Insights into the DNA damage recognition mechanism during nucleotide excision repair
소속 :
연사 : Jung-Hyun Min(Chemistry Department, University of Illinois at Chicago)
일시 : 2015-12-07 15:00 ~
장소 : 500동 1층 목암홀
DNA damage repair is central to maintaining the genome integrity and preventing cancer. The first step in DNA damage repair is the recognition of damaged sites from predominantly normal DNA. This task is especially formidable for the nucleotide excision repair (NER) which repairs structurally diverse lesions caused by various genotoxic sources such as UV irradiation, industrial pollutants and cigarette smoking. The xeroderma pigmentosum C (XPC) complex initiates nucleotide-excision repair by recognizing DNA lesions before recruiting downstream factors. How XPC detects structurally diverse lesions embedded within normal DNA is unknown. My group uses a combination of X-ray crystallography and time-resolved temperature-jump perturbation spectroscopy to unveil the molecular mechanism of DNA damage recognition by XPC.
In this talk, I will present a crystal structure that captures the yeast XPC orthologue (Rad4) on a single register of undamaged DNA. The structure shows that a disulphide-tethered Rad4 flips out normal nucleotides and adopts a conformation similar to that seen with damaged DNA. Contrary to many DNA repair enzymes that can directly reject non-target sites as structural misfits, our results suggest that Rad4/XPC uses a novel, ‘kinetic gating’ mechanism whereby lesion selectivity arises from the kinetic competition between DNA opening and the residence time of Rad4/XPC per site. This mechanism is further supported by measurements of Rad4-induced lesion-opening times using temperature-jump (T-jump) perturbation spectroscopy. In another set of study using T-jump combined with a novel FRET pair, we observed that Rad4 unwinds (‘twists’) nonspecific DNA in ~100–500s time-scales, showing that Rad4/XPC recognizes lesions in a step-wise ‘twist-open’ mechanism. These results represent the first direct observation of a sub-millisecond, nonspecific interrogation by a DNA repair protein. The research provides novel insights into the speed-and-stability paradox of specific DNA recognition, common to many DNA-binding proteins.
In this talk, I will present a crystal structure that captures the yeast XPC orthologue (Rad4) on a single register of undamaged DNA. The structure shows that a disulphide-tethered Rad4 flips out normal nucleotides and adopts a conformation similar to that seen with damaged DNA. Contrary to many DNA repair enzymes that can directly reject non-target sites as structural misfits, our results suggest that Rad4/XPC uses a novel, ‘kinetic gating’ mechanism whereby lesion selectivity arises from the kinetic competition between DNA opening and the residence time of Rad4/XPC per site. This mechanism is further supported by measurements of Rad4-induced lesion-opening times using temperature-jump (T-jump) perturbation spectroscopy. In another set of study using T-jump combined with a novel FRET pair, we observed that Rad4 unwinds (‘twists’) nonspecific DNA in ~100–500s time-scales, showing that Rad4/XPC recognizes lesions in a step-wise ‘twist-open’ mechanism. These results represent the first direct observation of a sub-millisecond, nonspecific interrogation by a DNA repair protein. The research provides novel insights into the speed-and-stability paradox of specific DNA recognition, common to many DNA-binding proteins.
