Abstract
Rad2/XPG belongs to the flap nuclease family and is responsible for a key step of the eukaryotic nucleotide excision DNA repair (NER) pathway. To elucidate the mechanism of DNA binding by Rad2/XPG, we solved crystal structures of the catalytic core of Rad2 in complex with a substrate. Rad2 utilizes three structural modules for recognition of the double-stranded portion of DNA substrate, particularly a Rad2-specific α-helix for binding the cleaved strand. The protein does not specifically recognize the single-stranded portion of the nucleic acid. Our data suggest that in contrast to related enzymes (FEN1 and EXO1), the Rad2 active site may be more accessible, which would create an exit route for substrates without a free 5′ end.
Highlights
Nucleotide excision repair (NER) is one of the main DNA repair pathways [1]
NER begins with the detection of the lesion, which in eukaryotes is performed by a complex of XPC and RAD23B proteins (Rad4 and Rad23 in Saccharomyces cerevisiae)
We report four structures of the Rad2 catalytic core solved in four different space groups, three of which represent the productive substrate binding mode and reveal that the protein recognizes a single 5 nucleotide of the single-stranded portion of the DNA and a 3 phosphate group of the ss/dsDNA junction
Summary
Nucleotide excision repair (NER) is one of the main DNA repair pathways [1]. Its characteristic feature is the ability to detect and remove a wide variety of DNA modifications with various chemical structures. NER begins with the detection of the lesion, which in eukaryotes is performed by a complex of XPC and RAD23B proteins (Rad and Rad in Saccharomyces cerevisiae). A general transcription factor, TFIIH, is recruited. It is a large complex of 10 subunits, including two helicases of opposite polarity: XPD and XPB. The XPA protein and RPA heterotrimer bind to form the pre-incision complex, and two nucleases are recruited to perform cuts on each side of the lesion, allowing the removal of the damaged DNA fragment of ∼30 nucleotides (nt). After the damaged DNA fragment is removed, DNA repair synthesis occurs
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