Stabilization of the genome of the mismatch repair deficient Mycobacterium tuberculosis by context-dependent codon choice

Background: The rate at which a stretch of DNA mutates is determined by the cellular systems for DNA replication and repair, and by the nucleotide sequence of the stretch itself. One sequence feature with a particularly strong influence on the mutation rate are nucleotide repeats. Some microbial pathogens use nucleotide repeats in their genome to stochastically vary phenotypic traits and thereby evade host defense. However, such unstable sequences also come at a cost, as mutations are often deleterious. Here, we analyzed how these opposing forces shaped genome stability in the human pathogen Mycobacterium tuberculosis. M. tuberculosis lacks a mismatch repair system, and this renders nucleotide repeats particularly unstable. Results: We found that proteins of M. tuberculosis are encoded by using codons in a context-dependent manner that prevents the emergence of nucleotide repeats. This context-dependent codon choice leads to a strong decrease in the estimated frame-shift mutation rate and thus to an increase in genome stability. Conclusion: These results indicate that a context-specific codon choice can partially compensate for the lack of a mismatch repair system, and helps to maintain genome integrity in this pathogen

The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of action

Mycobacterium tuberculosis is an extremely well adapted intracellular human pathogen that is exposed to multiple DNA damaging chemical assaults originating from the host defence mechanisms. As a consequence, this bacterium is thought to possess highly efficient DNA repair machineries, the nucleotide excision repair (NER) system amongst these. Although NER is of central importance to DNA repair in M. tuberculosis, our understanding of the processes in this species is limited. The conserved UvrABC endonuclease represents the multi-enzymatic core in bacterial NER, where the UvrA ATPase provides the DNA lesion-sensing function. The herein reported genetic analysis demonstrates that M. tuberculosis UvrA is important for the repair of nitrosative and oxidative DNA damage. Moreover, our biochemical and structural characterization of recombinant M. tuberculosis UvrA contributes new insights into its mechanism of action. In particular, the structural investigation reveals an unprecedented conformation of the UvrB-binding domain that we propose to be of functional relevance. Taken together, our data suggest UvrA as a potential target for the development of novel anti-tubercular agents and provide a biochemical framework for the identification of small-molecule inhibitors interfering with the NER activity in M. tuberculosi

Investigation of DNA repair pathways and the impact of SMC protein on chromosome stability in mycobacteria

Annually, Mycobacterium tuberculosis is responsible for nearly two million deaths worldwide. Part of its success lies in its ability to survive and replicate in macrophages, but the molecular mechanisms allowing it to do so are not yet understood. Once activated, macrophages produce reactive oxygen (ROI) and nitrogen (RNI) species, which amongst other targets damage DNA. M. tuberculosis seems to be superbly adapted to these unfavourable conditions, which demand efficient mechanisms to ensure genome stability. Mycobacteria are naturally devoid of the highly conserved mismatch repair (MMR) system, a crucial mechanism for mutation avoidance. Other DNA repair strategies, such as recombinational repair, base excision repair (BER), or nucleotide excision repair (NER), may compensate for some of the functions accomplished by the MMR system. In this study, the role of the NER pathway in mycobacteria was investigated. The bacterial NER pathway consists of the UvrABC endonuclease enzyme complex and a helicase named UvrD. Knockout mutants in the excinuclease component (uvrA or uvrB, respectively) and in the helicase (uvrD) were constructed in M. tuberculosis and in M. smegmatis. In addition, double mutants lacking both components were generated. Analyses of the NER mutants revealed that the mycobacterial NER system repairs a wide range of mutagenic DNA alterations and is an important defence mechanism against oxidative and nitrosative damage. In a gene conversion assay, the capability of M. smegmatis NER mutants to recognise and subsequently reject different base-pairing errors was assessed. Inactivation of uvrB and uvrD increased marker integration frequencies to various extents, with in part – and dependent on the mismatch studied - synergistic effects in the combined uvrB / uvrD mutant. Our results imply that NER and particularly the helicase UvrD in part compensate for the lack of MMR in mycobacteria. We also found that the helicase component UvrD is essential for long-term survival of M. tuberculosis in vitro and in vivo. Besides DNA repair, other mechanisms contribute to ensuring genome integrity. DNA binding proteins that establish chromosome architecture presumably are involved in stabilising the bacterial genome during persistence and to accomplish proper cell division during reactivation. SMC (structural maintenance of chromosomes) proteins play fundamental roles in various aspects of chromosome organisation and dynamics, including repair of DNA damage. Mutant strains of M. smegmatis and M. tuberculosis defective in SMC were constructed. Surprisingly, inactivation of smc did not result in a recognizable phenotype. This is in contrast to data on smc null mutants in other species. Our observations suggest that the maintenance of mycobacterial chromosome organisation differs from that of described models. Taken together, our observations provide evidence that despite the lack of a MMR system, mycobacteria possess efficient defence mechanisms against DNA damage. We found that the NER system has a fundamental role in mutation avoidance and that the helicase UvrD has an additional role besides participation in NER. In addition, our data suggest unique mechanisms for chromosome organisation and partitioning in mycobacteria. Overall, the obtained findings indicate that mechanisms that govern genome stability in mycobacteria differ significantly from those of described model species. Jedes Jahr ist das Bakterium Mycobacterium tuberculosis für weltweit fast zwei Millionen Todesfälle verantwortlich. Diese Bilanz liegt zum Teil darin begründet, dass das Bakterium in Makrophagen überleben und sich replizieren kann, wobei die molekularen Mechanismen für diese Fähigkeit noch nicht aufgeklärt sind. Aktivierte Makrophagen bilden reaktive Sauerstoff- (engl. reactive oxygen species, ROI) und Stickstoffmoleküle (engl. reactive nitrogen species, RNI), die unter anderem DNA-Schäden hervorrufen. Mycobacterium tuberculosis scheint hervorragend an diese Bedingungen angepasst zu sein, welche effiziente Mechanismen zur Sicherung der Genomstabilität erfordern. Mykobakterien fehlt das äusserst konservierte Basenfehlpaarungsreparatursystem (engl. mismatch repair, MMR), welches ein wichtiger Mechanismus zur Vermeidung von Mutationen ist. Es wird vermutet, dass andere DNA-Reparaturstrategien, wie Reparatur durch homologe Rekombination, Basenexzisionsreparatur (engl. base excision repair, BER) oder Nukleotidexzisionsreparatur (engl. nucleotide excision repair, NER) manche Funktionen des MMR übernehmen könnten. In dieser Arbeit wurde die Funktion des NER in Mykobakterien untersucht. Der bakterielle Nukleotidexzisionsreparaturweg besteht aus dem UvrABC Endonukleaseenzymkomplex und der Helikase UvrD. Die Exzinukleasekomponente uvrA bzw. uvrB sowie die Helikase uvrD von Mycobacterium tuberculosis und Mycobacterium smegmatis wurden durch gezielten Genaustausch inaktiviert. Zusätzlich wurden Doppelmutanten generiert, denen beide Komponenten fehlen. Die Charakterisierung der NER Mutanten zeigte, dass das mykobakterielle NER-System viele Arten von mutagenen DNA-Veränderungen reparieren kann und einen wichtigen Abwehrmechanismus gegen oxidative und nitrosative DNA-Schäden darstellt. In einem Genkonversionsassay wurde untersucht, ob die M. smegmatis NER-Mutanten verschiedene Basenfehlpaarungen erkennen und aussondern können. Verlust von uvrB und uvrD führte zu signifikant erhöhten Genkonversionsfrequenzen, sowie teilweise – abhängig von der untersuchten Basenfehlpaarung – zu einem synergistischen Effekt bei der uvrB / uvrD Doppelmutante. Unsere Ergebnisse zeigen, dass das NER- System und besonders die Helikase UvrD dazu beitragen, das Fehlen des MMR-Systems in Mykobakterien zu kompensieren. Ausserdem konnten wir in vitro und in vivo zeigen, dass die Helikase UvrD essentiell für die Persistenz von M. tuberculosis ist. Neben der DNA-Reparatur sind weitere Mechanismen an der Gewährleistung der Genomintegrität beteiligt. Es wird angenommen, dass Chromosomenstrukturproteine wesentlich dazu beitragen, dass das Genom während der Persistenz stabilisiert wird und dass die Zellteilung während der Reaktivierung korrekt verläuft. SMC (structural maintenance of chromosomes) Proteine haben grundlegende Funktionen in vielen Aspekten der Chromosomenorganisation und Chromosomendynamik. Mycobacterium smegmatis und Mycobacterium tuberculosis smc Mutanten wurden durch gezielten Genaustausch generiert. Erstaunlicherweise prägten die smc Mutanten keinen ersichtlichen Phänotyp aus. Dieses Ergebnis steht im Gegensatz zu bisher beschriebenen smc Mutanten in anderen Organismen. Unsere Beobachtungen deuten darauf hin, dass die mykobakterielle Chromosomorganisation von beschriebenen Modellen abweicht. Die Ergebnisse dieser Arbeit lassen sich wie folgt zusammenfassen. Obwohl Mykobakterien das MMR-System fehlt, verfügen sie über effiziente Abwehrmechanismen zur Vermeidung von DNA-Schäden. Das NER-System hat eine bedeutende Funktion in der Vermeidung von Mutationen und die Helikase UvrD hat neben der Beteiligung im NER-System weitere Funktionen. Des Weiteren weisen unsere Beobachtungen auf aussergewöhnliche Mechanismen der Chromosomenorganisation und Chromosomenteilung in Mykobakterien hin. Die Resultate zeigen, dass die Mechanismen zur Erhaltung der Genomstabilität in Mykobakterien signifikant von denen in bisher beschriebenen Modellorganismen abweichen

A Mycobacterial smc Null Mutant Is Proficient in DNA Repair and Long-Term Survival▿

SMC (structural maintenance of chromosomes) proteins play fundamental roles in various aspects of chromosome organization and dynamics, including repair of DNA damage. Mutant strains of Mycobacterium smegmatis and Mycobacterium tuberculosis defective in SMC were constructed. Surprisingly, inactivation of smc did not result in recognizable phenotypes in hallmark assays characteristic for the function of these genes. This is in contrast to data for smc null mutants in other species

The Uracil DNA Glycosylase UdgB of Mycobacterium smegmatis Protects the Organism from the Mutagenic Effects of Cytosine and Adenine Deamination▿

Spontaneous hydrolytic deamination of DNA bases represents a considerable mutagenic threat to all organisms, particularly those living in extreme habitats. Cytosine is readily deaminated to uracil, which base pairs with adenine during replication, and most organisms encode at least one uracil DNA glycosylase (UDG) that removes this aberrant base from DNA with high efficiency. Adenine deaminates to hypoxanthine approximately 10-fold less efficiently, and its removal from DNA in vivo has to date been reported to be mediated solely by alkyladenine DNA glycosylase. We previously showed that UdgB from Pyrobaculum aerophilum, a hyperthermophilic crenarchaeon, can excise hypoxanthine from oligonucleotide substrates, but as this organism is not amenable to genetic manipulation, we were unable to ascertain that the enzyme also has this role in vivo. In the present study, we show that UdgB from Mycobacterium smegmatis protects this organism against mutagenesis associated with deamination of both cytosine and adenine. Together with Ung-type uracil glycosylase, M. smegmatis UdgB also helps attenuate the cytotoxicity of the antimicrobial agent 5-fluorouracil

Codons consisting of three identical nucleotides ('homogeneous codons') are avoided at positions followed by one or more nucleotides of the same type

The under-representation increases with increasing number of identical nucleotides following. Each line represents data for one type of nucleotide (indicated by the line color).<p><b>Copyright information:</b></p><p>Taken from "Stabilization of the genome of the mismatch repair deficient by context-dependent codon choice"</p><p>http://www.biomedcentral.com/1471-2164/9/249</p><p>BMC Genomics 2008;9():249-249.</p><p>Published online 28 May 2008</p><p>PMCID:PMC2430213.</p><p></p

The figure shows the ratio between total number of observed and expected mononucleotide repeats in coding (filled circles and solid line) and intergenic regions (open circles and dashed line)

The expected values have been calculated under the null model that nucleotides are randomly distributed in each of these two compartments, and occur at the frequencies observed in each compartment. For repeats of length three to six nucleotides, the under-representation of mononucleotide repeats is significantly stronger in coding regions than in intergenic regions (at p < 0.0001, chi square test).<p><b>Copyright information:</b></p><p>Taken from "Stabilization of the genome of the mismatch repair deficient by context-dependent codon choice"</p><p>http://www.biomedcentral.com/1471-2164/9/249</p><p>BMC Genomics 2008;9():249-249.</p><p>Published online 28 May 2008</p><p>PMCID:PMC2430213.</p><p></p

We estimated the frame-shift mutation rate in the real genomes, and in randomized genomes without context-dependent codon choice

The ratio between these two values is a measure for the degree by which context-dependent codon choice stabilizes the genome, and is displayed on the z-axis. Increasing lightness of the colors indicate increasing values on the z-axis. This ratio was calculated for different combinations of two biological parameters – the increase in the frame-shift mutation rate per nucleotide added to a mononucleotide repeat (parameter , x-axis), and the minimal length for a mononucleotide repeat to exhibit a significant frame-shift mutation rate (parameter , y-axis; see Methods). Published data suggest that in MMR-deficient bacteria is close to 3 and is about 4. For this combination, context-dependent codon choice leads to a decrease in the frame-shift mutation rate in mononucleotide repeats of a factor of about 20 in and of a factor of 3 in .<p><b>Copyright information:</b></p><p>Taken from "Stabilization of the genome of the mismatch repair deficient by context-dependent codon choice"</p><p>http://www.biomedcentral.com/1471-2164/9/249</p><p>BMC Genomics 2008;9():249-249.</p><p>Published online 28 May 2008</p><p>PMCID:PMC2430213.</p><p></p

Characterization of the Mycobacterial NER System Reveals Novel Functions of the uvrD1 Helicase▿

In this study, we investigated the role of the nucleotide excision repair (NER) pathway in mycobacterial DNA repair. Mycobacterium smegmatis lacking the NER excinuclease component uvrB or the helicase uvrD1 gene and a double knockout lacking both genes were constructed, and their sensitivities to a series of DNA-damaging agents were analyzed. As anticipated, the mycobacterial NER system was shown to be involved in the processing of bulky DNA adducts and interstrand cross-links. In addition, it could be shown to exert a protective effect against oxidizing and nitrosating agents. Interestingly, inactivation of uvrB and uvrD1 significantly increased marker integration frequencies in gene conversion assays. This implies that in mycobacteria (which lack the postreplicative mismatch repair system) NER, and particularly the UvrD1 helicase, is involved in the processing of a subset of recombination-associated mismatches

The lines depict the ratio between the observed and expected number of mononucleotide repeats (summed over all genes in the genome) as a function of their length

The expected numbers were calculated with a null-model that conserved the amino acid sequence and the gene-specific codon frequencies. The areas comprise 95% of the data from the randomized genomes. For repeats of three (for A, C and G) or six (for T) nucleotides and longer, the lines lie below this area, indicating that such repeats are significantly under-represented.<p><b>Copyright information:</b></p><p>Taken from "Stabilization of the genome of the mismatch repair deficient by context-dependent codon choice"</p><p>http://www.biomedcentral.com/1471-2164/9/249</p><p>BMC Genomics 2008;9():249-249.</p><p>Published online 28 May 2008</p><p>PMCID:PMC2430213.</p><p></p