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)

DNA polymerase-alpha and -beta can be distinguished from one another by the differential effects of N-ethylmaleimide, KCl, ara-CTP and temperature, as well as on the basis of sedimentation. The sensitivity of DNA polymerase-beta to elevated temperatures as compared to DNA polymerase-alpha provides a new means of distinguishing between these two enzymes even in crude extracts and a possible probe for determining their function. DNA polymerase-alpha and -beta share several properties in common, including the ability to readily incorporate dUTP in place of dTTP. The Km for dUTP varies from 10 to 30 micron with different preparations of DNA polymerase-alpha and -beta. Thus, in mammalian cells, dUMP could be incorporated into DNA, and if excised by an endonuclease, would lead to discontinuities. Initial analyses of fidelity in direct comparative studies indicate that beta-class DNA polymerases are highly accurate in base selection when copying poly[d(A-T)]. Less than one molecule of dGMP is incorporated for every 12 000-45 000 molecules of dAMP and dTMP polymerized. DNA polymerase-alpha is somewhat less accurate, making one mistake for every 4000-10 000 correct nucleotides incorporated. Since both polymerases lack an exonucleolytic activity, this accuracy must be the result of selectivity for the complementary nucleotide by the polymerase.
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PMID:Distinctive properties of mammalian DNA polymerases. 28 7

The extent and location of DNA repair synthesis in a double-stranded oligonucleotide containing a single dUMP residue have been determined. Gently prepared Escherichia coli and mammalian cell extracts were employed for excision repair in vitro. The size of the resynthesized patch was estimated by restriction enzyme analysis of the repaired oligonucleotide. Following enzymatic digestion and denaturing gel electrophoresis, the extent of incorporation of radioactively labeled nucleotides in the vicinity of the lesion was determined by autoradiography. Cell extracts of E. coli and of human cell lines were shown to carry out repair mainly by replacing a single nucleotide. No significant repair replication on the 5' side of the lesion was observed. The data indicate that, after cleavage of the dUMP residue by uracil-DNA glycosylase and incision of the resultant apurinic-apyrimidinic site by an apurinic-apyrimidinic endonuclease activity, the excision step is catalyzed usually by a DNA deoxyribophosphodiesterase rather than by an exonuclease. Gap-filling and ligation complete the repair reaction. Experiments with enzyme inhibitors in mammalian cell extracts suggest that the repair replication step is catalyzed by DNA polymerase beta.
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PMID:Generation of single-nucleotide repair patches following excision of uracil residues from DNA. 154 15

Heat treatment of poly(deoxycytidylic acid)-[poly(dC)] induces the formation of dUMP residues, which code for dAMP when replicated by Escherichia coli DNA polymerases I and III. The specificity of dUMP coding properties is indicated by the quantitative relation between the dAMP incorporated and the frequency of dUMP residues in the heat-treated poly(dC). The dAMP incorporation is prevented by preincubation of uracil containing poly(dC) with uracil-DNA glycosylase. The excision of uracil by uracil-DNA glycosylase leads to the formation of apyrimidinic sites (AP sites), which are barely replicated in vitro under physiological conditions. However, the alteration of E. coli DNA polymerase I fidelity of replication by Mn2+ greatly stimulates the replication of AP sites. There is a preferential incorporation of dAMP, as compared to dTMP, opposite the AP sites. The dAMP incorporation is prevented by preincubation of poly(dC) containing AP sites with Micrococcus luteus AP endonuclease B. The results show a close association between DNA repair by base excision and the prevention of mutagenic processes in vitro. Furthermore, since the alteration of DNA polymerase fidelity allows some replication of the noncoding DNA lesion (AP site), this could imply a role in SOS-induced mutagenesis in vivo.
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PMID:Coding properties of poly(deoxycytidylic acid) templates containing uracil or apyrimidinic sites: in vitro modulation of mutagenesis by deoxyribonucleic acid repair enzymes. 676 Aug 93

Endonuclease V (deoxyinosine 3' endonuclease), the product of the nfi gene, has a specificity that encompasses DNAs containing dIMP, abasic sites, base mismatches, uracil, and even untreated single-stranded DNA. To determine its importance in DNA repair pathways, nfi insertion mutants and overproducers (strains bearing nfi plasmids) were constructed. The mutants displayed a twofold increase in spontaneous mutations for several markers and an increased sensitivity to killing by bleomycin and nitrofurantoin. An nfi mutation increased both cellular resistance to and mutability by nitrous acid. This agent should generate potential cleavage sites for the enzyme by deaminating dAMP and dCMP in DNA to dIMP and dUMP, respectively. Relative to that of a wild-type strain, an nfi mutant displayed a 12- to 1,000-fold increase in the frequency of nitrite-induced mutations to streptomycin resistance, which are known to occur in A x T base pairs. An nfi mutation also enhanced the lethality caused by a combined deficiency of exonuclease III and dUTPase, which has been attributed to unrepaired abasic sites. However, neither the deficiency nor the overproduction of endonuclease V affected the growth of the single-stranded DNA phages M13 or phiX174 nor of Uracil-containing bacteriophage lambda. These results suggest that endonuclease V has a significant role in the repair of deaminated deoxyadenosine (deoxyinosine) and abasic sites in DNA, but there was no evidence for its cleavage in vivo of single-stranded or uracil-containing DNA.
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PMID:Endonuclease V (nfi) mutant of Escherichia coli K-12. 942 91

Uracil is usually an inappropriate base in DNA, but it is also a normal intermediate during somatic hypermutation (SHM) and class switch recombination (CSR) in adaptive immunity. In addition, uracil is introduced into retroviral DNA by the host as part of a defence mechanism. The sources of uracil in DNA are spontaneous or enzymatic deamination of cytosine (U:G mispairs) and incorporation of dUTP (U:A pairs). Uracil in DNA is removed by a uracil-DNA glycosylase. The major ones are nuclear UNG2 and mitochondrial UNG1 encoded by the UNG-gene, and SMUG1 that also removes oxidized pyrimidines, e.g. 5-hydroxymethyluracil. The other ones are TDG that removes U and T from mismatches, and MBD4 that removes U from CpG contexts. UNG2 is found in replication foci during the S-phase and has a distinct role in repair of U:A pairs, but it is also important in U:G repair, a function shared with SMUG1. SHM is initiated by activation-induced cytosine deaminase (AID), followed by removal of U by UNG2. Humans lacking UNG2 suffer from recurrent infections and lymphoid hyperplasia, and have skewed SHM and defective CSR, resulting in elevated IgM and strongly reduced IgG, IgA and IgE. UNG-defective mice also develop B-cell lymphoma late in life. In the defence against retrovirus, e.g. HIV-1, high concentrations of dUTP in the target cells promotes misincorporation of dUMP-, and host cell APOBEC proteins may promote deamination of cytosine in the viral DNA. This facilitates degradation of viral DNA by UNG2 and AP-endonuclease. However, viral proteins Vif and Vpr counteract this defense by mechanisms that are now being revealed. In conclusion, uracil in DNA is both a mutagenic burden and a tool to modify DNA for diversity or degradation.
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PMID:DNA-uracil and human pathology. 1759 Apr 28

Protein p56 encoded by the Bacillus subtilis phage phi29 inhibits host uracil-DNA glycosylase (UDG) activity. In previous studies, we suggested that this inhibition is likely a defense mechanism developed by phage phi29 to prevent the action of UDG if uracilation occurs in DNA either from deamination of cytosine or the incorporation of dUMP during viral DNA replication. In this work, we analyzed the ability of phi29 DNA polymerase to insert dUMP into DNA. Primer extension analysis showed that viral DNA polymerase incorporates dU opposite dA with a catalytic efficiency only 2-fold lower than that for dT. Using the phi29 DNA amplification system, we found that phi29 DNA polymerase is also able to carry out the extension of the dA:dUMP pair and replicate past uracil. Additionally, UDG and apurinic-apyrimidinic endonuclease treatment of viral DNA isolated from phi29-infected cells revealed that uracil residues arise in phi29 DNA during replication, probably as a result of misincorporation of dUMP by the phi29 DNA polymerase. On the other hand, the action of UDG on uracil-containing phi29 DNA impaired in vitro viral DNA replication, which was prevented by the presence of protein p56. Furthermore, transfection activity of uracil-containing phi29 DNA was significantly higher in cells that constitutively synthesized p56 than in cells lacking this protein. Thus, our data support a model in which protein p56 ensures an efficient viral DNA replication, preventing the deleterious effect caused by UDG when it eliminates uracil residues present in the phi29 genome.
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PMID:Phage phi29 protein p56 prevents viral DNA replication impairment caused by uracil excision activity of uracil-DNA glycosylase. 1884 83