NHEJ protects mycobacteria in stationary phase against the harmful effects of desiccation

The physiological role of the non-homologous end-joining (NHEJ) pathway in the repair of DNA double-strand breaks (DSBs) was examined in Mycobacterium smegmatis using DNA repair mutants (DeltarecA, Deltaku, DeltaligD, Deltaku/ligD, DeltarecA/ku/ligD). Wild-type and mutant strains were exposed to a range of doses of ionizing radiation at specific points in their life-cycle. NHEJ-mutant strains (Deltaku, DeltaligD, Deltaku/ligD) were significantly more sensitive to ionizing radiation (IR) during stationary phase than wild-type M. smegmatis. However, there was little difference in IR sensitivity between NHEJ-mutant and wild-type strains in logarithmic phase. Similarly, NHEJ-mutant strains were more sensitive to prolonged desiccation than wild-type M. smegmatis. A DeltarecA mutant strain was more sensitive to desiccation and IR during both stationary and especially in logarithmic phase, compared to wild-type strain, but it was significantly less sensitive to IR than the DeltarecA/ku/ligD triple mutant during stationary phase. These data suggest that NHEJ and homologous recombination are the preferred DSB repair pathways employed by M. smegmatis during stationary and logarithmic phases, respectively

RNase HI Is Essential for Survival of Mycobacterium smegmatis

RNases H are involved in the removal of RNA from RNA/DNA hybrids. Type I RNases H are thought to recognize and cleave the RNA/DNA duplex when at least four ribonucleotides are present. Here we investigated the importance of RNase H type I encoding genes for model organism Mycobacterium smegmatis. By performing gene replacement through homologous recombination, we demonstrate that each of the two presumable RNase H type I encoding genes, rnhA and MSMEG4305, can be removed from M. smegmatis genome without affecting the growth rate of the mutant. Further, we demonstrate that deletion of both RNases H type I encoding genes in M. smegmatis leads to synthetic lethality. Finally, we question the possibility of existence of RNase HI related alternative mode of initiation of DNA replication in M. smegmatis, the process initially discovered in Escherichia coli. We suspect that synthetic lethality of double mutant lacking RNases H type I is caused by formation of R-loops leading to collapse of replication forks. We report Mycobacterium smegmatis as the first bacterial species, where function of RNase H type I has been found essential.The study was supported by POIG.01.01.02-10-107/09 project implemented under Innovative Economy Operational Programme, years 2007–2013 "Studies of the molecular mechanisms at the interface the human organism - the pathogen - environmental factors" and by grant of Polish National Center of Science 2011/01/N/NZ6/04186 “Identification of a novel mechanism of initiation of DNA replication in Mycobacterium smegmatis”

Direct and Inverted Repeats Elicit Genetic Instability by Both Exploiting and Eluding DNA Double-Strand Break Repair Systems in Mycobacteria

<div><p>Repetitive DNA sequences with the potential to form alternative DNA conformations, such as slipped structures and cruciforms, can induce genetic instability by promoting replication errors and by serving as a substrate for DNA repair proteins, which may lead to DNA double-strand breaks (DSBs). However, the contribution of each of the DSB repair pathways, homologous recombination (HR), non-homologous end-joining (NHEJ) and single-strand annealing (SSA), to this sort of genetic instability is not fully understood. Herein, we assessed the genome-wide distribution of repetitive DNA sequences in the <em>Mycobacterium smegmatis</em>, <em>Mycobacterium tuberculosis</em> and <em>Escherichia coli</em> genomes, and determined the types and frequencies of genetic instability induced by direct and inverted repeats, both in the presence and in the absence of HR, NHEJ, and SSA. All three genomes are strongly enriched in direct repeats and modestly enriched in inverted repeats. When using chromosomally integrated constructs in <em>M. smegmatis</em>, direct repeats induced the perfect deletion of their intervening sequences ∼1,000-fold above background. Absence of HR further enhanced these perfect deletions, whereas absence of NHEJ or SSA had no influence, suggesting compromised replication fidelity. In contrast, inverted repeats induced perfect deletions only in the absence of SSA. Both direct and inverted repeats stimulated excision of the constructs from the <em>attB</em> integration sites independently of HR, NHEJ, or SSA. With episomal constructs, direct and inverted repeats triggered DNA instability by activating nucleolytic activity, and absence of the DSB repair pathways (in the order NHEJ>HR>SSA) exacerbated this instability. Thus, direct and inverted repeats may elicit genetic instability in mycobacteria by 1) directly interfering with replication fidelity, 2) stimulating the three main DSB repair pathways, and 3) enticing L5 site-specific recombination.</p> </div

Southern blot confirming deletion of native <i>dnaA</i> gene in ∆<i>rnhA</i>/∆<i>dnaAattB</i>::<i>dnaA</i> mutant strain of <i>M</i>. <i>smegmatis</i>.

<p>We used gene replacement through homologous recombination to obtain mutants deficient in <i>rnhA</i>. Further, we used the same procedure to generate ∆<i>rnhA</i>/<i>dnaA</i>SCO strain where SCO signifies an intermediate step of gene replacement procedure. Next, through complementation procedure, we introduced an additional version of <i>dnaA</i> gene at the <i>attB</i> site of mycobacterial genome. Finally, we removed the native version of <i>dnaA</i>, thereby generating a mutant ∆<i>rnhA</i>/<i>dnaA</i>SCO<i>attB</i>::<i>dnaA</i>. For more information regarding plasmid construction and gene replacement procedure please refer to the text. Schematic representation of analyzed genomic region, including enzymes used for digestion, size of restriction fragments following digestion and the site of hybridization of hybridization probe, is presented in the upper part of the figure. Photographic film presenting results of Southern blot analysis is presented in the lower part of the figure. Bands corresponding to wild type genotype (wt) and mutant genotype (mut) are marked on the right side of the photograph.</p

Southern blots confirming deletions in single RNase H type I mutants of <i>M</i>. <i>smegmatis</i>.

<p>We used gene replacement through homologous recombination to obtain mutants deficient in either <i>rnhA</i> or MSMEG4305. Briefly, recombinant plasmids containing genomic regions of either <i>rnhA</i> or MSMEG4305 with large deletions within each gene were introduced into <i>M</i>. <i>smegmatis</i> mc² 155 cells. Following multistep selection we were able to identify clones where the native version of each gene has been replaced with manipulated sequence. Intermediate steps of gene replacement procedure are denoted SCO. For more information regarding plasmid construction and gene replacement procedure please refer to the text. Schematic representation of analyzed genomic regions, including enzymes used for digestion, size of restriction fragments following digestion and the site of hybridization of hybridization probe, is presented in the upper part of the figure. Photographic films presenting results of Southern blot analysis are presented in lower part of the figure. Bands corresponding to wild type genotype (wt) and mutant genotype (mut) are marked on the right side of each photograph.</p

Primers used in this study.

<p>Primers used in this study.</p

List of <i>M</i>. <i>smegmatis</i> strains used in this study.

<p>List of <i>M</i>. <i>smegmatis</i> strains used in this study.</p

Comparison of protein sequences of RNases H type I.

<p>Sequence alignment between RNases H type I of <i>E</i>. <i>coli</i> K12_MG1655 (RnhA), <i>M</i>. <i>smegmatis</i> mc² 155 (MSMEG4305 and RnhA) and <i>M</i>. <i>tuberculosis</i> H37Rv (Rv2228c) was performed using MultiAlin and visualized with ESPript 3.0. Highly similar or identical residues between protein sequences are written in bold. Identical residues across all analyzed sequences are shown in white on a black background. Similarities between protein sequences are marked by framing. The span of RNase H domains in each protein sequence, as defined by SMART, is highlighted in yellow.</p

Constitutive stable DNA replication of <i>E</i>. <i>coli</i> ∆<i>rnhA</i> mutants.

<p>An alternative mode of initiation of DNA replication was discovered in <i>E</i>. <i>coli</i> mutants lacking RNase H type I encoded by <i>rnhA</i>. (A,B) The initial strand opening involves RecA dependent hybridization of the RNA transcript to dsDNA, (C) The resulting R-loop is not resolved by RNase HI and therefore RNA persists on DNA strand and serves as a primer for elongation by PolI, (D,E) When the loop opens sufficiently, primosome is loaded on the leading strand. As replication continues, PolI removes persisting RNA transcript and primosome is loaded on the lagging strand.</p