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DNA Damage Recognition by the Uvrabc Nuclease System Bennett Van Houten A,∗, Deborah L

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DNA Damage Recognition by the Uvrabc Nuclease System Bennett Van Houten A,∗, Deborah L Mutation Research 577 (2005) 92–117 Review ‘Close-fitting sleeves’: DNA damage recognition by the UvrABC nuclease system Bennett Van Houten a,∗, Deborah L. Croteau a, Matthew J. DellaVecchia a, Hong Wang a, Caroline Kisker b a Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, 111 Alexander Drive, MD D3-01, Research Triangle Park, NC 27709, USA b Department of Pharmacology, Center for Structural Biology, SUNY, Stony Brook, NY, USA Received 28 January 2005; received in revised form 11 March 2005; accepted 11 March 2005 Available online 31 May 2005 Abstract DNA damage recognition represents a long-standing problem in the field of protein–DNA interactions. This article reviews our current knowledge of how damage recognition is achieved in bacterial nucleotide excision repair through the concerted action of the UvrA, UvrB, and UvrC proteins. Published by Elsevier B.V. Keywords: Protein–DNA interaction; Bacterial nucleotide excision repair; DNA damage recognition Contents Prologue ................................................................................................. 93 1. Introduction .............................................................................................. 93 2. Discovery of the genes .................................................................................... 93 3. General mechanism ....................................................................................... 94 4. Grasping the substrates: structures of some key DNA adducts.................................................. 94 5. UvrA .................................................................................................... 98 5.1. UvrA zinc fingers: DNA binding motifs ............................................................... 98 5.2. Structure and function of ATP binding domains ........................................................ 99 6. UvrB ................................................................................................... 100 6.1. UvrB crystal structure reveals a helicase fold ......................................................... 100 6.2. Role of domain 2 and the ␤-hairpin for UvrA interaction and its effect on UvrB’s ATPase activity.......... 100 ∗ Corresponding author. Tel.: +1 919 5417752; fax: +1 919 5417593. E-mail address: [email protected] (B. Van Houten). 0027-5107/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.mrfmmm.2005.03.013 B. Van Houten et al. / Mutation Research 577 (2005) 92–117 93 6.3. Assessing the DNA hand-off from UvrA to UvrB using photoaffinity DNA substrates .................... 103 6.4. UvrB as a model for the human repair protein, XPD................................................... 105 7. UvrC ................................................................................................... 105 7.1. Coordination between damage recognition and UvrC incision .......................................... 105 7.2. Catalytic mechanism of UvrC....................................................................... 106 7.3. UvrC homolog .................................................................................... 107 8. DNA damage recognition and processing by UvrA, UvrB and UvrC .......................................... 107 9. So what remains hidden higher up the sleeve: what is the nature of repair in vivo? .............................. 111 Acknowledgements ...................................................................................... 111 References .............................................................................................. 111 Prologue define the parameters that modulate DNA damage One of the key aspects of science is problem recognition, but large gaps in our knowledge still ex- definition. A few seminal papers in DNA repair and ist. In this article, we first explore the nature of the protein–DNA interactions have influenced much of my substrates that are acted on by the UvrABC system, thinking in DNA damage recognition. In particular, and then discuss each of the subunits in some detail. two papers by Hanawalt and coworkers [1,2] have had We consider how these three components work in con- a lasting impression on me. In 1965, Philip Hanawalt cert to identify and remove damage. Finally, we outline and Robert Haynes outlined an idea for damage what information is still beyond our grasp. recognition that was both elegant and prophetic: After observing that the Escherichia coli nucleotide excision repair system could act on a wide variety of DNA 2. Discovery of the genes adducts, they stated, “The recognition step in the repair mechanism could be formally equivalent to threading In the early days of DNA repair, investigators stud- the DNA through a close fitting ‘sleeve’ which gauges ied UV-induced mutagenesis and cell death in bacte- the closeness-of-fit to the Watson-Crick structure” ria. From these studies came the seminal observations [1]. The following review is dedicated to Professor that UV-induced DNA lesions were excised from DNA Hanawalt’s rich and lasting impact on my career as [3,4] and that repair synthesis occurred after excision my laboratory has struggled to understand what I have [5]. In time, the process collectively became known called the damage recognition problem: how does a as nucleotide excision repair (NER). The individual protein machine, made up of multiple protein subunits, bacterial genes responsible for the nucleotide excision first identify altered bases in a sea of non-damaged repair proteins were later discovered by complemen- DNA, and secondly, after marking the site for the tation studies, curiously, in exactly the same order in dual endonuclease activity of UvrC, faithfully remove which they operate in the NER pathway [6]. and replace a damaged stretch of 12–13 nucleotides? The NER genes are widely dispersed along the Professor Hanawalt has been a shining beacon of inspi- bacterial chromosome. The uvrA and uvrB genes are ration and enthusiasm, and I am greatly indebted to his under the control of the SOS response (for a review mentorship throughout my career. This review high- see [7]) while the uvrC gene is not. The SOS system lights recent work from my and several laboratories, but governs the up regulation of numerous genes in is not intended to be comprehensive, and I apologize response to DNA damaging agents. This is significant in advance for any pertinent work that is not cited. because the UvrA protein is normally expressed at very Ben Van Houten low levels, 20–25 copies per cell, while after induction with a DNA damaging agent, UvrA levels rise ten-fold. 1. Introduction Likewise, UvrB levels rise four-fold, from 250 copies per cell to 1000 copies (reviewed in [8]). Due to the Structure–function studies using the tools of NMR, labile character of UvrA and its low basal level of ex- X-ray crystallography and biochemistry have helped pression, it required 10 years to go from identification 94 B. Van Houten et al. / Mutation Research 577 (2005) 92–117 of the gene to purification and characterization of the tact with the DNA and then facilitates the transfer of protein by Seeberg and Steinum [9]. DNA to UvrB’s DNA binding domain [17]. The cryptic Nucleotide excision repair, mediated by the ATPase activity of UvrB (the red nodule on UvrB, see UvrABC type proteins, exists in bacteria and archaea, Fig. 1) is activated in the context of the UvrAB:DNA but not in eukaryotic cells. Today, the sequences complex and is required for damage verification. It is of more than 200 bacterial uvrA genes, 71 uvrB believed that UvrA hydrolyzes ATP, and affords its genes and 76 uvrC genes have been deposited in the self-dissociation from the recognition complex leav- Swiss-Protein database. These sequences provide a ing a salt resistant UvrB:DNA complex [18]. Before rich resource for comparative genomics. Thus, the 3 incision by UvrC, UvrB must be in its ATP bound identification of highly conserved residues becomes conformation [19]. UvrC catalyzes both incisions with immediately apparent as functional analysis can be the first incision four-phosphodiester bonds 3 to the le- explored by site-directed mutagenesis. sion, and the second, eight-phosphodiester bonds away from the DNA lesion on the 5 side. The dual inci- sions create a 12-nucleotide stretch of DNA containing 3. General mechanism the lesion [20–23]. Following incision, DNA helicase II (UvrD) is required to release UvrC and the incised A hypothetical scheme for key steps in the UvrABC oligonucleotide, while DNA polymerase I is thought to damage recognition and incision reaction is shown remove UvrB from the non-damaged DNA strand dur- (Fig. 1). In solution, UvrA dimer formation is driven by ing the repair synthesis [24,25]. DNA ligase I, encoded ATP [10]. While UvrA can recognize damaged DNA by the LigA gene, joins the newly synthesized DNA to independent of UvrB, we believe it is the complex the parent DNA, thus completing the NER pathway. of UvrA and UvrB together that provide this func- tion in vivo. UvrB interacts with the UvrA2 dimer in solution, creating an UvrA2B or UvrA2B2 complex. 4. Grasping the substrates:structures of some Based on X-ray crystallography results, UvrB appears key DNA adducts to be a monomer [11–13]; however, solution cross- linking studies and atomic force microscopy studies Long before the genes were cloned and the proteins have suggested that UvrB can form a dimer in solu- purified, it was recognized that UvrABC nucleotide ex- tion and on DNA ([14],
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