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

Gessner, Sophia; Martin, Zubair; Reiche, Michael; Santos, Joana A; Dinkele, Ryan; Ramudzuli, Atondaho; Dhar, Neeraj; Wet, Timothy J. de; Anoosheh, Saber; Lang, Dirk; Arron, Jesse; Chew, Teng Leong; Herrmann, Jennifer; Müller, Rolf; McKinney, John D.; Woodgate, Roger; Mizrahi, Valerie; Venclovas, C̆eslovas; Lamers, Meindert H.; Warner, Digby F.

doi:10.7554/elife.75628

Cited by 4 publications

(20 citation statements)

References 76 publications

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“…S4C). Others have reported similar results using N-terminal fusions (27). These data suggest that as in E. coli, the translesion polymerase abundance may be tightly regulated post-transcriptionally (29).…”

Section: Lrs2 Is Important For the Acquisition Of Mutations By Promot...supporting

confidence: 61%

“…When exposed to UV light, these strains acquired resistance to rifampin at a similar rate as wild type, showing that the fusion proteins retain the ability to repair damaged DNA (Fig. S3) (27).…”

Section: Lrs2 Is Important For the Acquisition Of Mutations By Promot...mentioning

confidence: 76%

“…ImuB recruits ImuA' and DnaE2 into an active complex by binding to the β-clamp subunit of the replisome. ImuA' and DnaE2 bind ImuB but not the β-clamp, and therefore require ImuB to associate with the replisome and catalyze mutations (8,11,27,28). Despite being cotranscribed, ImuA'-mScarlet and mScarlet-ImuB had dramatically different fluorescence intensities: ImuA'-mScarlet was barely detectable above background fluorescence levels, while mScarlet-ImuB was brightly fluorescent (Fig.…”

Section: Lrs2 Is Important For the Acquisition Of Mutations By Promot...mentioning

confidence: 99%

“…M. smegmatis and M. tuberculosis missing ImuA' and ImuB are more sensitive to MMC treatment and are less likely to mutate when exposed to DNAdamaging agents like ultraviolet light (Fig. S3A) (11,27). Thus, we hypothesized that cells missing lsr2 would be altered in these key phenotypes.…”

Section: Lrs2 Is Important For the Acquisition Of Mutations By Promot...mentioning

confidence: 99%

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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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Section: Lrs2 Is Important For the Acquisition Of Mutations By Promot...supporting

confidence: 61%

Section: Lrs2 Is Important For the Acquisition Of Mutations By Promot...mentioning

confidence: 76%

Section: Lrs2 Is Important For the Acquisition Of Mutations By Promot...mentioning

confidence: 99%

Section: Lrs2 Is Important For the Acquisition Of Mutations By Promot...mentioning

confidence: 99%

See 2 more Smart Citations

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

View full text Add to dashboard Cite

show abstract

“…In bacteria, such adaptation leads to increased survival rates and causes antimicrobial resistance. The importance of the TLS protein complex involving ImuA, ImuB, and ImuC in generating antimicrobial resistance is established from studies showing that deletions of imuA ( imuA ′ in mycobacteria), imuB or imuC in mycobacteria lead to reduced resistance to the antibiotic rifampicin after UV‐induced damage, as well as impaired survival after treatment with mitomycin C, a DNA‐damaging antibiotic (Erill et al, 2006 ; Gessner et al, 2023 ; Warner et al, 2010 ). Similarly, deletion of imuC in Pseudomonas aeruginosa increases sensitivity of the bacteria to alkylation damage (Jatsenko et al, 2017 ).…”

Section: Introductionmentioning

confidence: 99%

Myxococcus xanthus translesion DNA synthesis protein ImuA is an ATPase enhanced by DNA

Lichimo,

Sowa,

Tetenych

et al. 2024

Protein Science

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Translesion DNA synthesis pathways are necessary to ensure bacterial replication in the presence of DNA damage. Translesion DNA synthesis carried out by the PolV mutasome is well‐studied in Escherichia coli, but ~one third of bacteria use a functionally homologous protein complex, consisting of ImuA, ImuB, and ImuC (also called DnaE2). Numerous in vivo studies have shown that all three proteins are required for translesion DNA synthesis and that ImuC is the error‐prone polymerase, but the roles of ImuA and ImuB are unclear. Here we carry out biochemical characterization of ImuA and a truncation of ImuB from Myxococcus xanthus. We find that ImuA is an ATPase, with ATPase activity enhanced in the presence of DNA. The ATPase activity is likely regulated by the C‐terminus, as loss of the ImuA C‐terminus results in DNA‐independent ATP hydrolysis. We also find that ImuA binds a variety of DNA substrates, with DNA binding affinity affected by the addition of ADP or adenylyl‐imidodiphosphate. An ImuB truncation also binds DNA, with lower affinity than ImuA. In the absence of DNA, ImuA directly binds ImuB with moderate affinity. Finally, we show that ImuA and ImuB self‐interact, but that ImuA is predominantly a monomer, while truncated ImuB is a trimer in vitro. Together, with our findings and the current literature in the field, we suggest a model for translesion DNA synthesis, where a trimeric ImuB would provide sufficient binding sites for DNA, the β‐clamp, ImuC, and ImuA, and where ImuA ATPase activity may regulate assembly and disassembly of the translesion DNA synthesis complex.

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

Cited by 4 publications

References 76 publications

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

Myxococcus xanthus translesion DNA synthesis protein ImuA is an ATPase enhanced by DNA

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

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