A distinct physiological role of MutY in mutation prevention in mycobacteria

Abstract

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.

A combinatorial role for MutY and Fpg DNA glycosylases in mutation avoidance in Mycobacterium smegmatis

“…However, under oxidative stress conditions, generated by supplementation of cultures with H 2 O 2 , loss of MutY results in a significant decline in growth compared to the parental strain which is not exaggerated with the combinatorial loss of the Fpg or Nei homologues, suggesting that MutY is important for survival of M. smegmatis under oxidative stress conditions. These data are concordant with the loss of MutY function in M. tuberculosis [39], but conflict with a previous report which demonstrated that disruption of mutY in M. smegmatis does not lead to enhanced sensitivity to hydrogen peroxide [22]. In our work, we used the same parental strain of M. smegmatis [56] with similar experimental conditions hence, the reason for these discrepant results are unclear and may be due to the inherent variability in mutant generation, difference in quantitative survival assays or hydrogen peroxide decay.…”

“…The reduced frequency of the remaining mutations detected did not allow for meaningful comparisons to be made. Loss of Fpg and MutY function is expected to exacerbate G·C→T·A transversion mutagenesis due to persisting 8-oxoGua lesions that mispair with adenine [21,22]. Consistent with this, the Rif-resistant isolates derived from the fpg1 fpg2 mutY mutant showed approximately a 30-45% increase in the C → A (Gln513 → Lys) mutational genotype compared to the single mutY and the double fpg1 fpg2 deletion mutants respectively.…”

“…Biochemical characterization of MutY in M. smegmatis and M. tuberculosis showed substrate specificity to mispaired adenine opposite 8-oxoGua with weak excision activity for G and T paired with 8-oxoGua. As observed in other organisms, MutY does not process 8-oxoGua:C, pointing to a specific role in mutation avoidance in mycobacteria [22].…”

“…This plasticity may be a direct adaptation for mycobacterial survival in the hostile macrophage environment. MutY deficiency in M. smegmatis results in G → T transversions, with no increase in sensitivity to oxidative stress or elevation in mutation rates [22] suggesting that the two Fpg homologues present in this organism efficiently remove 8-oxoGua lesions. In contrast, disruption of mutY in M. tuberculosis leads to enhanced susceptibility to oxidative stress [39] which suggests that the single functional Fpg homologue is unable to maintain genome integrity and there is a greater dependency on MutY for genome maintenance.…”

“…In Escherichia coli, this mutagenic effect is prevented by the MutY DNA glycosylase from the 8-oxoGua (GO) repair system which, removes A mispaired with 8-oxoGua and allows a second chance for repair of 8-oxoGua [18][19][20][21][22][23][24] (Fig. S1A).…”