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1.
Proc Natl Acad Sci U S A ; 116(19): 9481-9490, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31019070

RESUMO

DNA double-strand breaks (DSBs) are serious genomic insults that can lead to chromosomal rearrangements if repaired incorrectly. To gain insight into the nuclear mechanisms contributing to these rearrangements, we developed an assay in yeast to measure cis (same site) vs. trans (different site) repair for the majority process of precise nonhomologous end joining (NHEJ). In the assay, the HO endonuclease gene is placed between two HO cut sites such that HO expression is self-terminated upon induction. We further placed an additional cut site in various genomic loci such that NHEJ in trans led to expression of a LEU2 reporter gene. Consistent with prior reports, cis NHEJ was more efficient than trans NHEJ. However, unlike homologous recombination, where spatial distance between a single DSB and donor locus was previously shown to correlate with repair efficiency, trans NHEJ frequency remained essentially constant regardless of the position of the two DSB loci, even when they were on the same chromosome or when two trans repair events were put in competition. Repair of similar DSBs via single-strand annealing of short terminal direct repeats showed substantially higher repair efficiency and trans repair frequency, but still without a strong correlation of trans repair to genomic position. Our results support a model in which yeast cells mobilize, and perhaps compartmentalize, multiple DSBs in a manner that no longer reflects the predamage position of two broken loci.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , Loci Gênicos/fisiologia , Genoma Fúngico/fisiologia , Saccharomyces cerevisiae , 3-Isopropilmalato Desidrogenase/biossíntese , 3-Isopropilmalato Desidrogenase/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética
2.
Biosci Biotechnol Biochem ; 73(11): 2541-3, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19897891

RESUMO

3-isopropylmalate dehydrogenase (IPMDH)-encoding leuB genes were obtained from the obligate piezophile Shewanella benthica DB21MT-2 and non-piezophile Shewanella oneidensis MR-1. The genes were expressed in Escherichia coli and the proteins were purified using His-tag. The estimated kinetic parameters of these enzymes indicated that IPMDH of S. benthica DB21MT-2 is more tolerant of high pressure than that of S. oneidensis MR-1. Thus such an adaptation is one of the mechanisms bacteria utilize for survival at high pressures.


Assuntos
3-Isopropilmalato Desidrogenase/metabolismo , Adaptação Fisiológica , Shewanella/enzimologia , Shewanella/isolamento & purificação , 3-Isopropilmalato Desidrogenase/biossíntese , 3-Isopropilmalato Desidrogenase/genética , 3-Isopropilmalato Desidrogenase/isolamento & purificação , Biocatálise , Cromatografia de Afinidade , Eletroforese , Histidina/metabolismo , Pressão Hidrostática , Oceano Pacífico , Shewanella/fisiologia
3.
Protein Pept Lett ; 14(8): 822-7, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17979826

RESUMO

3-isopropylmalate dehydrogenase (3IPMDH) is the third enzyme in leucine biosynthesis and a promising target for the development of broad-spectrum antibacterial agents. We report here the expression, purification and biochemical characterisation of Haemophilus influenzae 3-isopropylmalate dehydrogenase. The observed enzyme inhibition by the reaction product NADH could represent a regulatory mechanism for 3IPMDH.


Assuntos
3-Isopropilmalato Desidrogenase/biossíntese , Haemophilus influenzae/enzimologia , 3-Isopropilmalato Desidrogenase/isolamento & purificação , 3-Isopropilmalato Desidrogenase/metabolismo , Cátions Bivalentes/farmacologia , Cátions Monovalentes/farmacologia , Clonagem Molecular , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Temperatura
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