A distinct physiological role of MutY in mutation prevention in mycobacteria.
Oxidative damage to DNA results in the occurrence of 7,8-dihydro-8-oxoguanine (8-oxoG) in the genome. In eubacteria, repair of such damage is initiated by two major base-excision repair enzymes, MutM and MutY. We generated a MutY-deficient strain of Mycobacterium smegmatis to investigate the role of this enzyme in DNA repair. The MutY deficiency in M. smegmatis did not result in either a noteworthy susceptibility to oxidative stress or an increase in the mutation rate. However, rifampicin-resistant isolates of the MutY-deficient strain showed distinct mutations in the rifampicin-resistance-determining region of rpoB. Besides the expected C to A (or G to T) mutations, an increase in A to C (or T to G) mutations was also observed. Biochemical characterization of mycobacterial MutY (M. smegmatis and M. tuberculosis) revealed an expected excision of A opposite 8-oxoG in DNA. Additionally, excision of G and T opposite 8-oxoG was detected. MutY formed complexes with DNA containing 8-oxoG : A, 8-oxoG : G or 8-oxoG : T but not 8-oxoG : C pairs. Primer extension reactions in cell-free extracts of M. smegmatis suggested error-prone incorporation of nucleotides into the DNA. Based on these observations, we discuss the physiological role of MutY in specific mutation prevention in mycobacteria.
Kurthkoti
Krishna
K
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, India.
Srinath
Thiruneelakantan
T
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, India.
Kumar
Pradeep
P
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, India.
Malshetty
Vidyasagar S
VS
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, India.
Sang
Pau Biak
PB
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, India.
Jain
Ruchi
R
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, India.
Manjunath
Ramanathapuram
R
Department of Biochemistry, Indian Institute of Science, Bangalore, 560012, India.
Varshney
Umesh
U
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, India.
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004249:DNA Damage; D045647:DNA Glycosylases / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004269:DNA, Bacterial / Q000235:genetics; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D051379:Mice; D009154:Mutation; D020102:Mycobacterium smegmatis / Q000235:genetics* / Q000378:metabolism; D012293:Rifampin / Q000378:metabolism; D013379:Substrate Specificity
C453560:7,8-dihydro-8-oxoguanine; D001426:Bacterial Proteins; D004269:DNA, Bacterial; C024829:8-hydroxyguanine; D006147:Guanine; D006861:Hydrogen Peroxide; D045647:DNA Glycosylases; C061738:mutY adenine glycosylase; D012293:Rifampin