Roles for mycobacterial DinB2 in frameshift and substitution mutagenesis
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eLife assessment
This study uses a combination of compelling biochemical and genetic approaches to identify a highly mutagenic DNA polymerase, which drives a wide spectrum of mutations when overexpressed. The important findings advance the understanding of mutagenesis in mycobacteria. The work will be of interest to bacteriologists interested in mutagenesis and the emergence of drug resistance.
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Abstract
Translesion synthesis by translesion polymerases is a conserved mechanism of DNA damage tolerance. In bacteria, DinB enzymes are the widely distributed promutagenic translesion polymerases. The role of DinBs in mycobacterial mutagenesis was unclear until recent studies revealed a role for mycobacterial DinB1 in substitution and frameshift mutagenesis, overlapping with that of translesion polymerase DnaE2. Mycobacterium smegmatis encodes two additional DinBs (DinB2 and DinB3) and Mycobacterium tuberculosis encodes DinB2, but the roles of these polymerases in mycobacterial damage tolerance and mutagenesis is unknown. The biochemical properties of DinB2, including facile utilization of ribonucleotides and 8-oxo-guanine, suggest that DinB2 could be a promutagenic polymerase. Here, we examine the effects of DinB2 and DinB3 overexpression in mycobacterial cells. We demonstrate that DinB2 can drive diverse substitution mutations conferring antibiotic resistance. DinB2 induces frameshift mutations in homopolymeric sequences, both in vitro and in vivo. DinB2 switches from less to more mutagenic in the presence of manganese in vitro. This study indicates that DinB2 may contribute to mycobacterial mutagenesis and antibiotic resistance acquisition in combination with DinB1 and DnaE2.
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eLife assessment
This study uses a combination of compelling biochemical and genetic approaches to identify a highly mutagenic DNA polymerase, which drives a wide spectrum of mutations when overexpressed. The important findings advance the understanding of mutagenesis in mycobacteria. The work will be of interest to bacteriologists interested in mutagenesis and the emergence of drug resistance.
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Reviewer #1 (Public Review):
In this manuscript the authors overproduce two M. smegmatis DNA polymerases, DinB2 and DinB3, as a way to determine whether they may contribute to DNA damage tolerance and/or mutagenesis; the roles of these DNA polymerases in DNA damage tolerance and mutagenesis is currently unknown. The authors show that overproduced levels of DinB2, but not DinB3, impeded growth, and this inhibition was relieved by the disruption of DinB2 catalytic activity using the DinB-D109A mutation. They further demonstrate that the overproduction of DinB2 contributed to frameshift mutagenesis, while DinB3 did not. The contribution of overproduced levels of DinB2 to frameshift mutagenesis was studied in a careful and systematic way, convincingly showing that frameshifts correlated with DinB2 slipping while replicating homopolymeric …
Reviewer #1 (Public Review):
In this manuscript the authors overproduce two M. smegmatis DNA polymerases, DinB2 and DinB3, as a way to determine whether they may contribute to DNA damage tolerance and/or mutagenesis; the roles of these DNA polymerases in DNA damage tolerance and mutagenesis is currently unknown. The authors show that overproduced levels of DinB2, but not DinB3, impeded growth, and this inhibition was relieved by the disruption of DinB2 catalytic activity using the DinB-D109A mutation. They further demonstrate that the overproduction of DinB2 contributed to frameshift mutagenesis, while DinB3 did not. The contribution of overproduced levels of DinB2 to frameshift mutagenesis was studied in a careful and systematic way, convincingly showing that frameshifts correlated with DinB2 slipping while replicating homopolymeric nucleotide runs during dNTP and not rNTP incorporation. The authors also show that the metal cofactor (Mn vs Mg) contributes to the mutagenic behavior of DinB2. While this work is mostly compelling, the major concern is it fails to address the contribution of DinB2 and DinB3 to DNA damage tolerance and mutagenesis when they are expressed at normal levels from their respective chromosomal loci.
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Reviewer #2 (Public Review):
The role of the family IV polymerases in mycobacteria is only partly understood. In this work, the authors investigate the role of the M. smegmatis DinB2 and DinB3 polymerases by a combination of biochemical analysis of enzyme activity in vitro and mutational and phenotypic characterization of M. smegmatis strains during induced over-expression of these proteins. They show both polymerases to be mutagenic and uncover a distinct role for DinB2 in slippage on homopolymeric tracts that is dependent on manganese.
Previous work showed that DinB1 overexpression resulted in SOS induction. This work shows that DinB2 and DinB3 similarly increase RecA levels. Previous work also showed that DinB1 overexpression resulted in growth inhibition and loss of viability which was independent of its polymerase activity. In this …
Reviewer #2 (Public Review):
The role of the family IV polymerases in mycobacteria is only partly understood. In this work, the authors investigate the role of the M. smegmatis DinB2 and DinB3 polymerases by a combination of biochemical analysis of enzyme activity in vitro and mutational and phenotypic characterization of M. smegmatis strains during induced over-expression of these proteins. They show both polymerases to be mutagenic and uncover a distinct role for DinB2 in slippage on homopolymeric tracts that is dependent on manganese.
Previous work showed that DinB1 overexpression resulted in SOS induction. This work shows that DinB2 and DinB3 similarly increase RecA levels. Previous work also showed that DinB1 overexpression resulted in growth inhibition and loss of viability which was independent of its polymerase activity. In this work, overexpression on DinB2 but not DinB3 inhibits growth along with a loss in viability but in contrast to DinB1, this inhibitory effect is only seen with a polymerase-proficient enzyme and is even more enhanced in a steric gate mutant. Overexpression of DinB3 and DinB2 increases the frequency of Rif-resistant mutants independent of the SOS response and DnaE2. The mutation spectrum in DinB2-overexpressing cells was distinct from that caused by DinB1 or DinB3 overexpression. In vitro and in vivo experiments clearly demonstrate that DinB2 catalyzes frameshift mutagenesis on substrates with homopolymeric nucleotide stretches demonstrating enhanced slippage compared to the recent data with DinB1. Remarkably, this slippage is enhanced on homopolymeric runs of purines than pyrimidines in vitro. In vivo slippage by DinB2 was not enhanced by long G runs. The slippage in vitro was only evident in its DNA-dependent DNA polymerase mode and not during ribonucleotide incorporation. In addition, while magnesium alone was associated with mis-addition, the presence of manganese shifted the enzyme to slippage mode in vitro. The detrimental effect of DnaB2 over-expression on viability is, however, not related to its slippage activity since conditions that enhance slippage in vitro (specifically manganese) are associated with a greater detrimental effect on viability in vivo despite a lack of evidence of slippage using reporter constructs.
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Reviewer #3 (Public Review):
The work from Dupuy et al aims to characterize the mutagenic effects of two DinB homologs of Mycobacteria, DinB2, and DinB3. The manuscript shows solid and convincing biochemical data about slippage promoted by DinB 2 on various homopolymeric templates. Overall, this study makes a solid contribution to the understanding of the properties of polymerases from the different DinB subfamilies of bacteria, although some points of the in vivo experiments should be critically evaluated by the readers as described below.
In vivo DinB2 is the more mutagenic of the two and is toxic when overexpressed. Nevertheless, these results are obtained with the overexpression of the polymerases and should be interpreted with caution. In this sense, it would have been interesting to have a quantification of how much overexpression …
Reviewer #3 (Public Review):
The work from Dupuy et al aims to characterize the mutagenic effects of two DinB homologs of Mycobacteria, DinB2, and DinB3. The manuscript shows solid and convincing biochemical data about slippage promoted by DinB 2 on various homopolymeric templates. Overall, this study makes a solid contribution to the understanding of the properties of polymerases from the different DinB subfamilies of bacteria, although some points of the in vivo experiments should be critically evaluated by the readers as described below.
In vivo DinB2 is the more mutagenic of the two and is toxic when overexpressed. Nevertheless, these results are obtained with the overexpression of the polymerases and should be interpreted with caution. In this sense, it would have been interesting to have a quantification of how much overexpression the plasmids constructs achieve in the conditions used in the experiments, for a better assessment of the relevance of the data. For example, a physiological 10-fold increase in the expression of DinB2 is mentioned in the discussion - would that be close to what is achieved with plasmid-based overexpression?
The finding of kanR CFUs without any detectable mutations in the kan marker is worrisome and should be better discussed in the text. The same for sacB data in supplementary material. The explanation given in lines 216-218 does not make sense. Markers 7G and 8G clearly are barely measuring any mutagenesis. I think that the experiments in which most of the supposed KanR revertants actually have no Kan mutation should either be removed from the manuscript or better discussed, because it is uncertain what they are measuring, therefore no conclusion can be drawn from them. For the Kan markers, one possible explanation is that translational frameshifts are occurring and allow residual growth of some of the cells. Gene amplification as seen in the lac system of Cairns and Foster in E. coli could also promote growth without actual mutations. Is the KanR phenotype of these colonies heritable and stable?
Also, spontaneous mutagenesis should have been more precisely measured by using fluctuation analysis of larger sample sizes. In many instances, the results shown are the means of a few cultures with very large differences in mutant frequencies (several hundred-fold - e.g. Figures 4C, D and E, 5C and F, S3). Authors could discuss/explain their choice of statistical analysis and sample sizes.
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