A distinct role of formamidopyrimidine DNA glycosylase (MutM) in down-regulation of accumulation of G, C mutations and protection against oxidative stress in mycobacteria.
Reactive oxygen species produced as a part of cellular metabolism or environmental agent cause a multitude of damages in cell. Oxidative damages to DNA or the free nucleotide pool result in occurrence of 7,8-dihydro-8-oxoguanine (8-oxoG) in DNA, and failure to replace it with the correct base results in a variety of mutations in the genome. Formamidopyrimidine DNA glycosylase (Fpg/MutM), a functionally conserved repair enzyme initiates the 8-oxoG repair pathway in all eubacteria. DNA in mycobacteria with G+C rich genomes is particularly vulnerable to the oxidative damage. In this study, we disrupted fpg gene in Mycobacterium smegmatis to generate an Fpg deficient strain. The strain showed an enhanced mutator phenotype and susceptibility to hydrogen peroxide. Analyses of rifampicin resistance determining region (RRDR) revealed that, in contrast to Fpg deficient Escherichia coli where C to A mutations predominate, Fpg deficient M. smegmatis shows a remarkable increase in accumulation of A to G (or T to C) mutations. Interestingly, exposure of the mutant to sub-lethal level of hydrogen peroxide results in a major shift towards C to G (or G to C) mutations. Biochemical analysis showed that mycobacterial Fpg; and MutY (which excises misincorporated A against 8-oxoG) possess substrate specificities similar to their counterparts in E. coli. However, the DNA polymerase assays with cell-free extracts showed preferential incorporation of G in M. smegmatis as opposed to an A in E. coli. Our studies highlight the importance and the distinctive features of Fpg mediated DNA repair in mycobacteria.
Jain
Ruchi
R
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
Kumar
Pradeep
P
Varshney
Umesh
U
D000595:Amino Acid Sequence; D000995:Antitubercular Agents / Q000494:pharmacology; D001483:Base Sequence; D004249:DNA Damage; D017931:DNA Primers; D004269:DNA, Bacterial; D043382:DNA-Formamidopyrimidine Glycosylase / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D004352:Drug Resistance, Microbial; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D020102:Mycobacterium smegmatis / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D018384:Oxidative Stress; D012293:Rifampin / Q000494:pharmacology
D000995:Antitubercular Agents; D017931:DNA Primers; D004269:DNA, Bacterial; D006861:Hydrogen Peroxide; D043382:DNA-Formamidopyrimidine Glycosylase; D012293:Rifampin