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)

It is reported here that the rpr DNA repair gene of Serratia marcescens does not complement an Escherichia coli xth nfo AP endonuclease mutation for resistance to methyl methanesulphonate (MMS). Rather, rpr sensitized Escherichia coli wild-type, xth, and nfo strains to MMS. Also, it was found that rpr could not complement a triple tag alkA recA mutation in E. coli, indicating that there are limits to rpr complementing capabilities. It was determined that rpr gene dosage was not a factor in recA complementation. MMS sensitization of an E. coli wild-type strain, however, was directly related to rpr copy number. These data indicate that Rpr does not have an associated AP endonuclease activity, and that it is incapable of substituting for Tag I, Tag II, and RecA in a tag alkA recA background.
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PMID:Serratia marcescens rpr gene sensitizes Escherichia coli wild-type, xth, and nfo strains to methyl methanesulphonate. 169 47

DNA glycosylases, such as the Mag1 3-methyladenine (3MeA) DNA glycosylase, initiate the base excision repair (BER) pathway by removing damaged bases to create abasic apurinic/apyrimidinic (AP) sites that are subsequently repaired by downstream BER enzymes. Although unrepaired base damage may be mutagenic or recombinogenic, BER intermediates (e.g. AP sites and strand breaks) may also be problematic. To investigate the molecular basis for methylation-induced homologous recombination events in Saccharomyces cerevisiae, spontaneous and methylation-induced recombination were studied in strains with varied MAG1 expression levels. We show that cells lacking Mag1 have increased susceptibility to methylation-induced recombination, and that disruption of nucleotide excision repair (NER; rad4) in mag1 cells increases cellular susceptibility to these events. Furthermore, expression of Escherichia coli Tag 3MeA DNA glycosylase suppresses recombination events, providing strong evidence that unrepaired 3MeA lesions induce recombination. Disruption of REV3 (required for polymerase zeta (Pol zeta)) in mag1 rad4 cells causes increased susceptibility to methylation-induced toxicity and recombination, suggesting that Pol zeta can replicate past 3MeAs. However, at subtoxic levels of methylation damage, disruption of REV3 suppresses methylation-induced recombination, indicating that the effects of Pol zeta on recombination are highly dose-dependent. We also show that overproduction of Mag1 can increase the levels of spontaneous recombination, presumably due to increased levels of BER intermediates. However, additional APN1 endonuclease expression or disruption of REV3 does not affect MAG1-induced recombination, suggesting that downstream BER intermediates (e.g. single strand breaks) are responsible for MAG1-induced recombination, rather than uncleaved AP sites. Thus, too little Mag1 sensitizes cells to methylation-induced recombination, while too much Mag1 can put cells at risk of recombination induced by single strand breaks formed during BER.
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PMID:The S. cerevisiae Mag1 3-methyladenine DNA glycosylase modulates susceptibility to homologous recombination. 1250 87

Apolipoprotein A-I (apoA-I) serves critical functions in plasma lipoprotein metabolism as a structural component of high density lipoprotein, activator of lecithin:cholesterol acyltransferase, and acceptor of cellular cholesterol as part of the reverse cholesterol transport pathway. In an effort to facilitate structure:function studies of human apoA-I, we have optimized a plasmid vector for production of recombinant wild type (WT) and mutant apoA-I in bacteria. To facilitate mutagenesis studies, subcloning, and DNA manipulation, numerous silent mutations have been introduced into the apoA-I cDNA, generating 13 unique restriction endonuclease sites. The coding sequence for human apoA-I has been modified by the introduction of additional silent mutations that eliminate 18 separate codons that employ tRNAs that are of low or moderate abundance in Escherichia coli. Yields of recombinant apoA-I achieved using the optimized cDNA were 100+/-20 mg/L bacterial culture, more than fivefold greater than yields routinely obtained with the original cDNA. Site-directed mutagenesis of the apoA-I cDNA was performed to generate a Glu2Asp mutation in the N-terminal sequence of apoA-I. This modification, which creates an acid labile Asp-Pro peptide bond between amino acids 2 and 3, permits specific chemical cleavage of an N-terminal His-Tag fusion peptide used for rapid protein purification. The product protein's primary structure is identical to WT apoA-I in all other respects. Together, these changes in apoA-I cDNA and bacterial expression protocol significantly improve the yield of apoA-I protein without compromising the relative ease of purification.
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PMID:Optimized bacterial expression of human apolipoprotein A-I. 1250 90