Impact of DNA sequences in the DNA duplex opening by the Rad4/XPC nucleotide excision repair complex

Abstract

Rad4/XPC is a key DNA damage sensor for nucleotide excision repair (NER) in eukaryotes.Rad4/XPC recognizes diverse bulky lesions by flipping out two lesion-containing nucleotide pairs and inserting a -hairpin from the BHD3 domain (-hairpin3) into the DNA duplex. We have previously observed that Rad4 can form the same 'open' structure when covalently tethered to a normal DNA sequence containing consecutive C/G's (CCC/GGG) and that a similar open-like structure can be formed even when the -hairpin3 is lacking. Here, we report a crystal structure of the -hairpin3 mutant tethered to a sequence containing alternating C/G's (CGC/GCG). In contrast to the previous structures, Rad4 bound to CGC/GCG in a 180°-reversed manner, capping the end of the duplex without flipping out the nucleotides. MD simulations showed that CGC/GCG was inherently less 'openable' than CCC/GGG and that Rad4 failed to engage with its minor groove, a hallmark of productive binding towards 'opening'. These results reveal that DNA sequences significantly influence the thermodynamic barrier for DNA opening by Rad4, which may render certain DNA structures/sequences resistant to 'opening' despite a long residence time of Rad4. The reversemode may indicate unproductive binding for NER whereas the DNA end-binding may hint at Rad4/XPC's functions beyond NER.Yellow highlights indicate the CG repeats. The positions of the CGC/GCG 3-bp sequences after which the constructs are named are indicated in bold.