Structure of the gene complementing uvr-402 in Streptococcus pneumoniae: homology with Escherichia coli uvrB and the homologous gene in Micrococcus luteus

Sicard, N; Oreglia, Jacqueline; Estevenon, Anne-Marie

doi:10.1128/jb.174.7.2412-2415.1992

Cited by 16 publications

(12 citation statements)

References 36 publications

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“…2, the amino acid sequence of T. thermophilus UvrB protein is similar to that of other UvrB proteins. Comparison of the T. thermophilus UvrB protein with those of E. coli (19), M. luteus (20), Streptococcus pneumoniae (26), and Neisseria gonorrhoeae (27) revealed identities of 54, 52, 55, and 52% and similarities of 68, 66, 69, and 67%, respectively. The finding of the uvrB gene in T. thermophilus suggests that a UvrABC-like nucleotide excision repair system is present in this extremely thermophilic bacterium.…”

Section: Resultsmentioning

confidence: 99%

ATPase Activity of UvrB Protein from Thermus thermophilus HB8 and Its Interaction with DNA

Kato

Yamamoto

Kito

et al. 1996

Journal of Biological Chemistry

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Many living organisms remove wide range of DNA lesions from their genomes by the nucleotide excision repair system. The uvrB gene, which plays an essential role in the prokaryotic excision repair, was cloned from an extremely thermophilic bacterium, Thermus thermophilus HB8. Its nucleotide sequence was determined, and the deduced amino acid sequence showed it possessed a helicase motif, including a nucleotide-binding consensus sequence (Walker's A-type motif), which was also conserved in other UvrB proteins. The prokaryotic UvrB proteins and eukaryotic DNA repair helicases (Rad3 and XP-D) were classified into different groups by molecular phylogenetic analysis. The T. thermophilus uvrB gene product was overproduced in Escherichia coli and purified to apparent homogeneity. The purified T. thermophilus UvrB protein was stable up to 80°C at neutral pH. T. thermophilus UvrB protein showed ATPase activity at its physiological temperature, whereas the E. All living organisms have DNA repair systems to counteract the many forms of DNA damage due to sunlight, chemical agents, or ionizing radiation (1). If such damage is not repaired, mutagenesis or even cell death may occur. The DNA repair systems involve in situ repair (photoreactivation), base excision repair, nucleotide excision repair, mismatch repair, and recombinational repair (1). Of these, the nucleotide excision repair system can deal with a wide range of DNA lesions. In Escherichia coli of typical prokaryote, ABC excinuclease, which is encoded by the uvrA, B, and C genes, plays a major role in this system (2-5). The molecular mechanism of the nucleotide excision repair system, studied mainly in E. coli, is as follows. First, UvrA, a damage recognition protein, makes an UvrA 2 B complex with UvrB (6) and then this complex binds to the site of the DNA lesion, forming a UvrA 2 B-DNA complex (6, 7), in which, the DNA is unwound, creating a so-called "open complex." Next, UvrA dissociates from the complex and only UvrB is bound to the DNA. Then, UvrC becomes associated with the complex, forming an UvrBC-DNA complex (6). At this point, UvrB incises the 3Ј side of the DNA lesion and UvrC incises the 5Ј side (8). Finally, DNA helicase II, DNA polymerase I, and DNA ligase complete the repair (5).In the above model, UvrA recognizes the damaged site in the DNA and guides UvrB to it. However, Hsu et al. (9) showed that UvrB can bind to DNA in the absence of UvrA. Although this finding indicates that UvrB plays a central role in ABC excinuclease activity, the details of the reaction mechanism are not clear. In order to understand the molecular mechanism of this enzyme's action in detail, enzymatic studies and physicochemical approaches, including x-ray crystallography and nuclear magnetic resonance, are required.Thermus thermophilus HB8 is an aerobic, rod-shaped, nonsporulating, Gram-negative eubacterium, which can grow at temperatures over 75°C (10). Although hyper-thermophilic bacteria can grow at temperatures over 100°C (such as Pyrococcus fuliosus, 11), T. thermophilu...

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Section: Resultsmentioning

confidence: 99%

ATPase Activity of UvrB Protein from Thermus thermophilus HB8 and Its Interaction with DNA

Kato

Yamamoto

Kito

et al. 1996

Journal of Biological Chemistry

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“…Emission spectra of both UvrB mutants show a blue shift in max , either relative to tryptophan in water (355 nm), or compared with spectra obtained following denaturation of the proteins in a 6 M guanidine hydrochloride buffer (yielding a 356-nm maximum for both mutants; spectra not shown). The shift, generally indicative of a more hydrophobic environment, is greater for Trp 47 (338.5 nm) than for Trp 51 (345 nm). The quantum yield for Trp 47 , 0.15, is slightly greater than that for tryptophan in water, 0.14 (38), and comparable with the difference seen in emission intensity between native and guanidine-denatured UvrB-F47W (12% greater in native buffer).…”

Section: Preparation Of "Tryptophan Reporter" Mutants and Proteins-rementioning

confidence: 90%

“…This suggests that amino acid residue 477 is a histidine (CAC codon) rather than an arginine (CGC). Histidine occupies this position in the UvrB sequences of Micrococcus luteus (46), Streptococcus pneumoniae (47), and Neisseria gonorrheae (48).…”

Section: Preparation Of "Tryptophan Reporter" Mutants and Proteins-rementioning

confidence: 99%

“…UvrB* can interact with UvrA and form a preincision complex, but one that is less stable and with greatly reduced capacity for incision with added UvrC. Tryptophan (absent in the wild-type protein) has been introduced without significant alteration of function into the UvrB ATP binding motif, both at a site shown earlier to be mutable without phenotypic effect (Asn 51 ) and at a residue nearer the essential and conserved lysine (Lys 45 ) of the Walker motif (Phe 47 ). Both "reporter mutants" reveal conformational changes, localized to the binding site, that accompany proteolytic activation.…”

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confidence: 99%

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Introduction of a Tryptophan Reporter Group into the ATP Binding Motif of the Escherichia coli UvrB Protein for the Study of Nucleotide Binding and Conformational Dynamics

Hildebrand

Grossman

1998

Journal of Biological Chemistry

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The DNA-dependent ATPase activity of UvrB is required to support preincision steps in nucleotide excision repair in Escherichia coli. This activity is, however, cryptic. Elicited in nucleotide excision repair by association with the UvrA protein, it may also be unmasked by a specific proteolysis eliminating the C-terminal domain of UvrB (generating UvrB*). We introduced fluorescent reporter groups (tryptophan replacing Phe 47 or Asn 51 ) into the ATP binding motif of UvrB, without significant alteration of behavior, to study both nucleotide binding and those conformational changes expected to be essential to function. The inserted tryptophans occupy moderately hydrophobic, although potentially heterogeneous, environments as evidenced by fluorescence emission and time-resolved decay characteristics, yet are accessible to the diffusible quencher acrylamide. Activation, via specific proteolysis, is accompanied by conformational change at the ATP binding site, with multiple changes in emission spectra and a greater shielding of the tryptophans from diffusible quencher. Titration of tryptophan fluorescence with ATP has revealed that, although catalytically incompetent, UvrB can bind ATP and bind with an affinity equal to that of the active UvrB* form (K d of ϳ1 mM). The ATP binding site of UvrB is therefore functional and accessible, suggesting that conformational change either brings amino acid residues into proper alignment for catalysis and/or enables response to effector DNA.

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“…It is mediated by the UvrABC excinuclease enzyme complex and the helicase UvrD (43), a system capable of dealing with a broad range of bulky and/or helix-distorting lesions, such as UV-induced photodimers and oxidized thymines, intraand interstrand cross-links, and other large base modifications formed through exposure to DNA-modifying agents (2). Analysis of NER in Bacillus subtilis (43), Streptococcus pneumoniae (47), Mycoplasma genitalium (43), and Deinococcus radiodurans (33) indicated that the repair mechanism originally characterized in E. coli is highly conserved in all prokaryotes. Interestingly, the mycobacterial UvrD1 protein has recently been shown to possess an additional role in DNA metabolism outside of NER; it was shown to physically and functionally interact with Ku, a protein participating in the nonhomologous-end-joining pathway of DNA double-strand break repair.…”

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confidence: 99%

Characterization of the Mycobacterial NER System Reveals Novel Functions of theuvrD1Helicase

et al. 2009

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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 success of Mycobacterium tuberculosis as a human pathogen lies to some extent in its ability to survive and replicate in macrophages (23). It is currently unknown how it manages to overcome the assault on its DNA by macrophagegenerated reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI), which represent an otherwise very effective defense against intracellular pathogens (5,8,35). One possibility is that M. tuberculosis effectively detoxifies ROI and RNI. Alternatively, it may possess highly effective DNA repair machineries. In silico analyses of mycobacterial genomes including M. tuberculosis (6, 34), Mycobacterium leprae (7), Mycobacterium bovis (15), Mycobacterium avium, Mycobacterium paratuberculosis, and Mycobacterium smegmatis (The Institute for Genome Research [http://www.tigr.org]) revealed the presence of genes encoding enzymes involved in DNA damage reversal, nucleotide excision repair (NER), base excision repair (BER), recombinational repair, nonhomologous end joining, and SOS repair. Surprisingly, mycobacteria are devoid of the mutLS-based postreplicative mismatch repair (MMR) system (34, 51), which is otherwise highly conserved throughout evolution and contributes to mutation avoidance by correcting replication errors resulting from nucleotide misincorporation and polymerase slippage (27,46). In addition, MMR inhibits recombination between nonidentical (homeologous) sequences and thus helps to control the fidelity of recombination (32, 41, 56).The finding that mycobacteria exhibit a general mutation rate comparable to that of MMR-proficient species suggests that they possess alternative or compensating strategies for mismatch recognition and MMR (51). It is conceivable, for example, that their replication and recombination fidelity is higher than in other prokaryotes. Alternatively, it is possible that the fidelity of these processes is controlled by one of the other DNA metabolic pathways, such as NER.Prokaryotic NER has been extensively studied in Escherichia coli. It is mediated by the UvrABC excinuclease enzyme complex and the helicase UvrD (43), a system cap...

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Structure of the gene complementing uvr-402 in Streptococcus pneumoniae: homology with Escherichia coli uvrB and the homologous gene in Micrococcus luteus

Cited by 16 publications

References 36 publications

ATPase Activity of UvrB Protein from Thermus thermophilus HB8 and Its Interaction with DNA

ATPase Activity of UvrB Protein from Thermus thermophilus HB8 and Its Interaction with DNA

Introduction of a Tryptophan Reporter Group into the ATP Binding Motif of the Escherichia coli UvrB Protein for the Study of Nucleotide Binding and Conformational Dynamics

Characterization of the Mycobacterial NER System Reveals Novel Functions of theuvrD1Helicase

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