Identification of Residues within UvrB That Are Important for Efficient DNA Binding and Damage Processing

Skorvaga, Milan; DellaVecchia, Matthew J.; Croteau, Deborah L.; Theis, Karsten; Truglio, J.J.; Mandavilli, Bhaskar S.; Kisker, Caroline; Houten, Bennett Van

doi:10.1074/jbc.m409266200

Cited by 37 publications

(55 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…No stable preincision complex of UvrB⅐DNA was observed here (Fig. S4), consistent with previous work (28). Because the mutant UvrBY96A has lost its damage-specific recognition ability and strand-destabilizing activity, the observed DNA wrapping of UvrBY96A may suggest that DNA wrapping takes place before damage-specific recognition, indicating that DNA wrapping may be involved in the process of searching for the damage along DNA.…”

Section: Dna Wrapping Of Mutant Uvrb Proteinssupporting

confidence: 79%

“…Mutant UvrBY96A can bind UvrA, can form the UvrAB⅐DNA complex, and has high DNA-stimulated ATPase, but it exhibits defects in strand-destabilizing activity (28). No stable preincision complex of UvrB⅐DNA was observed here (Fig.…”

Section: Dna Wrapping Of Mutant Uvrb Proteinsmentioning

confidence: 80%

“…The E. coli UvrA, UvrB, and UvrC proteins were produced and purified as previously described (34), and B. caldotenax UvrA, wtUvrB, UvrBY96A, and UvrB⌬4 proteins were prepared following published protocols (28,30,35). The damage probes and the oligonucleotides for their synthesis are listed in Tables S1 and S2.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

DNA wrapping is required for DNA damage recognition in the Escherichia coli DNA nucleotide excision repair pathway

Wang

Lü

Tang

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Localized DNA melting may provide a general strategy for recognition of the wide array of chemically and structurally diverse DNA lesions repaired by the nucleotide excision repair (NER) pathway. However, it is not clear what causes such DNA melting and how it is driven. Here, we show a DNA wrapping-melting model supported by results from dynamic monitoring of the key DNA-protein and protein-protein interactions involved in the early stages of the Escherichia coli NER process. Using an analytical technique involving capillary electrophoresis coupled with laser-induced fluorescence polarization, which combines a mobility shift assay with conformational analysis, we demonstrate that DNA wrapping around UvrB, mediated by UvrA, is an early event in the damage-recognition process during E. coli NER. DNA wrapping of UvrB was confirmed by Fö rster resonance energy transfer and fluorescence lifetime measurements. This wrapping did not occur with readily denaturable damaged DNA substrates (''bubble'' DNA), suggesting that DNA wrapping of UvrB plays an important role in the induction of DNA melting around the damage site. Analysis of DNA wrapping of mutant UvrB Y96A further suggests that a cooperative interaction between DNA wrapping of UvrA2B and contact of the ␤-hairpin of UvrB with the bulky damage moiety may be involved in the local DNA melting at the damage site.capillary electrophoresis ͉ laser induced fluorescence polarization ͉ Bacillus caldotenax ͉ fluorescence resonance energy transfer N ucleotide excision repair (NER) displays a unique capability of recognizing and processing a broad spectrum of bulky lesions, which are chemically and structurally diverse (1, 2). In Escherichia coli, the complex of UvrA and UvrB is responsible for damage search and recognition along DNA (1, 3). A number of models have been proposed to explain the repair of the broad spectrum of substrates acted on by the E. coli NER, including the helicasescanning model (4-6), the damage-processing model (7), and the padlock model (8, 9). The key difference among these models is the mechanism of damage recognition. In the helicase-scanning model, the UvrA 2 B complex locates damage through a helicase-driven translocation step. In the damage-processing model, the UvrA 2 B complex finds damage by random diffusion. Accumulating evidence shows that local DNA melting around the damage site induced by the UvrAB complex provides a structural basis for efficient incision of damaged nucleotides by the UvrB and UvrC proteins (10-12). These observations form the basis for the padlock model. Recently, crystal structure analysis of UvrB-DNA complexes has provided evidence that also supports the padlock model (13,14). However, the mechanism responsible for DNA melting remains to be elucidated. It has been proposed that DNA wrapping around NER proteins may be pertinent to the recognition of the broad spectrum of repairable substrates (15-17). There is a need to confirm this proposition with experiments conducted under dynamic conditions.In the present study, ...

show abstract

Section: Dna Wrapping Of Mutant Uvrb Proteinssupporting

confidence: 79%

Section: Dna Wrapping Of Mutant Uvrb Proteinsmentioning

confidence: 80%

See 1 more Smart Citation

DNA wrapping is required for DNA damage recognition in the Escherichia coli DNA nucleotide excision repair pathway

Wang

Lü

Tang

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…While UvrB does contain helicase domains, ATP hydrolysis by UvrB is only associated with limited DNA unwinding (29). UvrB's helicase action is therefore more accurately described as 'helix destabilization' (30), and may fulfill a role similar to that of the weak eukaryotic helicase XPB. UvrB therefore locally distorts the DNA at lesion sites, which promotes the binding and endonuclease activity of UvrC (21).…”

Section: Bacterial Ner: No Gimmicksmentioning

confidence: 99%

Nucleotide Excision Repair in the Three Domains of Life

Farnell¹

2011

WURJ:HNS

View full text Add to dashboard Cite

Nucleotide excision repair (NER) is a vital DNA repair pathway which acts on a wide range of helixdistorting lesions. The importance of this pathway is highlighted by its functional conservation throughout evolution and by several human diseases, such as xeroderma pigmentosum, which are caused by a defective NER pathway. This review summarizes the NER mechanisms present in all three domains of life: eukaryotes, bacteria, and archaea.

show abstract

“…29 and 30). Secondary DNA damage recognition by UvrB is thought to be achieved by ATP-driven translocation of UvrB (31,32), with damaged nucleotides sterically hindering this translocation process (33)(34)(35)(36)(37). However, the UvrAB complex has very limited strand displacement activity (32) which probably reflects a strand opening function rather than processive translocation in the manner of a typical helicase.…”

mentioning

confidence: 99%

Stimulation of UvrD Helicase by UvrAB

Atkinson

Guy

Cadman

et al. 2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

Helicases play critical roles in all aspects of nucleic acid metabolism by catalyzing the remodeling of DNA and RNA structures. UvrD is an abundant helicase in Escherichia coli with well characterized functions in mismatch and nucleotide excision repair and a possible role in displacement of proteins such as RecA from single-stranded DNA. Helicases and translocases use the energy derived from NTP hydrolysis to translocate along single-stranded or doublestranded nucleic acids to remodel nucleic acid structures. Many of these enzymes have RecA-like motor domains, reflecting close similarities in translocation mechanisms (1-3). Specificity is often, therefore, conferred by additional domains within these motor enzymes (4). It is also becoming apparent that helicases and translocases often function as part of larger multisubunit complexes rather than in isolation and that such interactions impact on motor function and specificity. The complex interactions between replicative helicases and other proteins acting at the replication fork have long been known to be critical for replisome function (5-7). Many helicases also interact with, and their activities modulated by, ssDNA 2 -binding proteins (8 -13), while helicase/translocase interactions with RNA polymerases are emerging (14, 15).The Escherichia coli 3Ј-5Ј helicase UvrD (16) has roles in mismatch (17) and nucleotide excision repair (18) and may also act to displace proteins such as RecA at replication forks or ssDNA gaps in duplex DNA (19 -21). UvrD is likely the most abundant helicase in E. coli (22). There is also a second helicase in E. coli, Rep, that shares 40% identity with UvrD but has no known role in mismatch or nucleotide excision repair or in protein displacement in vivo. Employment of UvrD, but not Rep, in a diversity of roles in vivo might, therefore, demand specific physical or functional interactions between UvrD and other proteins in each system. However, specific interaction of UvrD has only been documented with a component of the mismatch repair system, MutL (23). This interaction appears to be essential in allowing a motor with limited dsDNA processivity to unwind the large tracts of DNA necessary during mismatch repair (23)(24)(25)(26).Little is known concerning the protein displacement function of UvrD in vivo. Lack of UvrD leads to a hyperrecombination phenotype (27), whereas UvrD can both promote (20,21) and inhibit (20) RecA-catalyzed strand exchange in vitro, depending on reaction conditions. UvrD might, therefore, promote turnover of RecA-ssDNA complexes at blocked forks and gaps in duplex DNA. Inhibition of RecA function at blocked forks might facilitate other pathways of fork repair that do not rely on strand exchange, possibly minimizing the risks to genome stability that blocked forks present (28). However, whether abortion of strand exchange by UvrD in vivo requires other proteins is unknown. In contrast, the role of UvrD in nucleotide excision repair is well characterized. Nucleotide excision repair is needed to remove and replace ...

show abstract

Identification of Residues within UvrB That Are Important for Efficient DNA Binding and Damage Processing

Cited by 37 publications

References 14 publications

DNA wrapping is required for DNA damage recognition in the Escherichia coli DNA nucleotide excision repair pathway

DNA wrapping is required for DNA damage recognition in the Escherichia coli DNA nucleotide excision repair pathway

Nucleotide Excision Repair in the Three Domains of Life

Stimulation of UvrD Helicase by UvrAB

Contact Info

Product

Resources

About