Crystal structure of the catalytic core of Rad2: insights into the mechanism of substrate binding.

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.

Ty3 reverse transcriptase complexed with an RNA-DNA hybrid shows structural and functional asymmetry.

Retrotransposons are a class of mobile genetic elements that replicate by converting their single-stranded RNA intermediate to double-stranded DNA through the combined DNA polymerase and ribonuclease H (RNase H) activities of the element-encoded reverse transcriptase (RT). Although a wealth of structural information is available for lentiviral and gammaretroviral RTs, equivalent studies on counterpart enzymes of long terminal repeat (LTR)-containing retrotransposons, from which they are evolutionarily derived, is lacking. In this study, we report the first crystal structure of a complex of RT from the Saccharomyces cerevisiae LTR retrotransposon Ty3 in the presence of its polypurine tract-containing RNA-DNA hybrid. In contrast to its retroviral counterparts, Ty3 RT adopts an asymmetric homodimeric architecture whose assembly is substrate dependent. Moreover, our structure and biochemical data suggest that the RNase H and DNA polymerase activities are contributed by individual subunits of the homodimer.