Faulty DNA repair can lead to increased mutations, formation of cancers, and cell death. The process by which repair proteins find damaged bases within the DNA represents an important type of protein-DNA interaction, which is not well-understood. The UvrA, UvrB,and UvrC proteins work together to identify and remove DNA damage in a process called nucleotide excision repair. One of the most remarkable aspects of NER is that it can remove a wide range of DNA lesions that differ in chemistry and structure. The UvrABC proteins are believed to recognize the damage-induced distortion in the DNA helix rather than the lesion per se. However, detailed studies of the kinetics,thermodynamics and structural aspects of the Uvr proteins have been limited due to the lability and instability of the proteins. To overcome this problem we have recently cloned and overexpressed UvrA, UvrB,and UvrC from the thermophile, Bacillus caldotenax. The proteins maintain their activity at 65oC and are more amenable to structural and biophysical studies. Work is underway to understand the structure and function of these proteins using x-ray crystallography, stopped-flow fluorescence and site-directed mutagenesis. 1. Characterization of a beta-hairpin deletion mutant of UvrB. This motif is believed to be involved in DNA binding. UvrB plays a major role in recognition and processing of DNA lesions during nucleotide excision repair. The crystal structure of UvrB revealed a similar fold as found in monomeric DNA helicases. Homology modeling suggested that the b-hairpin motif of UvrB might be involved in DNA binding. To determine a role of the b-hairpin of Bacillus caldotenax UvrB, we have constructed a deletion mutant, Dbh UvrB, which lacks residues Q97-D112 of the b-hairpin. Dbh UvrB does not form a stable UvrB-DNA pre-incision complex and is inactive in UvrABC-mediated incision. However, Dbh UvrB is able to bind to UvrA and form a complex with UvrA, and damaged DNA, competing with wild type UvrB. In addition, Dbh UvrB shows wild type-like ATPase activity in complex with UvrA that is stimulated by damaged DNA. In contrast to wt UvrB, the ATPase activity of mutant UvrB does not lead to a destabilization of the damaged duplex. These results indicate that the conserved b-hairpin motif is a major factor in DNA binding. 2. Characterization of the UvrA protein from Bacillus caldotenax. The UvrA gene, which plays an essential role in the prokaryotic nucleotide excision repair, was cloned from a thermophilic eubacterium, Bacillus caldotenax (Bca), and its nucleotide sequence was determined. The nucleotide sequence showed 71% identity with that of the B. subtilis uvrA gene. The deduced amino acid sequence contains a characteristic duplicated structure, including two Walker A-type ATP-binding sites and two zinc finger DNA-binding motifs. The predicted amino acid sequence of Bca UvrA protein showed ~82% and ~62% identity with that of B. subtilis and E. coli, respectively. The Bca UvrA protein was purified to apparent homogeneity and showed thermostability at 65oC for extended periods of time, a DNA-stimulated ATPase, and complements E.coli UvrB and UvrC in an in vitro incision reaction. While Bca UvrA binds damaged DNA 3-4 times less efficiently than Eco UvrA, Bca UvrA protein promotes efficient loading of UvrB and supports higher amounts of incision. These results provide evidence for complementation of E. coli UvrA by B. caldotenax UvrA both in vitro and in vivo, indicating a remarkable evolutionary conservation of nucleotide excision repair systems 3. A molecular model for the human nucleotide excision repair protein, XPD, was developed based on the protein?s structural and functional relationship with a bacterial nucleotide excision repair protein, UvrB. While XPD does not share significant sequence identity with UvrB, the proteins share an evolutionary relationship in possessing seven highly conserved helicase motifs that define a common protein structural template. They also have similar functional roles in their ATPase activity and ability to unwind DNA in the process of NER. The validity of using the crystal structure of UvrB as a template for the development of an XPD model was tested by mimicking human disease causing mutations, (XPD: R112H, D234N, R601L) in UvrB (E110R, D338N, R506A), and mutation of conserved residues (XPD: H237, D609; UvrB: H341A, D510A). The XPD structural model can be employed in understanding the molecular mechanism of XPD human disease causing mutations. The value of this XPD model demonstrates the power of the generalized approach for the prediction of the structure of a mammalian protein based on the crystal structure of an orthologous bacterial protein. Cross-linking UvrA, UvrB and UvrC to DNA adducts. We are taking two complementary approaches to identify the domains of the proteins that are making contact at or near the DNA lesion. The first approach uses the reactivity of azidophenacyl bromide to phosphothioates to introduce azidophenacyl into any sequence context. Our goal is to move this cross linking reagent in both the 5 and 3 directions, relative to where the DNA lesion is, and cross link UvrB to DNA then submit the UvrB-DNA for sequence analysis. To date, the efficiency of cross-linking as a function of compound concentration, UV fluence and wavelength have been tested. In preliminary studies, we have identified the reaction conditions that are necessary for proper UvrA mediated loading of UvrB onto DNA. We have tested several UvrB mutants and compared the cross linking results we obtained using the Electrophoretic Mobility Shift Assay (EMSA). Our cross-linking results agreed with the previous results of the EMSA assays and unequivocally demonstrated that the DNA was in contact with UvrB within the UvrA2B complex that was detected by the EMSA assay. We have also demonstrated that we can cross-link all of the DNA repair components UvrA, UvrB and UvrC, albeit with different efficiencies. UvrA cross-links to both single- and double-stranded DNA. At high concentrations of UvrB it will cross-link to single-stranded DNA but not double-stranded DNA. However, at low concentrations of UvrB, UvrA readily loads UvrB onto double-stranded DNA and we can detect a UvrB:DNA cross-link. In addition, UvrC can be cross-linked to both single- and double-stranded DNA. A second novel approach utilizes a novel cross-linking reagent (through collaboration with Dr. Olga I. Lavrik, Novosibirsk Institute, Russia) to """"""""trap"""""""" UvrA, UvrB, and UvrC in their natural proximity to damaged DNA. This unique reagent, 5-{[N-(4-azidotetrafluorobenzylideneaminooxy)-methylcarbamoyl]-trans-3-aminopropenyl-1]-2?-deoxyuridine-5?-triphosphate} (lithium salt) (FABC-dUTP), when incorporated into a double stranded oligonucleotide, serves as both the DNA lesion and the cross-linking agent. It was found that incision of a 50 bp substrate containing this adduct is ~60-70% which is as good as the best substrates tested in the laboratory. Approximately 15-25% of the DNA (32P-labeled) was observed to cross-link to UvrA . The addition of UvrB to the reaction mixture at concentrations ranging from 200 nM to 2 ?M (UvrA:UvrB 1:1 ? 1:10) caused the overall cross linking of UvrA to decrease with increasing concentration of UvrB. The highest percentage of UvrB cross-linked in the presence of UvrA and 2 nM DNA was approximately 9%. UvrC only (5 ?M) was also cross-linked in the presence of 2 nM DNA (7%). Structure-function studies of UvrB.
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