Characterization of the Mycobacterial NER System Reveals Novel Functions of the<i>uvrD1</i>Helicase

Güthlein, Carolin; Wanner, Roger M.; Sander, Peter; Davis, E. A.; Bosshard, Martin; Jiricny, Josef; Böttger, Erik C.; Springer, Burkhard

doi:10.1128/jb.00216-08

Cited by 34 publications

(28 citation statements)

References 56 publications

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“…Allelic replacement techniques were used to generate M. smegmatis knockout mutants (4). Using genomic DNA, 1-to 1.5-kb fragments upstream (5Ј) and downstream (3Ј) of the target genes ung (MSMEG_2399) and udgB (MSMEG_5031) were amplified by PCR and cloned into pMCS5-rpsL-hyg.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2009

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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.

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Section: Methodsmentioning

confidence: 99%

“…The vectors were transformed into M. smegmatis mc 2 155 derivative SMR5 rrnB as previously described (4).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2009

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“…Thus, a promoter matching the consensus for SigA is responsible for at least 95% of the expression of lexA and the remaining 5% of expression is not due to SigG. The sensitivity of the wild-type and ⌬sigG2 strains to DNA damage caused by UV irradiation and different DNA-damaging drugs was assessed as described previously (3,13). Survival rates after exposure to different doses of UV irradiation were similar for the ⌬sigG and the wild-type strains; in contrast, enhanced susceptibility was seen with a uvrB mutant used as a control (Fig.…”

Section: Vol 193 2011mentioning

confidence: 99%

SigG Does Not Control Gene Expression in Response to DNA Damage in Mycobacterium tuberculosis H37Rv

Smollett¹,

Dawson²,

Davis³

2011

J Bacteriol

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Expression of the Mycobacterium tuberculosis sigG sigma factor was induced by a variety of DNA-damaging agents, but inactivation of sigG did not affect induction of gene expression or bacterial survival under these conditions. Therefore, SigG does not control the DNA repair response of M. tuberculosis H37Rv.Mycobacterium tuberculosis has at least two mechanisms regulating gene expression following DNA damage (6): the SOS response mediated by LexA and RecA (5,9,17) and an alternative mechanism independent of both RecA and LexA (6, 21). It is not known what controls the RecA-independent response, although a potential promoter motif common to a number of genes in this regulon that suggests the possible involvement of an alternative sigma factor has been identified (11). Of the 13 sigma factors in M. tuberculosis (2), SigG was the most highly induced following DNA damage and macrophage infection (1, 21). Moreover, a recent study suggested that SigG was responsible for transcription of lexA in a clinical isolate, CDC1551 (14).Here, we addressed the role of SigG in M. tuberculosis H37Rv in response to DNA damage by comparing the global transcriptional profile of a sigG mutant strain with that of the wild type. Our results indicate that the absence of SigG does not affect the induction of known DNA damage response genes. In addition, we could find no evidence for the involvement of SigG in the transcription of lexA in vivo. Furthermore, the sigG mutant was no more susceptible to DNA damage than the parental strain. Therefore, we conclude that SigG does not control expression of either the RecA-dependent or the RecAindependent genes in response to DNA damage in M. tuberculosis H37Rv.A potential role for SigG in regulating the response to DNA damage was initially proposed based on its induction following exposure to mitomycin C (21), which causes interstrand crosslinks and alkylation damage. Basing our analysis on the pelleting properties of the bacteria, we suspect mitomycin C also affects the mycobacterial cell surface. Therefore, we measured the levels of sigG mRNA following other treatments predicted to damage DNA, namely, bleomycin exposure, ofloxacin exposure, and UV irradiation, resulting in breaks in DNA, inhibition of DNA gyrase, and induction of the formation of thymine dimers, respectively. The effect of cell surface stress on sigG expression was also assessed by treatment with ethambutol and exposure to sodium dodecyl sulfate (SDS). M. tuberculosis H37Rv cultures (See Table 1 for strains and plasmids used) were grown under Advisory Committee on Dangerous Pathogens (ACDP) containment level 3 conditions as described previously (6). Cultures were divided into aliquots at an optical density at 600 nm (OD 600 ) of 0.3 to 0.4; samples were induced with mitomycin C (0.2 g ml Ϫ1 ), ofloxacin (7.5 g ml Ϫ1 ), or bleomycin (1.5 g ml Ϫ1 ) for 5 h at 37°C, exposed to 23.5 J m Ϫ2UV irradiation followed by 5 h of recovery at 37°C, or induced with SDS (0.05% for 5 h at 37°C) or ethambutol (15 g ml Ϫ1for 24 h at 37°C). Anothe...

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“…The findings that HelY ATPase is unresponsive to a DNA polynucleotide cofactor and that HelY is unable to unwind a 3=-tailed duplex in which the loading strand is DNA distinguish HelY from all other mycobacterial NTPases/helicases characterized previously. Whereas many of the other mycobacterial NTPases/helicases are implicated biochemically and genetically in DNA replication, recombination, or repair (2,5,7,14,26,27,28,29), the biochemical activities of HelY are consistent with a role in mycobacterial RNA metabolism.…”

Section: Discussionmentioning

confidence: 88%

Mycobacterium smegmatis HelY Is an RNA-Activated ATPase/dATPase and 3′-to-5′ Helicase That Unwinds 3′-Tailed RNA Duplexes and RNA:DNA Hybrids

2015

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Mycobacteria have a large and distinctive ensemble of DNA helicases that function in DNA replication, repair, and recombination. Little is known about the roster of RNA helicases in mycobacteria or their roles in RNA transactions. The 912-amino-acid Mycobacterium smegmatis HelY (MSMEG_3885) protein is a bacterial homolog of the Mtr4 and Ski2 helicases that regulate RNA 3= processing and turnover by the eukaryal exosome. Here we characterize HelY as an RNA-stimulated ATPase/dATPase and an ATP/dATP-dependent 3=-to-5= helicase. HelY requires a 3= single-strand RNA tail (a loading RNA strand) to displace the complementary strand of a tailed RNA:RNA or RNA:DNA duplex. The findings that HelY ATPase is unresponsive to a DNA polynucleotide cofactor and that HelY is unable to unwind a 3=-tailed duplex in which the loading strand is DNA distinguish HelY from other mycobacterial nucleoside triphosphatases/helicases characterized previously. The biochemical properties of HelY, which resemble those of Mtr4/Ski2, hint at a role for HelY in mycobacterial RNA catabolism. H elicases are nucleic acid-dependent nucleoside triphosphatases (NTPases) that play important roles in diverse nucleic acid transactions. DNA helicases orchestrate replication, recombination, and repair. RNA helicases choreograph transcription, RNA processing, ribosome biogenesis, translation, and RNA turnover. Helicases use the chemical energy of nucleoside triphosphate (NTP) hydrolysis either to effect mechanical changes in the secondary structure of nucleic acids or to remodel (or disrupt) the structures of protein-nucleic acid complexes. Helicases are classified into superfamilies, families, and subfamilies according to their distinctive primary, tertiary, and quaternary structures and their biochemical specificities, i.e., NTP preference, nucleic acid preference (duplex DNA, duplex RNA, or RNA:DNA hybrids), and directionality of translocation or unwinding (5= to 3= or 3= to 5=) (1).Differences in the roster of helicases between taxa offer useful clues to the evolution and diversification of DNA replication/repair and RNA synthesis/processing strategies. Where the helicase rosters diverge in animals versus pathogens, they can suggest antiinfective drug targets. With this in mind, we are focused on the helicases of members of the genus Mycobacterium, a genus of the phylum Actinobacteria that includes the human pathogen Mycobacterium tuberculosis and its avirulent relative, M. smegmatis. To date, we have purified and characterized six mycobacterial DNA helicases: AdnAB (2, 3, 4), UvrD1 (5, 6), UvrD2 (7), SftH (8), RqlH (9), and Lhr (10). Mycobacterial DNA helicases XBP (11, 12), RecG (13,14,15), RecD (16), RuvB (17), and DnaB (18) have been purified and characterized by other investigators.Comparatively little attention has been paid to the mycobacterial RNA helicase repertoire. The transcription termination factor Rho is, to our knowledge, the lone example of a biochemically characterized NTP-driven RNA translocase/helicase from a Mycobacterium spec...

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Characterization of the Mycobacterial NER System Reveals Novel Functions of theuvrD1Helicase

Cited by 34 publications

References 56 publications

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

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

SigG Does Not Control Gene Expression in Response to DNA Damage in Mycobacterium tuberculosis H37Rv

Mycobacterium smegmatis HelY Is an RNA-Activated ATPase/dATPase and 3′-to-5′ Helicase That Unwinds 3′-Tailed RNA Duplexes and RNA:DNA Hybrids

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