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 (EMBOJ 18:6899-6907, 1999).  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.