Genome Editing Outcomes Reveal Mycobacterial NucS Participates in a Short-Patch Repair of DNA Mismatches

Abstract

In the canonical DNA mismatch repair (MMR) mechanism in bacteria, if during replication a nucleotide is incorrectly mis-paired with the template strand, the resulting repair of this mis-pair can result in the degradation and re-synthesis of hundreds or thousands of nucleotides on the newly-replicated strand (long-patch repair). While mycobacteria, which include important pathogens such asMycobacterium tuberculosis, lack the otherwise highly-conserved enzymes required for the canonical MMR reaction, it was found that disruption of a mycobacterial mismatch-sensitive endonuclease NucS results in a hyper-mutative phenotype, which has led to the idea that NucS might be involved in a cryptic, independently-evolved DNA MMR mechanism. It has been proposed that nuclease activity at a mismatch might result in correction by homologous recombination (HR) with a sister chromatid. Using oligonucleotide recombination, which allows us to introduce mismatches during replication specifically into the genomes of a model forM. tuberculosis,Mycobacterium smegmatis, we find that NucS participates in a direct repair of DNA mismatches where the patch of excised nucleotides is largely confined to within ~5 - 6 bp of the mis-paired nucleotides, which is inconsistent with mechanistic models of canonical mycobacterial HR or other double-strand break (DSB) repair reactions. The results presented provide evidence of a novel NucS-associated mycobacterial MMR mechanism occurring in vivo to regulate genetic mutations in mycobacteria.

Correction of non-random mutational biases along a linear bacterial chromosome by the mismatch repair endonuclease NucS

“…Such striking preference was also observed with NucS from T. kodakarensis ( 9 ) and C. glutamicum ( 20 , 21 ). Furthermore, recent oligonucleotide recombination experiments in M. smegmatis yet to be published ( 70 ) revealed that in vivo NucS would only correct G/T but not A/C mismatches in this species. In this case, transition events in MMR-deficient Δ nucS lines are due to uncorrected G/T mismatches.…”

“…Hence, NucS-dependent DSBs may also be repaired by NHEJ in Streptomyces . Very recently, a preprint manuscript ( 70 ) suggested that mycobacterial NucS participates in a MMR reaction involving short patch repair confined within 5–6 bp of the mispaired nucleotides, which appears to be mechanistically distinct from repair by homologous recombination or NHEJ DSB repair mechanisms. A similar study in M. smegmatis , not approved by peer review yet, has also shown that a NucS-promoted DSB is processed by a 5′–3′ exonuclease, but is independent of both RecA, RadA and NHEJ functions Ku and LigD ( 93 ).…”

Correction of non-random mutational biases along a linear bacterial chromosome by the mismatch repair endonuclease NucS

“…Such striking preference was also observed with NucS from T. kodakarensis ( 9 ) and C. glutamicum ( 20 ,21 ). Furthermore, recent oligonucleotide recombination experiments in M. smegmatis yet to be published ( 70 ) revealed that in vivo NucS would only correct G / T but not A / C mismatches in this species. In this case, transition events in MMR-deficient nucS lines are due to uncorrected G / T mismatches.…”

“…This suggests that PCNA, which interacts with NucS and the Mre11/Rad50 complexes, may act as a master regulator of the MMR and homologous recombination pathways. Interestingly, in human cells, it has been proposed by (63) suggested that mycobacterial NucS participates in a MMR reaction involving short patch repair confined within 5-6 bp of the mispaired nucleotides, which appears to be mechanistically distinct from repair by homologous recombination or NHEJ DSB repair mechanisms. A similar study in M. smegmatis, not approved by peer review yet, has also shown that a NucS-promoted DSB is processed by a 5'-3' exonuclease, but is independent of both RecA, RadA, and NHEJ functions Ku and LigD (89).…”