Biochemical characterization of plant Rad52 protein from rice (Oryza sativa)

Plant Physiol Biochem. 2016 Sep;106:108-17. doi: 10.1016/j.plaphy.2016.04.048. Epub 2016 Apr 29.

Abstract

DNA damage in living cells is repaired by two main pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). Of all the genes promoting HR, Rad52 (Radiation sensitive 52) is an important gene which is found to be highly conserved across different species. It was believed that RAD52 is absent in plant systems until lately. However, recent genetic studies have shown the presence of RAD52 homologues in plants. Rad52 homologues in plant systems have not yet been characterized biochemically. In the current study, we bring out the biochemical properties of rice Rad52-2a protein. OsRad52-2a was over-expressed in Escherichia coli BL21 (DE3) cells and the protein was purified. The identity of purified OsRad52-2a protein was confirmed via peptide mass fingerprinting. Gel filtration and native PAGE analysis indicated that the OsRad52-2a protein in its native state probably formed an undecameric structure. Purified OsRad52-2a protein showed binding to single stranded DNA, double stranded DNA. Protein also mediated the renaturation of complementary single strands into duplex DNA in both agarose gel and FRET based assays. Put together, OsRad52-2a forms oligomeric structures and binds to ssDNA/dsDNA for mediating an important function like renaturation during homologous recombination. This study represents the first report on biochemical properties of OsRad52-2a protein from important crop like rice. This information will help in dissecting the recombination and repair machinery in plant systems.

Keywords: DNA binding; Oryza sativa; OsRad52; Rad52 homologues; Renaturation; Strand annealing.

MeSH terms

  • Amino Acid Sequence
  • Chromatography, Gel
  • Circular Dichroism
  • Cloning, Molecular
  • DNA, Plant / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Oryza / metabolism*
  • Plant Proteins / chemistry
  • Plant Proteins / isolation & purification
  • Plant Proteins / metabolism*
  • Protein Binding
  • Protein Denaturation
  • Rad52 DNA Repair and Recombination Protein / chemistry
  • Rad52 DNA Repair and Recombination Protein / isolation & purification
  • Rad52 DNA Repair and Recombination Protein / metabolism*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Substances

  • DNA, Plant
  • Plant Proteins
  • Rad52 DNA Repair and Recombination Protein