Paper Citing NAMD - Abstract
Lu, Zheng; Hou, Jingli; Wang, You; Liu, Jianhua
Involvement of Ser94 in RNase HIII from Chlamydophila pneumoniae in the recognition of a single ribonucleotide misincorporated into double-stranded DNA
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, 1824:859-865, JUL 2012
We recently provided the first report that RNase Hill can cleave a DNA-rN(1)-DNA/DNA substrate (rN(1), one ribonucleotide) in vitro. In the present study, mutagenesis analyses and molecular dynamics (MD) simulations were performed on RNase Hill from Chlamydophila pneumoniae AR39 (CpRNase Hill). Our results elucidate the mechanism of ribonucleotide recognition employed by CpRNase HIII, indicating that the G95/K96/G97 motif of CpRNase Hill represents the main surface interacting with single ribonucleotides, in a manner similar to that of the GR(K)G motif of RNase HI's. However, CpRNase Hill lacks the specific tyrosine required for RNase HII to recognize single ribonucleotides in double-stranded DNA (dsDNA). Interestingly, MD shows that Ser94 of CpRNase HIII forms a stable hydrogen bond with the deoxyribonucleotide at the (5')RNA-DNA(3') junction, moving this nucleotide away from the chimeric ribonucleotide. This movement appears to deform the nucleic acid backbone at the RNA-DNA junction and allows the ribonucleotide to interact with the GKG motif. Based on the inferences drawn from MD simulations, biochemical results indicated that Ser94 was necessary for catalytic activity on the DNA-rN(1)-DNA/DNA substrate; mutant S94V could bind this substrate but exhibited no cleavage. Mismatches opposite the single ribonucleotide misincorporated in dsDNA inhibited cleavage by CpRNase Hill to varying degrees but did not interfere with CpRNase/substrate binding. Further MD results implied that mismatches impair the interaction between Ser94 and the deoxyribonucleotide at the RNA-DNA junction. Consequently, recognition of the misincorporated ribonucleotide was disturbed. Our results may help elucidate the distinct substrate-recognition properties of different RNase Hs. (C) 2012 Elsevier B.V All rights reserved.
