The Structure and Mechanism of DNA Damage Recognition by XPC/Rad4 Nucleotide Excision Repair Complex
thesisposted on 01.07.2016 by Xuejing Chen
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Nucleotide excision repair (NER) is an important pathway that repairs structurally diverse DNA lesions in the cell, to maintain genome stability. XPC-Rad23B-Cen2 (yeast ortholog Rad4-Rad23-Cdc31/Rad33) complex initiates NER by binding to lesions, and recruits downstream NER factors. The crystal structure of Rad4-Rad23 bound to damaged DNA revealed that Rad4 recognizes lesions in an indirect manner, and forms an ‘open’ complex with DNA. However, it is still unknown how XPC efficiently distinguish lesions embedded in large excess of normal DNA. In this dissertation, the mechanism of damage recognition of Rad4 was investigated first by solving crystal structure of Rad4 complex bound to undamaged DNA. To prevent Rad4 binding non-specifically in multiple registers which inhibits crystallization, the protein was covalently tethered to DNA. This crystal structure revealed the same ‘open’ conformation as the damaged DNA bound structure. Next, the kinetics of DNA base opening and twisting were studied by temperature jump spectroscopy. First, using 2-aminopurine (2AP) as a probe, a full nucleotide flipping relaxation rate of ~7 ms was observed, which is specific to damaged DNA and requires the -hairpin3 that inserts into DNA duplex in the open conformation. Second, using a novel FRET pair tCO/tCnitro as probes, two distint phases of kinetics were observed: The slow phase, which overlapped on the same time scale as the nucleotide flipping kinetics captured by 2AP, was observed only when bound to specific, damaged DNA, but did not require -hairpin3, thus representing the rate-limiting step during the fomation of the open conformation. On the other hand, the fast phase did not require a DNA lesion or -hairpin3, and represents an nonspecific interrogation step preceding open conformation formation. Taken altogether, a “kinetic gating” mechanism for lesion recognition has been proposed, where Rad4/XPC complex interogates the DNA for distortion in duplex structure by fast twisting, and becomes “trapped” at lesion site, possibly due to an increase in residence time, leading to fully opening the DNA and stable protein-DNA complex formation. This hypothesis could explain how Rad4/XPC is able to detect thermodynamically destablized lesions without recognizing specific structure of the lesion.