DNA-Dependent ATPase Activity of Bacterial XPB Helicases

Biswas, Tapan; Pero, Jessica M; Joseph, Caleb G.; Tsodikov, Oleg V.

doi:10.1021/bi8022416

Cited by 25 publications

(26 citation statements)

References 55 publications

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“…1A showed the absorption spectra of standard sodium dihydrogen phosphate, in complex with dye reagent mentioned above, from the wavelength of 550e750 nm. The spectra exhibited a characteristic absorption peak approximately at 650 nm, which deviated slightly from previously proposed 620 nm [18,19] and 630 nm [20]. Then the standard curve was determined in the 0e300 mM range of sodium dihydrogen phosphate (Fig.…”

Section: Optimization Of the Parameters Of Malachite Green Methodsmentioning

confidence: 68%

ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method

Yang

Wang

2016

Analytical Biochemistry

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Section: Optimization Of the Parameters Of Malachite Green Methodsmentioning

confidence: 68%

ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method

Yang

Wang

2016

Analytical Biochemistry

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“…The TFIIH complex comprises up to 10 protein subunits and contains two helicases: XPB and XPD. XPB is missing in the genomes of many prokaryotes, but several actinobacterial species, including M. leprae, M. tuberculosis and Kineococcus radiotolerans express a XPB homolog [10], [11]. In contrast, the TFIIH XPD/ERCC2 homolog DinG, (damage-inducible G) is commonly expressed in many bacterial species [12].…”

Section: Introductionmentioning

confidence: 99%

“…Bacterial XPB also contains the main helicase core and a shorter N-terminal domain, but lacks the C-terminal domain [10], [11]. The N-terminal domain of eukaryotic XPB interacts with the p52 subunit of TFIIH which stimulates the ATPase activity of XPB [13].…”

Section: Introductionmentioning

confidence: 99%

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Enzymatic Activities and DNA Substrate Specificity of Mycobacterium tuberculosis DNA Helicase XPB

et al. 2012

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XPB, also known as ERCC3 and RAD25, is a 3′→5′ DNA repair helicase belonging to the superfamily 2 of helicases. XPB is an essential core subunit of the eukaryotic basal transcription factor complex TFIIH. It has two well-established functions: in the context of damaged DNA, XPB facilitates nucleotide excision repair by unwinding double stranded DNA (dsDNA) surrounding a DNA lesion; while in the context of actively transcribing genes, XPB facilitates initiation of RNA polymerase II transcription at gene promoters. Human and other eukaryotic XPB homologs are relatively well characterized compared to conserved homologs found in mycobacteria and archaea. However, more insight into the function of bacterial helicases is central to understanding the mechanism of DNA metabolism and pathogenesis in general. Here, we characterized Mycobacterium tuberculosis XPB (Mtb XPB), a 3′→5′ DNA helicase with DNA-dependent ATPase activity. Mtb XPB efficiently catalyzed DNA unwinding in the presence of significant excess of enzyme. The unwinding activity was fueled by ATP or dATP in the presence of Mg2+/Mn2+. Consistent with the 3′→5′ polarity of this bacterial XPB helicase, the enzyme required a DNA substrate with a 3′ overhang of 15 nucleotides or more. Although Mtb XPB efficiently unwound DNA model substrates with a 3′ DNA tail, it was not active on substrates containing a 3′ RNA tail. We also found that Mtb XPB efficiently catalyzed ATP-independent annealing of complementary DNA strands. These observations significantly enhance our understanding of the biological roles of Mtb XPB.

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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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DNA-Dependent ATPase Activity of Bacterial XPB Helicases

Cited by 25 publications

References 55 publications

ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method

ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method

Enzymatic Activities and DNA Substrate Specificity of Mycobacterium tuberculosis DNA Helicase XPB

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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