Methanosarcina acetivorans flap endonuclease 1 activity is inhibited by a cognate single-stranded-DNA-binding protein

J Bacteriol. 2006 Sep;188(17):6153-67. doi: 10.1128/JB.00045-06.


The oligonucleotide/oligosaccharide-binding (OB) fold is central to the architecture of single-stranded- DNA-binding proteins, which are polypeptides essential for diverse cellular processes, including DNA replication, repair, and recombination. In archaea, single-stranded DNA-binding proteins composed of multiple OB folds and a zinc finger domain, in a single polypeptide, have been described. The OB folds of these proteins were more similar to their eukaryotic counterparts than to their bacterial ones. Thus, the archaeal protein is called replication protein A (RPA), as in eukaryotes. Unlike most organisms, Methanosarcina acetivorans harbors multiple functional RPA proteins, and it was our interest to determine whether the different proteins play different roles in DNA transactions. Of particular interest was lagging-strand DNA synthesis, where recently RPA has been shown to regulate the size of the 5' region cleaved during Okazaki fragment processing. We report here that M. acetivorans RPA1 (MacRPA1), a protein composed of four OB folds in a single polypeptide, inhibits cleavage of a long flap (20 nucleotides) by M. acetivorans flap endonuclease 1 (MacFEN1). To gain a further insight into the requirement of the different regions of MacRPA1 on its inhibition of MacFEN1 endonuclease activity, N-terminal and C-terminal truncated derivatives of the protein were made and were biochemically and biophysically analyzed. Our results suggested that MacRPA1 derivatives with at least three OB folds maintained the properties required for inhibition of MacFEN1 endonuclease activity. Despite these interesting observations, further biochemical and genetic analyses are required to gain a deeper understanding of the physiological implications of our findings.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Archaeal Proteins / chemistry
  • Archaeal Proteins / genetics
  • Archaeal Proteins / metabolism
  • Archaeal Proteins / pharmacology*
  • DNA, Single-Stranded / metabolism
  • Electrophoretic Mobility Shift Assay
  • Flap Endonucleases / antagonists & inhibitors*
  • Flap Endonucleases / metabolism
  • Methanosarcina / metabolism*
  • Models, Molecular
  • Molecular Sequence Data
  • Oligonucleotides / metabolism
  • Oligosaccharides / metabolism
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / pharmacology
  • Replication Protein A / chemistry
  • Replication Protein A / genetics
  • Replication Protein A / metabolism
  • Replication Protein A / pharmacology*
  • Sequence Alignment


  • Archaeal Proteins
  • DNA, Single-Stranded
  • Oligonucleotides
  • Oligosaccharides
  • Recombinant Proteins
  • Replication Protein A
  • Flap Endonucleases