Molecular and Functional Characterization of RecD, a Novel Member of the SF1 Family of Helicases, from Mycobacterium tuberculosis

Dewhare, Shivendra Singh; Umesh, T. G.; Muniyappa, K.

doi:10.1074/jbc.m114.619395

Cited by 4 publications

(8 citation statements)

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“…A point mutation at position 146 of the MtUvrB polypeptide from cysteine to alanine was generated by an overlapping PCR-based strategy in the M. tuberculosis uvrB gene. , First, two primers were designed by incorporating the mutation from cysteine to alanine at residue 146. Vector pET28a(+) harboring the MtuvrB gene was used as a template, and then two independent PCRs were performed.…”

Section: Methodsmentioning

confidence: 99%

“…Synthetic oligonucleotides used in this study are listed in Table S1, and DNA substrates are listed in Table . The ODNs were radiolabeled at the 5′ end by using [γ- 32 P]ATP and T4 polynucleotide kinase. , Subsequently, DNA substrates were annealed by mixing an appropriate combination of ODNs. For this purpose, stoichiometric amounts of purified ODNs were added to 100 μL of 0.3 M sodium citrate buffer (pH 7) containing 3 M NaCl.…”

Section: Methodsmentioning

confidence: 99%

“…Reaction mixtures containing 25 mM Tris-HCl (pH 7.5), 1 mM MgCl 2 , 2.5 mM DTT, the indicated 32 P-labeled substrate at 0.5 nM, 0.1 μg/mL BSA, and the indicated concentrations of MtUvrB were preincubated at 37 °C for 10 min as previously described . The reaction was started by the addition of 3 mM ATP and 5 nM unlabeled ODN, and incubation was continued for an additional 90 min.…”

Section: Methodsmentioning

confidence: 99%

“…The biotin/streptavidin assay was performed as previously described . Briefly, the reaction mixture contained 25 mM Tris-HCl (pH 7.5), 1 mM MgCl 2 , 2.5 mM DTT, 0.1 μg/mL BSA, 0.5 nM substrate, and 100 nM streptavidin.…”

Section: Methodsmentioning

confidence: 99%

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Mycobacterium tuberculosis UvrB Is a Robust DNA-Stimulated ATPase That Also Possesses Structure-Specific ATP-Dependent DNA Helicase Activity

2016

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Much is known about the Escherichia coli nucleotide excision repair (NER) pathway; however, very little is understood about the proteins involved and the molecular mechanism of NER in mycobacteria. In this study, we show that Mycobacterium tuberculosis UvrB (MtUvrB), which exists in solution as a monomer, binds to DNA in a structure-dependent manner. A systematic examination of MtUvrB substrate specificity reveals that it associates preferentially with single-stranded DNA, duplexes with 3' or 5' overhangs, and linear duplex DNA with splayed arms. Whereas E. coli UvrB (EcUvrB) binds weakly to undamaged DNA and has no ATPase activity, MtUvrB possesses intrinsic ATPase activity that is greatly stimulated by both single- and double-stranded DNA. Strikingly, we found that MtUvrB, but not EcUvrB, possesses the DNA unwinding activity characteristic of an ATP-dependent DNA helicase. The helicase activity of MtUvrB proceeds in the 3' to 5' direction and is strongly modulated by a nontranslocating 5' single-stranded tail, indicating that in addition to the translocating strand it also interacts with the 5' end of the substrate. The fraction of DNA unwound by MtUvrB decreases significantly as the length of the duplex increases: it fails to unwind duplexes longer than 70 bp. These results, on one hand, reveal significant mechanistic differences between MtUvrB and EcUvrB and, on the other, support an alternative role for UvrB in the processing of key DNA replication intermediates. Altogether, our findings provide insights into the catalytic functions of UvrB and lay the foundation for further understanding of the NER pathway in M. tuberculosis.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Mycobacterium tuberculosis UvrB Is a Robust DNA-Stimulated ATPase That Also Possesses Structure-Specific ATP-Dependent DNA Helicase Activity

2016

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“…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.…”

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confidence: 99%

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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A model of DNA unwinding dynamics by the RecBCD complex and its regulation by Chi recognition

Xie¹

2018

Journal of Theoretical Biology

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Molecular and Functional Characterization of RecD, a Novel Member of the SF1 Family of Helicases, from Mycobacterium tuberculosis

Cited by 4 publications

References 76 publications

Mycobacterium tuberculosis UvrB Is a Robust DNA-Stimulated ATPase That Also Possesses Structure-Specific ATP-Dependent DNA Helicase Activity

Mycobacterium tuberculosis UvrB Is a Robust DNA-Stimulated ATPase That Also Possesses Structure-Specific ATP-Dependent DNA Helicase Activity

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

A model of DNA unwinding dynamics by the RecBCD complex and its regulation by Chi recognition

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