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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)

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

To understand the mechanism involved in the coordination of the sequential repair reactions that lead to long-patch BER, we have investigated interactions between proteins involved in this pathway. We find that human AP endonuclease 1 (APE1) physically interacts with flap endonuclease 1 (FEN1) and with proliferating cell nuclear antigen. An oligonucleotide substrate containing a reduced abasic site, which was pre-incised with APE1, was employed to reconstitute the excision step of long-patch BER with purified human DNA polymerase beta and FEN1. We demonstrate that addition of APE1 to the excision reaction mixture slightly (1.5-2-fold) stimulates the removal of the displaced flap by FEN1. These results suggest the possibility that long-patch BER is coordinated and directed by protein-protein interactions.
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PMID:Interaction of human AP endonuclease 1 with flap endonuclease 1 and proliferating cell nuclear antigen involved in long-patch base excision repair. 1160 88

Many types of DNA damage induce a cellular response that inhibits replication but allows repair by up-regulating the p53 pathway and inducing p21(Cip1, Waf1, Sdi1). The p21 regulatory protein can bind proliferating cell nuclear antigen (PCNA) and prohibit DNA replication. We show here that p21 also inhibits PCNA stimulation of long patch base excision repair (BER) in vitro. p21 disrupts PCNA-directed stimulation of flap endonuclease 1 (FEN1), DNA ligase I, and DNA polymerase delta. The dilemma is to understand how p21 prevents DNA replication but allows BER in vivo. Differential regulation by p21 is likely to relate to the utilization of DNA polymerase beta, which is not sensitive to p21, in the repair pathway. We have also found that apurinic/apyrimidinic endonuclease 1 (APE1) stimulates long patch BER. Furthermore, neither APE1 activity nor its ability to stimulate long patch BER is significantly affected by p21 in vitro. We propose that APE1 serves as an assembly and coordination factor for long patch BER proteins. APE1 initially cleaves the DNA and then facilitates the sequential binding and catalysis by DNA polymerase beta, DNA polymerase delta, FEN1, and DNA ligase I. This model implies that BER can be regulated differentially, based upon the assembly of relevant proteins around APE1 in the presence or absence of PCNA.
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PMID:Regulatory roles of p21 and apurinic/apyrimidinic endonuclease 1 in base excision repair. 1164 13

DNA polymerase (pol) delta is essential for both leading and lagging strand DNA synthesis during chromosomal replication in eukaryotes. Pol delta has been implicated in the Okazaki fragment maturation process for the extension of the newly synthesized fragment and for the displacement of the RNA/DNA segment of the preexisting downstream fragment generating an intermediate flap structure that is the target for the Dna2 and flap endonuclease-1 (Fen 1) endonucleases. Using a single-stranded minicircular template with an annealed RNA/DNA primer, we could measure strand displacement by pol delta coupled to DNA synthesis. Our results suggested that pol delta alone can displace up to 72 nucleotides while synthesizing through a double-stranded DNA region in a distributive manner. Proliferating cell nuclear antigen (PCNA) reduced the template dissociation rate of pol delta, thus increasing the processivity of both synthesis and strand displacement, whereas replication protein A (RP-A) limited the size of the displaced fragment down to 20-30 nucleotides, by generating a "locked" flap DNA structure, which was a substrate for processing of the displaced fragment by Fen 1 into a ligatable product. Our data support a model for Okazaki fragment processing where the strand displacement activity of DNA polymerase delta is modulated by the concerted action of PCNA, RP-A and Fen 1.
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PMID: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. 1172 25

RNA primer removal from Okazaki fragments during lagging-strand replication and the excision of damaged DNA bases requires the action of structure-specific nucleases, such as the mammalian flap endonuclease 1 (FEN-1). This nuclease contains two conserved motifs enriched with acidic amino acid residues that are important for catalytic function. Similar motifs have been identified in nucleases found in viruses, archebacteria, eubacteria, and in eukaryotes ranging from yeast to humans. Unique among these proteins, the putative FEN-1 homologue in Escherichia coli is contained within the N-terminal region of the DNA polymerase I (PolN). To demonstrate that the cellular functions of FEN-1 reside in PolN, we cloned and expressed the amino terminal domain (323 amino acid residues) of PolI in a Saccharomyces cerevisiae strain lacking the FEN-1 homologue RAD27. Overexpression of PolN suppressed, to varying degrees, phenotypes associated with a rad27 null strain. These include temperature sensitivity, Okazaki fragment processing, a mutator phenotype, a G2/M cell cycle arrest, minichromosome loss, and methyl methane sulfonate sensitivity. We purified Rad27 and PolN proteins in order to determine whether differences in their intrinsic nuclease activities or interaction with proliferating cell nuclear antigen (PCNA) could explain the partial suppression of some phenotypes. We found that the in vitro nuclease activities of Rad27 were more potent than those of PolN and the activity of Rad27, but not PolN, was stimulated by PCNA. We conclude that the N-terminal nuclease domain of E. coli polymerase I encodes a functional homologue of FEN-1.
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PMID:Suppression of Saccharomyces cerevisiae rad27 null mutant phenotypes by the 5' nuclease domain of Escherichia coli DNA polymerase I. 1222 7

Nicks and flaps are intermediates in various processes of DNA metabolism, including replication and repair. Photoaffinity modification was employed in studying the interaction of the replication protein A (RPA) and flap endonuclease 1 (FEN-1) with DNA duplexes similar to structures arising during long-patch base excision repair. The proteins were also tested for effect on DNA polymerase beta (Pol beta) interaction with DNA. Using Pol beta, a photoreactive dTTP analog was added to the 3' end of an oligonucleotide flanking a nick or a flap in DNA intermediates. The character and intensity of protein labeling depended on the type of intermediates and on the presence of the phosphate or tetrahydrofuran at the 5' end of a nick or a flap. Photoaffinity labeling of Pol beta substantially (up to three times) increased in the presence of RPA or FEN-1. Various DNA substrates were used to study the effects of RPA and FEN-1 on Pol beta-mediated DNA synthesis with displacement of a downstream primer. In contrast to FEN-1, RPA had no effect on DNA repair synthesis by Pol beta during long-patch base excision repair.
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PMID:[Interaction of replication protein A and flap endonuclease 1 with DNA duplexes containing a nick or flap]. 1250 May 43

DNA base excision repair (BER) constitutes a major mechanism to restore the integrity of the genome following modifications of nucleobases. Although it is well established that poly(ADP-ribosylation) facilitates BER, the mechanism of this stimulation has remained unknown. Previous observations suggested that poly(ADP-ribose), which is synthesised from NAD(+), could serve as a unique source of ATP required for the ligation step in BER. This pathway of ATP generation is thought to compensate ATP shortage and relies on the release of pyrophosphate during DNA repair synthesis. Here, we present evidence that, in situations of cellular energy depletion, the synthesis of poly(ADP-ribose) is indeed stimulated. Simultaneously, single nucleotide repair is reduced. Rather, the number of nucleotides incorporated by DNA polymerase beta (Pol beta) during DNA repair synthesis is increased. Using a reconstituted system including the recombinant BER proteins Pol beta, AP endonuclease 1 (APE 1), X-ray repair cross-complementing group-1 (XRCC1), DNA ligase III (Lig III), flap endonuclease 1 (FEN 1), and poly(ADP-ribose) polymerase-1 (PARP-1), it is demonstrated that in the absence of ATP, both long patch DNA synthesis by Pol beta and poly(ADP-ribosylation) catalysed by PARP-1 are stimulated. Consequently, the preferred use of either long patch or single nucleotide BER depends on the availability of ATP. It is proposed that long patch BER is required for ATP generation from poly(ADP-ribose) and, therefore, predominant under conditions of ATP shortage.
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PMID:ATP-dependent selection between single nucleotide and long patch base excision repair. 1367 48

The toroidal damage checkpoint complex Rad9-Rad1-Hus1 (9-1-1) has been characterized as a sensor of DNA damage. Flap endonuclease 1 (FEN1) is a structure-specific nuclease involved both in removing initiator RNA from Okazaki fragments and in DNA repair pathways. FEN1 activity is stimulated by proliferating cell nuclear antigen (PCNA), a toroidal sliding clamp that acts as a platform for DNA replication and repair complexes. We show that 9-1-1 also binds and stimulates FEN1. Stimulation is observed on a variety of flap, nick, and gapped substrates simulating repair intermediates. Blocking 9-1-1 entry to the double strands prevents a portion of the stimulation. Like PCNA stimulation, 9-1-1 stimulation cannot circumvent the tracking mechanism by which FEN1 enters the substrate; however, 9-1-1 does not substitute for PCNA in the stimulation of DNA polymerase beta. This suggests that 9-1-1 is a damage-specific activator of FEN1.
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PMID:The human Rad9-Rad1-Hus1 checkpoint complex stimulates flap endonuclease 1. 1555 96

DNA polymerase beta (pol beta) and flap endonuclease 1 (FEN1) are key players in pol beta-mediated long-patch base excision repair (LP-BER). It was proposed that this type of LP-BER is accomplished through FEN1 removal of a 2- to 11-nucleotide flap created by pol beta strand displacement DNA synthesis. To understand how these enzymes might cooperate during LP-BER, we characterized purified human pol beta DNA synthesis by utilizing various BER intermediates, including single-nucleotide-gapped DNA, nicked DNA, and nicked DNA with various lengths of flaps all with a 5'-terminal tetrahydrofuran (THF) residue. We observed that nicked DNA and nicked-THF flap DNA were poor substrates for pol beta-mediated DNA synthesis; yet, DNA synthesis was strongly stimulated by purified human FEN1. FEN1 did not improve pol beta substrate binding. FEN1 cleavage activity was required for the stimulation, suggesting that FEN1 removed a barrier to pol beta DNA synthesis. In addition, FEN1 cleavage on both nicked and nicked-THF flap DNA resulted in a one-nucleotide gapped DNA molecule that was an ideal substrate for pol beta. This study demonstrates that pol beta cooperates with FEN1 to remove DNA damage via a "Hit and Run" mechanism, involving alternating short gap production by FEN1 and gap filling by pol beta, rather than through coordinated formation and removal of a strand-displaced flap.
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PMID:DNA polymerase beta and flap endonuclease 1 enzymatic specificities sustain DNA synthesis for long patch base excision repair. 1556 6

The human checkpoint sensor and alternative clamp Rad9-Rad1-Hus1 can interact with and specifically stimulate DNA ligase I. The very recently described interactions of Rad9-Rad1-Hus1 with MutY DNA glycosylase, DNA polymerase beta and Flap endonuclease 1 now complete our view that the long-patch base excision machinery is an important target of the Rad9-Rad1-Hus1 complex, thus enhancing the quality control of DNA.
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PMID:The human checkpoint sensor and alternative DNA clamp Rad9-Rad1-Hus1 modulates the activity of DNA ligase I, a component of the long-patch base excision repair machinery. 1587 98


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