Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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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)

The influence of poly(ADP-ribose) polymerase (PARP) on the replication of DNA containing the SV40 origin of replication has been examined. Extensive replication of SV40 DNA can be carried out in the presence of T antigen, topoisomerase I, the multimeric human single strand DNA-binding protein (HSSB), and DNA polymerase alpha-DNA primase (pol alpha-primase) complex (the monopolymerase system). In the monopolymerase system, both small products (Okazaki fragments), arising from lagging strand synthesis, and long products, arising from leading strand synthesis, are formed. The synthesis of long products requires the presence of relatively high levels of pol alpha-primase complex. In the presence of PARP, the synthesis of long products was blocked and only small Okazaki fragments accumulated, arising from the replication of the lagging strand template. The inhibition of leading strand synthesis by PARP can be effectively reversed by supplementing the monopolymerase system with the multimeric activator 1 protein (A1), the proliferating cell nuclear antigen (PCNA) and PCNA-dependent DNA polymerase delta (the dipolymerase system). The inhibition of leading strand synthesis in the monopolymerase system was caused by the binding of PARP to the ends of DNA chains, which blocked their further extension by pol alpha. The selective accumulation of Okazaki fragments was shown to be due to the coupled synthesis of primers by DNA primase and their immediate extension by pol alpha complexed to primase. PARP had little effect on this coupled reaction, but did inhibit the subsequent elongation of products, presumably after pol alpha dissociated from the 3'-end of the DNA fragments. PARP inhibited several other enzymatic reactions which required free ends of DNA chains. PARP inhibited exonuclease III, DNA ligase, the 5' to 3' exonuclease, and the elongation of primed DNA templates by pol alpha. In contrast, PARP only partly competed with the elongation of primed DNA templates by the pol delta elongation system which required SSB, A1, and PCNA. These results suggest that the binding of PARP at the ends of nascent DNA chains can be displaced by the binding of A1 and PCNA to primer ends. HSSB can be poly(ADP-ribosylated) in vivo as well as in vitro. However, the selective effect of PARP in blocking leading strand synthesis in the monopolymerase system was shown to depend primarily on its DNA binding property rather than on its ability to synthesize poly(ADP-ribose).
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PMID:Influence of poly(ADP-ribose) polymerase on the enzymatic synthesis of SV40 DNA. 167 70

To determine the contribution that DNA polymerase alpha makes to the overall DNA replication fidelity in mammalian systems, we measured the fidelity of replication of the SV40-based shuttle vector, pZ189, in a reconstituted in vitro DNA replication system which contained purified HeLa DNA polymerase alpha (in addition to single-stranded DNA binding protein, topoisomerase II, DNA ligase, 5'----3' exonuclease, ribonuclease H, and SV40 T-antigen). We found that DNA polymerase alpha is highly accurate when carrying out bidirectional replication in this system. This high fidelity of replication by DNA polymerase alpha in the reconstituted replication system contrasts with a relatively low fidelity of gap-filling DNA synthesis on the same target gene by purified HeLa cell DNA polymerase alpha in the absence of other replication factors. The fidelity of DNA replication by DNA polymerase alpha, although relatively high in the reconstituted system, is about 4-fold lower than DNA replication in a crude HeLa cell extract which contains additional replication factors including DNA polymerase delta. These results demonstrate that DNA polymerase alpha has the capacity to replicate DNA with high fidelity when carrying out semiconservative DNA replication in a minimal reconstituted replication system, but additional cellular factors not present in the reconstituted system may contribute to the higher replication fidelity of the crude system.
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PMID:DNA polymerase alpha from HeLa cells synthesizes DNA with high fidelity in a reconstituted replication system. 221 24

A complex network of interacting proteins and enzymes is required for DNA replication. Much of our present understanding is derived from studies of the bacterium Escherichia coli and its bacteriophages T4 and T7. These results served as a guideline for the search and the purification of analogous proteins in eukaryotes. model systems for replication, such as the simian virus 40 DNA, lead the way. Generally, DNA replication follows a multistep enzymatic pathway. Separation of the double-helical DNA is performed by DNA helicases. Synthesis of the two daughter strands is conducted by two different DNA polymerases: the leading strand is replicated continuously by DNA polymerase delta and the lagging strand discontinuously in small pieces by DNA polymerase alpha. The latter is complexed to DNA primase, an enzyme in charge of frequent RNA primer syntheses on the lagging strand. Both DNA polymerases require several auxiliary proteins. They appear to make the DNA polymerases processive and to coordinate their functional tasks at the replication fork. 3'----5'-exonuclease, mostly part of the DNA polymerase delta polypeptide, can perform proof-reading by excising incorrectly base-paired nucleotides. The short DNA pieces of the lagging strand, called Okazaki fragments, are processed to a long DNA chain by the combined action of RNase H and 5'----3'-exonuclease, removing the RNA primers, DNA polymerase alpha or beta, filling the gap, and DNA ligase, sealing DNA pieces by phosphodiester bond formation. Torsional stress during DNA replication is released by DNA topoisomerases. In contrast to prokaryotes, DNA replication in eukaryotes not only has to create two identical daughter strands but also must conserve higher-order structures like chromatin.
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PMID:Eukaryotic DNA replication. Enzymes and proteins acting at the fork. 226 94

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

Previous investigations have revealed that the human TE-671 MR human rhabdomyosarcoma xenograft selected in vivo for melphalan resistance (M. C. Rosenberg, et al., Cancer Res., 49: 6917-6922, 1989) is cross-resistant to a wide variety of alkylating agents and to bleomycin, but is collaterally sensitive to etoposide. Although glutathione levels were noted to be elevated in TE-671 MR compared to the melphalan-sensitive parental TE-671 xenograft, treatment with buthionine sulfoximine to deplete glutathione levels did not fully restore melphalan sensitivity in the TE-671 MR xenograft. The present studies were undertaken to search for additional mechanisms of resistance in the TE-671 MR xenograft. Drug sensitivity testing performed at the dose of agents that was lethal to 10% of the animals revealed that the TE-671 MR xenograft maintained resistance to the bifunctional cross-linking agent 1,3-bis(2-chloroethyl)-1-nitrosourea and was cross-resistant to the topoisomerase I poison topotecan. Treatment with buthionine sulfoximine did not sensitize the TE-671 MR xenograft to 1,3-bis(2-chloroethyl)-1-nitrosourea. Further, even though O6-alkylguanine-DNA alkyltransferase levels were high in both the TE-671 and TE-671 MR xenografts, depletion of O6-alkylguanine-DNA alkyltransferase activity by treatment with O6-benzylguanine substantially sensitized the TE-671 xenografts but not the TE-671 MR xenografts, suggesting an additional mechanism of resistance. Measurement of additional enzyme activities that might be involved in DNA repair revealed significant elevations in DNA polymerase alpha (46 +/- 8 (SD) units/mg protein in TE-671, 69 +/- 6 units/mg protein in TE-671 MR, P < 0.05) and DNA polymerase beta (0.43 +/- 0.01 units/mg protein in TE-671, 0.78 +/- 0.12 units/mg protein in TE-671 MR, P < 0.05) but not DNA polymerase delta or total DNA ligase. Examination of topoisomerases by activity assays and Western blotting revealed a 2-fold increase in topoisomerase II and a 2-fold decrease in topoisomerase I in the TE-671 MR xenograft compared to the parental xenograft, apparently explaining the collateral sensitivity to etoposide and cross-resistance to topotecan. These results suggest that TE-671 MR xenografts contain multiple changes in activities of DNA repair-related proteins and other nuclear proteins that could contribute to alkylating agent resistance.
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PMID:Elevated DNA polymerase alpha, DNA polymerase beta, and DNA topoisomerase II in a melphalan-resistant rhabdomyosarcoma xenograft that is cross-resistant to nitrosoureas and topotecan. 801 71

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

Human DNA polymerase and DNA ligase utilization for the repair of a major class of ionizing radiation-induced DNA lesion [DNA single-strand breaks containing 3'-phosphoglycolate (3'-PG)] was examined using a novel, chemically defined vector substrate containing a single, site-specific 3'-PG single-strand break lesion. In addition, the major human AP endonuclease, HAP1 (also known as APE1, APEX, Ref-1), was tested to determine if it was involved in initiating repair of 3'-PG-containing single-strand break lesions. DNA polymerase beta was found to be the primary polymerase responsible for nucleotide incorporation at the lesion site following excision of the 3'-PG blocking group. However, DNA polymerase delta/straightepsilon was also capable of nucleotide incorporation at the lesion site following 3'-PG excision. In addition, repair reactions catalyzed by DNA polymerase beta were found to be most effective in the presence of DNA ligase III, while those catalyzed by DNA polymerase delta/straightepsilon appeared to be more effective in the presence of DNA ligase I. Also, it was demonstrated that the repair initiating 3'-PG excision reaction was not dependent upon HAP1 activity, as judged by inhibition of HAP1 with neutralizing HAP1-specific polyclonal antibody.
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PMID:Determination of human DNA polymerase utilization for the repair of a model ionizing radiation-induced DNA strand break lesion in a defined vector substrate. 1032 34

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

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

In the presence of proliferating cell nuclear antigen, yeast DNA polymerase delta (Pol delta) replicated DNA at a rate of 40-60 nt/s. When downstream double-stranded DNA was encountered, Pol delta paused, but most replication complexes proceeded to carry out strand-displacement synthesis at a rate of 1.5 nt/s. In the presence of the flap endonuclease FEN1 (Rad27), the complex carried out nick translation (1.7 nt/s). The Dna2 nuclease/helicase alone did not efficiently promote nick translation, nor did it affect nick translation with FEN1. Maturation in the presence of DNA ligase was studied with various downstream primers. Downstream DNA primers, RNA primers, and small 5'-flaps were efficiently matured by Pol delta and FEN1, and Dna2 did not stimulate maturation. However, maturation of long 5'-flaps to which replication protein A can bind required both DNA2 and FEN1. The maturation kinetics were optimal with a slight molar excess over DNA of Pol delta, FEN1, and proliferating cell nuclear antigen. A large molar excess of DNA ligase substantially enhanced the rate of maturation and shortened the nick-translation patch (nucleotides excised past the RNA/DNA junction before ligation) to 4-6 nt from 8-12 nt with equimolar ligase. These results suggest that FEN1, but not DNA ligase, is a stable component of the maturation complex.
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PMID:Okazaki fragment maturation in yeast. I. Distribution of functions between FEN1 AND DNA2. 1242 38


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