryotes, base excision repair can proceed by two alternative pathways: a DNA polymerase beta-dependent pathway and a proliferating cell nuclear antigen (PCNA)-dependent pathway. Recently, we have reconstituted the PCNA-dependent AP site repair reaction with six purified human proteins: AP endonuclease, replication factor C (RFC), PCNA, flap endonuclease 1 (FEN1), DNA polymerase delta (pol delta), and DNA ligase I. In this reconstituted system, the number of nucleotides replaced during the repair reaction (patch size) was predominantly two nucleotides. PCNA can directly interact with RFC, pol delta, FEN1 and DNA ligase I. These interactions are partly through a consensus motif, QXX(I/L/M)XX(F/H)(F/Y), found in each of the four proteins. PCNA functions as a molecular adaptor for recruiting these factors to the site of DNA repair. Two DNA-N-glycosylases among those so far cloned from human, UNG2 and MYH, are found to have the same PCNA-binding motif. Major substrates of these enzymes, a uracil opposite an adenine for UNG2 and an adenine opposite an 8-oxoguanine for MYH, are formed during DNA replication. Therefore, UNG2 and MYH may serve for replication-coupled base excision repair through the direct interaction with PCNA in the replication machinery.
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PMID:Molecular mechanism of PCNA-dependent base excision repair. 1155 92

The Escherichia coli beta dimer is a ring-shaped protein that encircles DNA and acts as a sliding clamp to tether the replicase, DNA polymerase III holoenzyme, to DNA. The gamma complex (gammadeltadelta'chipsi) clamp loader couples ATP to the opening and closing of beta in assembly of the ring onto DNA. These proteins are functionally and structurally conserved in all cells. The eukaryotic equivalents are the replication factor C (RFC) clamp loader and the proliferating cell nuclear antigen (PCNA) clamp. The delta subunit of the E. coli gamma complex clamp loader is known to bind beta and open it by parting one of the dimer interfaces. This study demonstrates that other subunits of gamma complex also bind beta, although weaker than delta. The gamma subunit like delta, affects the opening of beta, but with a lower efficiency than delta. The delta' subunit regulates both gamma and delta ring opening activities in a fashion that is modulated by ATP interaction with gamma. The implications of these actions for the workings of the E. coli clamp loading machinery and for eukaryotic RFC and PCNA are discussed.
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PMID:Interplay of clamp loader subunits in opening the beta sliding clamp of Escherichia coli DNA polymerase III holoenzyme. 1157 66

DNA replication efficiency is dictated by DNA polymerases (pol) and their associated proteins. The recent discovery of DNA polymerase Y family (DinB/UmuC/RAD30/REV1 superfamily) raises a question of whether the DNA polymerase activities are modified by accessory proteins such as proliferating cell nuclear antigen (PCNA). In fact, the activity of DNA pol IV (DinB) of Escherichia coli is enhanced upon interaction with the beta subunit, the processivity factor of DNA pol III. Here, we report the activity of Sso DNA pol Y1 encoded by the dbh gene of the archaeon Sulfolobus solfataricus is greatly enhanced by the presence of PCNA and replication factor C (RFC). Sso pol Y1 per se was a distributive enzyme but a substantial increase in the processivity was observed on poly(dA)-oligo(dT) in the presence of PCNA (039p or 048p) and RFC. The length of the synthesized DNA product reached at least 200 nucleotides. Sso pol Y1 displayed a higher affinity for DNA compared with pol IV of E. coli, suggesting that the two DNA polymerases have distinct reason(s) to require the processivity factors for efficient DNA synthesis. The abilities of pol Y1 and pol IV to bypass DNA lesions and their sensitive sites to protease are also discussed.
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PMID:Synthetic activity of Sso DNA polymerase Y1, an archaeal DinB-like DNA polymerase, is stimulated by processivity factors proliferating cell nuclear antigen and replication factor C. 1158 Dec 67


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