Engineering of functional replication protein a homologs based on insights into the evolution of oligonucleotide/oligosaccharide-binding folds

J Bacteriol. 2008 Sep;190(17):5766-80. doi: 10.1128/JB.01930-07. Epub 2008 Jun 27.


The bacterial single-stranded DNA-binding protein (SSB) and the archaeal/eukaryotic functional homolog, replication protein A (RPA), are essential for most aspects of DNA metabolism. Structural analyses of the architecture of SSB and RPA suggest that they are composed of different combinations of a module called the oligonucleotide/oligosaccharide-binding (OB) fold. Members of the domains Bacteria and Eukarya, in general, contain one type of SSB or RPA. In contrast, organisms in the archaeal domain have different RPAs made up of different organizations of OB folds. Interestingly, the euryarchaeon Methanosarcina acetivorans harbors multiple functional RPAs named MacRPA1 (for M. acetivorans RPA 1), MacRPA2, and MacRPA3. Comparison of MacRPA1 with related proteins in the publicly available databases suggested that intramolecular homologous recombination might play an important role in generating some of the diversity of OB folds in archaeal cells. On the basis of this information, from a four-OB-fold-containing RPA, we engineered chimeric modules to create three-OB-fold-containing RPAs to mimic a novel form of RPA found in Methanococcoides burtonii and Methanosaeta thermophila. We further created two RPAs that mimicked the RPAs in Methanocaldococcus jannaschii and Methanothermobacter thermautotrophicus through fusions of modules from MacRPA1 and M. thermautotrophicus RPA. Functional studies of these engineered proteins suggested that fusion and shuffling of OB folds can lead to well-folded polypeptides with most of the known properties of SSB and RPAs. On the basis of these results, different models that attempt to explain how intramolecular and intermolecular homologous recombination can generate novel forms of SSB or RPAs are proposed.

Publication types

  • 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*
  • Binding Sites
  • Electrophoretic Mobility Shift Assay
  • Evolution, Molecular
  • Fluorescence Polarization
  • Methanococcaceae / genetics
  • Methanococcaceae / metabolism
  • Methanosarcina / genetics
  • Methanosarcina / metabolism
  • Methanosarcinaceae / genetics
  • Methanosarcinaceae / metabolism
  • Models, Genetic
  • Molecular Sequence Data
  • Oligonucleotides / metabolism*
  • Oligosaccharides / metabolism*
  • Protein Binding
  • Protein Engineering / methods*
  • Protein Structure, Secondary
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Replication Protein A / chemistry
  • Replication Protein A / genetics
  • Replication Protein A / metabolism*
  • Sequence Homology, Amino Acid


  • Archaeal Proteins
  • Oligonucleotides
  • Oligosaccharides
  • Recombinant Fusion Proteins
  • Replication Protein A