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

Arch Biochem Biophys. 2016 Sep 15:606:143-50. doi: 10.1016/j.abb.2016.08.001. Epub 2016 Aug 3.

Abstract

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.

Keywords: A-form; Exonuclease; RNase H; ssDNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / chemistry*
  • Catalytic Domain
  • Crystallization
  • Crystallography, X-Ray
  • DNA Repair
  • DNA, Single-Stranded / chemistry*
  • Exonucleases / chemistry
  • Hydrogen-Ion Concentration
  • Mutation
  • Nucleic Acid Conformation
  • Oligodeoxyribonucleotides
  • Pyrococcus furiosus / enzymology*
  • Ribonuclease H / chemistry*
  • Substrate Specificity
  • X-Ray Diffraction

Substances

  • Bacterial Proteins
  • DNA, Single-Stranded
  • Oligodeoxyribonucleotides
  • Exonucleases
  • Ribonuclease H