Processing of A-form ssDNA by cryptic RNase H fold exonuclease PF2046

•Crystal structure of PF2046 in complex with ssDNA is determined.•PF2046 which has RNase H fold may function in DNA repair.•4-nucleotide long groove binds ssDNA in A-form.•Expanding the groove by mutation in Phe220 alters specificity.

RNase H fold protein PF2046 of Pyrococcus furiosus is a 3′-5′ ssDNA exonuclease that cleaves after the second nucleotide from the 3′ end of ssDNA and prefers poly-dT over poly-dA as a substrate. In our crystal structure of PF2046 complexed with an oligonucleotide of four thymidine nucleotides (dT4), PF2046 accommodates dT4 tightly in a groove and imposes steric hindrance on dT4 mainly by Phe220 such that dT4 assumes the A-form. As poly-dA prefer B-form due to the stereochemical restrictions, the A-form ssDNA binding by PF2046 should disfavor the processing of poly-dA. Phe220 variants display reduced activity toward poly-dA and the A-form appears to be a prerequisite for the processing by PF2046.