Distinctive roles of translesion polymerases DinB1 and DnaE2 in diversification of the mycobacterial genome through substitution and frameshift mutagenesis

Abstract

Antibiotic resistance of Mycobacterium tuberculosis is exclusively a consequence of chromosomal mutations. Translesion synthesis (TLS) is a widely conserved mechanism of DNA damage tolerance and mutagenesis, executed by translesion polymerases such as DinBs. In mycobacteria, DnaE2 is the only known agent of TLS and the role of DinB polymerases is unknown. Here we demonstrate that mycobacterial DinB1 abets insertion and deletion frameshift mutagenesis in homo-oligonucleotide runs. DinB1 is the primary mediator of spontaneous -1 frameshift mutations in homo-oligonucleotide runs whereas DnaE2 and DinBs are redundant in DNA damage-induced -1 frameshift mutagenesis. DinB1 also promotes missense mutations conferring resistance to rifampicin, with a mutational signature distinct from that of DnaE2. These results highlight DinB1 and DnaE2 as drivers of mycobacterial genome diversification with relevance to antimicrobial resistance and host adaptation.

Pleiotropic roles for mycobacterial DinB2 in frameshift and substitution mutagenesis

“…The predominant missense change at this codon was CAC>CGC (His>Arg) (Fig. 2D), the same mutation stimulated by DinB1 (Dupuy et al , 2022). The absolute frequency of this mutation was increased 12-fold after DinB3 overexpression (Figure 2D).…”

“…In addition, a previously undetected mutation type emerged: a trinucleotide mutation (G TC>T GT) spanning two codons and detected in 6.8% of sequenced rif R colonies. Overall, the absolute frequency of all mutation types was increased at least 3-fold after DinB2 overexpression, revealing the ability of the polymerase to stimulate diverse mutation types, in contrast to DinB1 (Dupuy et al , 2022).…”

“…Our prior work showed that overexpression of DinB1 in M. smegmatis also induces cell death, probably by interacting with the replicative machinery and competing with the replicative DNA polymerase (Dupuy et al , 2022), a phenotype that was exacerbated by a polymerase-dead active site mutation in DinB1. Unlike DinB1, neither DinB2 nor DinB3 have a predicted β clamp binding motif (Kana et al , 2010).…”

“…Previous studies showed that mycobacterial DnaE2 and DinB1 confer antibiotic resistance by stimulating chromosomal substitution mutations with distinct mutation signatures (Boshoff et al , 2003; Dupuy et al , 2022). To investigate if DinB2 and DinB3 are similarly mutagenic, we measured the rifampicin resistance (rif R ) frequency in M. smegmatis strains overexpressing DinB2 or DinB3.…”

“…Mycobacterial DinBs exemplify three phylogenetic subfamilies found in many actinobacteria: DinB1/DinX, DinB2/DinP and DinB3/msDinB3 (Cole et al , 1998; Timinskas & Venclovas, 2019). In a recent study, we revealed that Mycobacterium smegmatis and M. tuberculosis DinB1 contribute to alkylation damage tolerance, antibiotic resistance though a characteristic mutagenic spectrum, and chromosome diversification through frameshift mutations in homo-oligonucleotide runs (Dupuy et al , 2022). Some of these DinB1 activities, particularly homopolymeric run frameshift mutagenesis, are redundant with DnaE2 during exposure to DNA damage, suggesting that DinB1 and DnaE2, both of which interact with the β-clamp, exert their overlapping activities at the replication fork.…”