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Query: EC:6.5.1.2 (DNA ligase)
 2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In eukaryotes, nucleotide excision repair of DNA is a complex process that requires many polypeptides to perform dual incision and remove a segment of about 30 nucleotides containing the damage, followed by repair DNA synthesis to replace the excised segment. Nucleotide excision repair DNA synthesis is dependent on proliferating cell nuclear antigen (PCNA). To study gap-filling DNA synthesis during DNA nucleotide excision repair, UV-damaged DNA was first incubated with PCNA-depleted human cell extracts to create repair incisions. Purified DNA polymerase delta or epsilon, with DNA ligase, was then used to form the repair patch. DNA polymerase delta could perform repair synthesis and was strictly dependent on the presence of both PCNA and replication factor C, but gave rise to a very low proportion of complete, ligated circles. The presence of replication protein A (which is also required for nucleotide excision repair) did not alter this result, while addition of DNase IV increased the fraction of ligated products. DNA polymerase epsilon, on the other hand, could fill the repair patch in the absence of PCNA and replication factor C, and most of the products were ligated circles. Addition of replication protein A changed the situation dramatically, and synthesis by polymerase epsilon became dependent on both PCNA and replication factor C. A combination of DNA polymerase epsilon, PCNA, replication factor C, replication protein A, and DNA ligase I appears to be well-suited to the task of creating nucleotide excision repair patches.
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PMID:Nucleotide excision repair DNA synthesis by DNA polymerase epsilon in the presence of PCNA, RFC, and RPA. 771 Oct 23

The identification and purification of human cell proteins required for the production of form I DNA following DNA replication from the simian virus 40 (SV40) origin is described. Using these proteins, complete SV40 DNA replication was reconstituted with only purified DNA replication factors: SV40 large tumor antigen (TAg), replication protein A (RPA), DNA topoisomerases I and II, DNA polymerase alpha-primase, replication factor C (RFC), the proliferating cell nuclear antigen (PCNA), DNA polymerase delta, maturation factor 1 (MF1), and DNA ligase I. MF1, a 5' to 3' exonuclease and DNA ligase I were both identified as essential components for production of covalently closed circular relaxed (form I) DNA. MF1 is probably the same exonuclease previously shown by others to function during DNA synthesis on artificial DNA templates or in conjunction with DNA polymerase alpha from the SV40 origin. Combined with these previous studies, our results suggest that MF1 functions to remove an RNA primer attached to every Okazaki fragment during lagging strand DNA synthesis. Interestingly, whereas mammalian DNA ligase I functioned in the reconstituted replication system, mammalian DNA ligase III did not substitute and the phage T4 DNA ligase functioned inefficiently, suggesting that DNA ligase I has a specific role as a replicative DNA ligase in eukaryotic cells.
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PMID:Reconstitution of complete SV40 DNA replication with purified replication factors. 814 77

We have cloned a gene encoding a DNA-binding protein by Southwestern screening of a murine cDNA library with a double-stranded oligonucleotide containing the sequence from the bidirectional promoter of the alpha 1 and alpha 2 collagen IV genes. The middle portion of this 1131-amino acid protein has a region homologous to bacterial DNA ligases, and the more carboxyl portion contains several domains homologous to p40, p38, p37, and p36.5 subunits of activator 1 (A1, also called replication factor C), a human replication protein complex. Western blotting revealed that antiserum generated against part of the recombinant protein reacted specifically with the 145-kDa component of the purified human A1 complex, indicating that it is the murine counterpart of the A1 p145. Characterization of the DNA-binding activity of the recombinant fusion protein by gel mobility-shift assay revealed that it had a preference for a run of pyrimidines on one strand. Deletion analysis using recombinant proteins revealed that the DNA ligase-like domain was required for DNA-binding activity. The finding that the region required for the binding of murine A1 p145 to DNA has similarity to a domain found in DNA ligases suggests that this region may be utilized by both proteins in recognizing DNA.
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PMID:Cloning of the large subunit of activator 1 (replication factor C) reveals homology with bacterial DNA ligases. 826 86

Schizosaccharomyces pombe alternative excision repair has been shown genetically and biochemically to be involved in the repair of a wide variety of DNA lesions. AER is initiated by a damage-specific endonuclease (Uve1p) that recognizes UV-induced photoproducts, base mispairs, abasic sites, and platinum G-G diadducts and cleaves the DNA phosphodiester backbone 5' to a lesion. Several models exist that employ various mechanisms for damage removal based on the activities of Rad2p, a nuclease thought to be responsible for damage excision in AER. This study represents the first report of the biochemical reconstitution of the AER pathway. A base mispair-containing substrate is repaired in a reaction requiring S. pombe Uve1p, Rad2p, DNA polymerase delta, replication factor C, proliferating cell nuclear antigen, and T4 DNA ligase. Surprisingly, damage is removed exclusively by the 5' to 3' exonuclease activity of Rad2p and not its "flap endonuclease" activity and is absolutely dependent upon the presence of the 5'-phosphoryl moiety at the Uve1p cleavage site.
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PMID:In vitro reconstitution of the Schizosaccharomyces pombe alternative excision repair pathway. 1070 16

DNA ligase I is responsible for joining Okazaki fragments during DNA replication. An additional proposed role for DNA ligase I is sealing nicks generated during excision repair. Previous studies have shown that there is a physical interaction between DNA ligase I and proliferating cell nuclear antigen (PCNA), another important component of DNA replication and repair. The results shown here indicate that human PCNA enhances the reaction rate of human DNA ligase I up to 5-fold. The stimulation is specific to DNA ligase I because T4 DNA ligase is not affected. Electrophoretic mobility shift assays indicate that PCNA improves the binding of DNA ligase I to the ligation site. Increasing the DNA ligase I concentration leads to a reduction in PCNA stimulation, consistent with PCNA-directed improvement of DNA ligase I binding to its DNA substrate. Two experiments show that PCNA is required to encircle duplex DNA to enhance DNA ligase I activity. Biotin-streptavidin conjugations at the ends of a linear substrate inhibit PCNA stimulation. PCNA cannot enhance ligation on a circular substrate without the addition of replication factor C, which is the protein responsible for loading PCNA onto duplex DNA. These results show that PCNA is responsible for the stable association of DNA ligase I to nicked duplex DNA.
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PMID:DNA ligase I and proliferating cell nuclear antigen form a functional complex. 1133 Dec 87

Human immunodeficiency virus type 1 (HIV-1) DNA integration intermediates consist of viral and host DNA segments separated by a 5-nucleotide gap adjacent to a 5'-AC unpaired dinucleotide. These short-flap (pre-repair) integration intermediates are structurally similar to DNA loci undergoing long-patch base excision repair in mammalian cells. The cellular proteins flap endonuclease 1 (FEN-1), proliferating cell nuclear antigen, replication factor C, DNA ligase I and DNA polymerase delta are required for the repair of this type of DNA lesion. The role of FEN-1 in the base excision repair pathway is to cleave 5'-unpaired flaps in forked structures so that DNA ligase can seal the single-stranded breaks that remain following gap repair. The rate of excision by FEN-1 of 5'-flaps from short- and long-flap oligonucleotide substrates that mimic pre- and post-repair HIV-1 integration intermediates, respectively, and the effect of HIV-1 integrase on these reactions were examined in the present study. Cleavage of 5'-flaps by FEN-1 in pre-repair HIV-1 integration intermediates was relatively inefficient and was further decreased 3-fold by HIV-1 integrase. The rate of removal of 5'-flaps by FEN-1 from post-repair HIV-1 integration intermediates containing relatively long (7-nucleotide) unpaired 5'-tails and short (1-nucleotide) gaps was increased 3-fold relative to that seen with pre-repair substrates and was further stimulated 5- to 10-fold by HIV-1 integrase. Overall, post-repair structures were cleaved 18 times more effectively in the presence of HIV-1 integrase than pre-repair structures. The site of cleavage was 1 or 2 nucleotides 3' of the branch point and was unaffected by HIV-1 integrase. Integrase alone had no detectable activity in removing 5'-flaps from either pre- or post-repair substrates.
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PMID:Stimulation of human flap endonuclease 1 by human immunodeficiency virus type 1 integrase: possible role for flap endonuclease 1 in 5'-end processing of human immunodeficiency virus type 1 integration intermediates. 1206 2

The total cDNA obtained through reverse transcription of F. oxysporum CGMCC 0536 mRNA used as template, a fragment about 1.5kb was amplied with oligo(dT)15 primer and a gene specific primer designed on the base of the sequence of both NH2-terminus and the cDNA sequence encoding D-lactonohydrolase of Fusarium oxysporum reported on the NCBI, then the fragment was cloned to the pMD18-T vector and sequenced. The sequence encoding D-lactonohydrolase of F. moniliforme CGMCC 0536 shows a high homology of 90.06% with that of F. oxysporum indicating that the gene encoding D-lactonohydrolase is highly conservative. Two specific primers were designed according to the sequence result, and a fragment, 1146bp, was amplied using hot start PCR with these two specific primers. Subsequently, the resulting products were digested with EcoR I and Sal I and ligated to the pTrc99a vector digested with the same enzymes using T4 DNA ligase. the recombinant plasmid, pTrc99a-LAC, was transformed into Escherichia coli JM109. The two positive clones were induced with IPTG, and enzymes expressed in Escherichia coli JM109, the enzyme activity was about 37U and 41U respectively. The expression products were analyzed by SDS-polyacrylamide gel electrophoresis indicating that about 40kD protein was obtained.
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PMID:[Cloning and expression of Fusarium moniliforme CGMCC 0536 D-lactonohydrolase gene in Escherichia coli]. 1610 62

DNA ligase I joins Okazaki fragments during DNA replication and completes certain excision repair pathways. The participation of DNA ligase I in these transactions is directed by physical and functional interactions with proliferating cell nuclear antigen, a DNA sliding clamp, and, replication factor C (RFC), the clamp loader. Here we show that DNA ligase I also interacts with the hRad17 subunit of the hRad17-RFC cell cycle checkpoint clamp loader, and with each of the subunits of its DNA sliding clamp, the heterotrimeric hRad9-hRad1-hHus1 complex. In contrast to the inhibitory effect of RFC, hRad17-RFC stimulates joining by DNA ligase I. Similar results were obtained with the homologous Saccharomyces cerevisiae proteins indicating that the interaction between the replicative DNA ligase and checkpoint clamp is conserved in eukaryotes. Notably, we show that hRad17 preferentially interacts with and specifically stimulates dephosphorylated DNA ligase I. Moreover, there is an increased association between DNA ligase I and hRad17 in S phase following DNA damage and replication blockage that occurs concomitantly with DNA damage-induced dephosphorylation of chromatin-associated DNA ligase I. Thus, our results suggest that the in vivo interaction between DNA ligase I and the checkpoint clamp loader is regulated by post-translational modification of DNA ligase I.
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PMID:A conserved physical and functional interaction between the cell cycle checkpoint clamp loader and DNA ligase I of eukaryotes. 1756 5

The sliding clamp PCNA is a crucial component of the DNA replication machinery. Timely PCNA loading and unloading are central for genome integrity and must be strictly coordinated with other DNA processing steps during replication. Here, we show that the S. cerevisiae Elg1 replication factor C-like complex (Elg1-RLC) unloads PCNA genome-wide following Okazaki fragment ligation. In the absence of Elg1, PCNA is retained on chromosomes in the wake of replication forks, rather than at specific sites. Degradation of the Okazaki fragment ligase Cdc9 leads to PCNA accumulation on chromatin, similar to the accumulation caused by lack of Elg1. We demonstrate that Okazaki fragment ligation is the critical prerequisite for PCNA unloading, since Chlorella virus DNA ligase can substitute for Cdc9 in yeast and simultaneously promotes PCNA unloading. Our results suggest that Elg1-RLC acts as a general PCNA unloader and is dependent upon DNA ligation during chromosome replication.
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PMID:Replication-Coupled PCNA Unloading by the Elg1 Complex Occurs Genome-wide and Requires Okazaki Fragment Ligation. 2621 19