Roles for mycobacterial DinB2 in frameshift and substitution mutagenesis

Dupuy, Pierre; Ghosh, Shreya; Fay, Allison; Adefisayo, Oyindamola; Gupta, Richa; Shuman, Stewart; Glickman, Michael S.

doi:10.7554/elife.83094

Cited by 3 publications

(1 citation statement)

References 36 publications

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“…In mycobacteria, the fidelity of chromosome replication is established by the major replicative polymerase DnaE1, which possesses endogenous proofreading activity ( 4 ). In addition to DnaE1, mycobacteria encode a suite of other polymerases with specialized repair functions that are only expressed under certain conditions, such as oxidative or DNA-damaging stress ( 5 – 7 ). For example, the mycobacterial specialist polymerases DinB1, DinB2, DinB3, and DnaE2 are induced under different conditions and have varying repair activities and mutational signatures ( 5 – 10 ).…”

Section: Introductionmentioning

confidence: 99%

A nucleoid-associated protein is involved in the emergence of antibiotic resistance by promoting the frequent exchange of the replicative DNA polymerase in Mycobacterium smegmatis

Ng,

Rego

2024

mSphere

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Antibiotic resistance in Mycobacterium tuberculosis exclusively originates from chromosomal mutations, either during normal DNA replication or under stress, when the expression of error-prone DNA polymerases increases to repair damaged DNA. To bypass DNA lesions and catalyze error-prone DNA synthesis, translesion polymerases must be able to access the DNA, temporarily replacing the high-fidelity replicative polymerase. The mechanisms that govern polymerase exchange are not well understood, especially in mycobacteria. Here, using a suite of quantitative fluorescence imaging techniques, we discover that in Mycobacterium smegmatis , as in other bacterial species, the replicative polymerase, DnaE1, exchanges at a timescale much faster than that of DNA replication. Interestingly, this fast exchange rate depends on an actinobacteria-specific nucleoid-associated protein (NAP), Lsr2. In cells missing lsr2 , DnaE1 exchanges less frequently, and the chromosome is replicated more faithfully. Additionally, in conditions that damage DNA, cells lacking lsr2 load the complex needed to bypass DNA lesions less effectively and, consistently, replicate with higher fidelity but exhibit growth defects. Together, our results show that Lsr2 promotes dynamic flexibility of the mycobacterial replisome, which is critical for robust cell growth and lesion repair in conditions that damage DNA. IMPORTANCE Unlike many other pathogens, Mycobacterium tuberculosis has limited ability for horizontal gene transfer, a major mechanism for developing antibiotic resistance. Thus, the mechanisms that facilitate chromosomal mutagenesis are of particular importance in mycobacteria. Here, we show that Lsr2, a nucleoid-associated protein, has a novel role in DNA replication and mutagenesis in the model mycobacterium Mycobacterium smegmatis . We find that Lsr2 promotes the fast exchange rate of the replicative DNA polymerase, DnaE1, at the replication fork and is important for the effective loading of the DnaE2-ImuA′-ImuB translesion complex. Without lsr2 , M. smegmatis replicates its chromosome more faithfully and acquires resistance to rifampin at a lower rate, but at the cost of impaired survival to DNA damaging agents. Together, our work establishes Lsr2 as a potential factor in the emergence of mycobacterial antibiotic resistance.

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Section: Introductionmentioning

confidence: 99%

A nucleoid-associated protein is involved in the emergence of antibiotic resistance by promoting the frequent exchange of the replicative DNA polymerase in Mycobacterium smegmatis

Ng,

Rego

2024

mSphere

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A nucleoid-associated protein is involved in the emergence of antibiotic resistance by promoting the frequent exchange of the replicative DNA polymerase inM. smegmatis

Ng¹,

Park²,

Rego³

2023

Preprint

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Antibiotic resistance inM. tuberculosisexclusively originates from chromosomal mutations, either during normal DNA replication or under stress, when the expression of error-prone DNA polymerases increases to repair damaged DNA. The mechanisms by which polymerases switch at the replication fork are unknown. Here, we discover that a mutation in the nucleoid-associated protein, Lsr2, produces several hallmarks of an impaired DNA damage response, including a severe growth defect when exposed to genotoxic drugs and reduced mutation frequency. The transcription factor function of Lsr2 cannot explain these phenotypes. Instead, through a series of genetic and fluorescence imaging experiments, we find that Lsr2 is important for modulating the dynamics of polymerases at the replication fork. The major replicative DNA polymerase, DnaE1, exchanges more frequently in the presence of Lsr2, while, during DNA damage, cells lackinglsr2cannot effectively load the complex needed to bypass DNA lesions, and thus replicate with higher fidelity but exhibit growth defects. Together, our results show that Lsr2 promotes dynamic flexibility of the mycobacterial replisome, which is critical for robust cell growth and lesion repair in conditions that damage DNA.

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Investigating the composition and recruitment of the mycobacterial ImuA′–ImuB–DnaE2 mutasome

Gessner

Martin

Reiche

et al. 2023

eLife

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A DNA damage-inducible mutagenic gene cassette has been implicated in the emergence of drug resistance in Mycobacterium tuberculosis during anti-tuberculosis (TB) chemotherapy. However, the molecular composition and operation of the encoded 'mycobacterial mutasome' - minimally comprising DnaE2 polymerase and ImuA′ and ImuB accessory proteins - remain elusive. Following exposure of mycobacteria to DNA damaging agents, we observe that DnaE2 and ImuB co-localize with the DNA polymerase III β subunit (β clamp) in distinct intracellular foci. Notably, genetic inactivation of the mutasome in an imuBAAAAGG mutant containing a disrupted β clamp-binding motif abolishes ImuB-β clamp focus formation, a phenotype recapitulated pharmacologically by treating bacilli with griselimycin and in biochemical assays in which this β clamp-binding antibiotic collapses pre-formed ImuB-β clamp complexes. These observations establish the essentiality of the ImuB-β clamp interaction for mutagenic DNA repair in mycobacteria, identifying the mutasome as target for adjunctive therapeutics designed to protect anti-TB drugs against emerging resistance.

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Roles for mycobacterial DinB2 in frameshift and substitution mutagenesis

Cited by 3 publications

References 36 publications

A nucleoid-associated protein is involved in the emergence of antibiotic resistance by promoting the frequent exchange of the replicative DNA polymerase in Mycobacterium smegmatis

A nucleoid-associated protein is involved in the emergence of antibiotic resistance by promoting the frequent exchange of the replicative DNA polymerase in Mycobacterium smegmatis

A nucleoid-associated protein is involved in the emergence of antibiotic resistance by promoting the frequent exchange of the replicative DNA polymerase inM. smegmatis

Investigating the composition and recruitment of the mycobacterial ImuA′–ImuB–DnaE2 mutasome

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