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:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Oxidative damage to DNA includes diverse lesions in the sugar-phosphate backbone. The chemical "nuclease" bis(1,10-phenanthroline)copper complex [(OP)(2)Cu] is believed to generate a mixture of direct oxidative strand breaks and C1'-oxidized abasic sites (2-deoxyribonolactone; dL). We found that, under our conditions, the lesions produced by (OP)(2)Cu (50 microM) in synthetic duplex DNA were predominantly dL, accompanied by approximately 30% direct strand breaks with 3'-phosphates. For enzymatic studies, (OP)(2)Cu was used to introduce damage with limited sequence-selectivity, while photolysis of a site-specific 2'-deoxyuridine-1'-t-butyl ketone generated dL at a defined position. The results showed that Ape1, the major human abasic endonuclease, catalyzed 5'-incision of dL sites, but acted at least 10-fold less effectively to remove the 3'-phosphates at direct strand breaks. Kinetic analysis of Ape1 incision using the site-specific dL substrate revealed the same k(cat) for dL and regular (glycosylase-generated) abasic sites, but with K(m) approximately five-fold higher for dL substrate. The efficiency of Ape1 acting on dL, and the abundance of this enzyme in vivo, indicate that dL sites in vivo would be rapidly processed by the endonuclease. The recent observation that Ape1-cleaved dL sites can covalently trap DNA polymerase beta during the abasic excision process suggests that efficient incision of dL by Ape1 may potentiate further problems in DNA repair.
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PMID:Action of human apurinic endonuclease (Ape1) on C1'-oxidized deoxyribose damage in DNA. 1253 88

Damaged DNA bases are removed from mammalian genomes by base excision repair (BER). Single nucleotide BER requires several enzymatic activities, including DNA polymerase and 5',2'-deoxyribose-5-phosphate lyase. Both activities are intrinsic to four human DNA polymerases whose base substitution error rate during gap-filling DNA synthesis varies by more than 10,000-fold. This suggests that BER fidelity could vary over a wide range in an enzyme dependent manner. To investigate this possibility, here we describe an assay to measure the fidelity of BER reactions reconstituted with purified enzymes. When human uracil DNA glycosylase, AP endonuclease, DNA polymerase beta, and DNA ligase 1 replace uracil opposite template A or G, base substitution error rates are <or=0.3 to <or=2.8 x 10-4. BER error rates are higher when excess incorrect dNTPs are included in the reaction or when wild type DNA polymerase beta is replaced by DNA polymerase beta variants that fill single nucleotide gaps with lower fidelity. Under these conditions, the base substitution fidelity of polymerase beta-dependent BER is 3-8-fold higher than is single nucleotide gap filling by polymerase beta alone. Thus other proteins in the BER reaction may enhance the base substitution fidelity of DNA polymerase beta during single nucleotide BER.
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PMID:The base substitution fidelity of DNA polymerase beta-dependent single nucleotide base excision repair. 1273 1

The major abasic endonuclease of human cells, Ape1 protein, is a multifunctional enzyme with critical roles in base excision repair (BER) of DNA. In addition to its primary activity as an apurinic/apyrimidinic endonuclease in BER, Ape1 also possesses 3'-phosphodiesterase, 3'-phosphatase, and 3'-->5'-exonuclease functions specific for the 3' termini of internal nicks and gaps in DNA. The exonuclease activity is enhanced at 3' mismatches, which suggests a possible role in BER for Ape1 as a proofreading activity for the relatively inaccurate DNA polymerase beta. To elucidate this role more precisely, we investigated the ability of Ape1 to degrade DNA substrates that mimic BER intermediates. We found that the Ape1 exonuclease is active at both mismatched and correctly matched 3' termini, with preference for mismatches. In our hands, the exonuclease activity of Ape1 was more active at one-nucleotide gaps than at nicks in DNA, even though the latter should represent the product of repair synthesis by polymerase beta. However, the exonuclease activity was inhibited by the presence of nearby 5'-incised abasic residues, which result from the apurinic/apyrimidinic endonuclease activity of Ape1. The same was true for the recently described exonuclease activity of Escherichia coli endonuclease IV. Exonuclease III, the E. coli homolog of Ape1, did not discriminate among the different substrates. Removal of the 5' abasic residue by polymerase beta alleviated the inhibition of the Ape1 exonuclease activity. These results suggest roles for the Ape1 exonuclease during BER after both DNA repair synthesis and excision of the abasic deoxyribose-5-phosphate by polymerase beta.
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PMID:Modulation of the 3'-->5'-exonuclease activity of human apurinic endonuclease (Ape1) by its 5'-incised Abasic DNA product. 1285 37

Ape1 is the major human abasic endonuclease, initiating repair of this common DNA lesion by incising the phosphodiester backbone 5' to the damage site. This enzyme also functions in specific contexts to excise 3'-blocking termini, e.g. phosphate and phosphoglycolate residues, from DNA. Recently, the comparatively "minor" 3' to 5' exonuclease activity of Ape1 was found to contribute to the excision of certain 3'-mismatched nucleotides. In this study, I characterize more thoroughly the 3'-nuclease properties of Ape1 and define the effects of specific DNA determinants on this function. Data within shows that Ape1 is a non- or poorly processive exonuclease, which degrades one nucleotide gap, 3'-recessed, and nicked DNAs, but exhibits no detectable activity on blunt end or single-stranded DNA. A 5'-phosphate, compared to a 5'-hydroxyl group, reduced Ape1 degradation activity roughly tenfold, suggesting that the biological impact of certain DNA single strand breaks may be influenced by the terminal chemistry. In the context of a base excision repair-like DNA intermediate, a 5'-abasic residue exerted an about tenfold attenuation on the 3' to 5' exonuclease efficiency of Ape1. A 3'-phosphate group had little impact on Ape1 exonuclease activity, and oligonucleotides harboring these blocking termini were activated by Ape1 for DNA polymerase beta extension. Ape1 was also found to remove 3'-tyrosyl residues from 3'-recessed and nicked DNAs, suggesting a potential role in processing covalent topoisomerase I-DNA intermediates formed during chromosome relaxation. While exhibiting preferential excision of thymine in a T:G mismatch context, Ape1 was unable to degrade a triple 3'-thymine mispair. However, Ape1 was able to excise double nucleotide mispairs, apparently through a novel 3'-flap-type endonuclease activity, again activating these substrates for polymerase beta extension.
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PMID:Properties of and substrate determinants for the exonuclease activity of human apurinic endonuclease Ape1. 1286 Jan 25

XRCC1 participates in DNA single strand break and base excision repair (BER) to preserve genetic stability in mammalian cells. XRCC1 participation in these pathways is mediated by its interactions with several of the acting enzymes. Here, we report that XRCC1 interacts physically and functionally with hOGG1, the human DNA glycosylase that initiates the repair by BER of the mutagenic oxidized base 8-oxoguanine. This interaction leads to a 2- to 3-fold stimulation of the DNA glycosylase activity of hOGG1. XRCC1 stimulates the formation of the hOGG1 Schiff-base DNA intermediate without interfering with the endonuclease activity of APE1, the second enzyme in the pathway. On the contrary, the stimulation in the appearance of the incision product seems to reflect the addition of the effects of XRCC1 on the two first enzymes of the pathway. The data presented support a model by which XRCC1 will pass on the DNA intermediate from hOGG1 to the endonuclease APE1. This results in an acceleration of the overall repair process of oxidized purines to yield an APE1-cleaved abasic site, which can be used as a substrate by DNA polymerase beta. More importantly, the results unveil a highly coordinated mechanism by which XRCC1, through its multiple protein-protein interactions, extends its orchestrating role from the base excision step to the resealing of the repaired DNA strand.
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PMID:Role of XRCC1 in the coordination and stimulation of oxidative DNA damage repair initiated by the DNA glycosylase hOGG1. 1293 15

Base excision repair (BER) of damaged deoxyribonucleic acid (DNA) is a multistep process during which potentially lethal abasic sites temporarily exist. Repair of these lesions is greatly stimulated by heat shock protein 70 (Hsp70), which enhances strand incision and removal of the abasic sites by human apurinic-apyrimidinic endonuclease (HAP1). The resulting single-strand gaps must then be filled in. Here, we show that Hsp70 and its 48- and 43-kDa N-terminal domains greatly stimulated filling in the single-strand gaps by DNA polymerase beta, a novel finding that extends the role of Hsps in DNA repair. Incorporation of deoxyguanosine monophosphate (dGMP) to fill in single-strand gaps in DNA phagemid pBKS by DNA polymerase beta was stimulated by Hsp70. Truncated proteins derived from the C-terminus of Hsp70 as well as unrelated proteins were less effective, but proteins derived from the N-terminus of Hsp70 remained efficient stimulators of DNA polymerase beta repair of DNA single-strand gaps. In agreement with these results, repair of a gap in a 30-bp oligonucleotide by polymerase beta also was strongly stimulated by Hsp70 although not by a truncated protein from the C-terminus of Hsp70. Sealing of the repaired site in the oligonucleotide by human DNA ligase 1 was not specifically stimulated by Hsp-related proteins. Results presented here now implicate and extend the role of Hsp70 as a partner in the enzymatic repair of damaged DNA. The participation of Hsp70 jointly with base excision enzymes improves repair efficiency by mechanisms that are not yet understood.
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PMID:Heat shock protein 70 stimulation of the deoxyribonucleic acid base excision repair enzyme polymerase beta. 1462 1

Repair of both normal and reduced AP sites is activated by AP endonuclease, which recognizes and cleaves a phosphodiester bond 5' to the AP site. For a short period of time an incised AP site is occupied by poly(ADP-ribose) polymerase and then DNA polymerase beta adds one nucleotide into the repair gap and simultaneously removes the 5'-sugar phosphate. Finally, the DNA ligase III/XRCC1 complex accomplishes repair by sealing disrupted DNA ends. However, long-patch BER pathway, which is involved in the removal of reduced abasic sites, requires further DNA synthesis resulting in strand displacement and the generation of a damage-containing flap that is later removed by the flap endonuclease. Strand-displacement DNA synthesis is accomplished by DNA polymerase delta/epsilon and DNA ligase I restores DNA integrity. DNA synthesis by DNA polymerase delta/epsilon is dependent on proliferating cell nuclear antigen, which also stimulates the DNA ligase I and flap endonuclease. These repair events are supported by multiple protein-protein interactions.
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PMID:Repair of abasic sites in DNA. 1463 52

Apurinic/apyrimidinic (AP) endonuclease (APE) is a multifunctional protein possessing both DNA repair and redox regulatory activities. In base excision repair (BER), APE is responsible for processing spontaneous, chemical, or monofunctional DNA glycosylase-initiated AP sites via its 5'-endonuclease activity and 3'-"end-trimming" activity when processing residues produced as a consequence of bifunctional DNA glycosylases. In this study, we have fully characterized a mammalian model of APE haploinsufficiency by using a mouse containing a heterozygous gene-targeted deletion of the APE gene (Apex(+/-)). Our data indicate that Apex(+/-) mice are indeed APE-haploinsufficient, as exhibited by a 40-50% reduction (p < 0.05) in APE mRNA, protein, and 5'-endonuclease activity in all tissues studied. Based on gene dosage, we expected to see a concomitant reduction in BER activity; however, by using an in vitro G:U mismatch BER assay, we observed tissue-specific alterations in monofunctional glycosylase-initiated BER activity, e.g. liver (35% decrease, p < 0.05), testes (55% increase, p < 0.05), and brain (no significant difference). The observed changes in BER activity correlated tightly with changes in DNA polymerase beta and AP site DNA binding levels. We propose a mechanism of BER that may be influenced by the redox regulatory activity of APE, and we suggest that reduced APE may render a cell/tissue more susceptible to dysregulation of the polymerase beta-dependent BER response to cellular stress.
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PMID:Apurinic/apyrimidinic endonuclease (APE/REF-1) haploinsufficient mice display tissue-specific differences in DNA polymerase beta-dependent base excision repair. 1497 23

Kinetoplast DNA (kDNA), the form of mitochondrial DNA in trypanosomatids, consists of thousands of interlocked circular DNAs organized into a compact disk structure. A type II DNA topoisomerase, a DNA polymerase beta, and a structure-specific endonuclease have been localized to antipodal sites flanking the kDNA disk along with nascent DNA minicircles. We have cloned a gene (LIG k) encoding a mitochondrial DNA ligase in the trypanosomatid Crithidia fasciculata, and we show that an epitope-tagged form of the ligase colocalizes with the other replication proteins at the antipodal sites and also at the two faces of the kDNA disk. DNA LIG k becomes adenylated in reactions with ATP, and the adenylate moiety is removed by incubation with pyrophosphate or nicked DNA. The ligase interacts physically with the beta polymerase and is proposed to be involved in the repair of gaps in the newly synthesized minicircles. In yeast and mammals, a single gene encodes both nuclear and mitochondrial forms of DNA ligase. The LIG K protein sequence has low similarity to mitochondrial DNA ligases in other eukaryotes and is distinct from the C. fasciculata nuclear DNA ligase (LIG I).
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PMID:Mitochondrial DNA ligase in Crithidia fasciculata. 1507 Jul 15

The paradigm for repair of oxidized base lesions in genomes via the base excision repair (BER) pathway is based on studies in Escherichia coli, in which AP endonuclease (APE) removes all 3' blocking groups (including 3' phosphate) generated by DNA glycosylase/AP lyases after base excision. The recently discovered mammalian DNA glycosylase/AP lyases, NEIL1 and NEIL2, unlike the previously characterized OGG1 and NTH1, generate DNA strand breaks with 3' phosphate termini. Here we show that in mammalian cells, removal of the 3' phosphate is dependent on polynucleotide kinase (PNK), and not APE. NEIL1 stably interacts with other BER proteins, DNA polymerase beta (pol beta) and DNA ligase IIIalpha. The complex of NEIL1, pol beta, and DNA ligase IIIalpha together with PNK suggests coordination of NEIL1-initiated repair. That NEIL1/PNK could also repair the products of other DNA glycosylases suggests a broad role for this APE-independent BER pathway in mammals.
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PMID:AP endonuclease-independent DNA base excision repair in human cells. 1526 Sep 72


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