Characterization of two highly similar Rad51 homologs of Physcomitrella patens.
The moss Physcomitrella patens, which is a land plant with efficient homologous recombination, encodes two Rad51 proteins (PpaRad51.1 and PpaRad51.2). The PpaRad51.1 and PpaRad51.2 proteins, which share 94 % identity between them, interact with themselves and with each other. Both proteins bind ssDNA and dsDNA in a Mg(2+) and pH-dependent manner, with a stoichiometry of one PpaRad51.1 monomer per 3(+/-1) nt or bp and one PpaRad51.2 monomer per 1(+/-0.5) nt or bp, respectively. At neutral pH, a 1.6-fold excess of both proteins is required for ssDNA and dsDNA binding. PpaRad51.1 and PpaRad51.2 show ssDNA-dependent ATPase activity and efficiently promote strand annealing in a nucleotide-independent but in a Mg(2+)-dependent manner. Both proteins promote joint-molecule formation, DNA strand invasion and are able to catalyse strand exchange in the presence of Mg(2+) and ATP. No further increase in the activities is observed when both proteins are present in the same reaction. None of the PpaRad51 gene products complement the DNA repair and recombination phenotype of Saccharomyces cerevisiae rad51delta mutants. However, PpaRad51.1 confers a dominant-negative DNA repair phenotype, and both PpaRad51 proteins reduce the levels of double-strand break-induced recombination when overexpressed in S. cerevisiae wt cells. These results suggest that both PpaRad51 proteins are bona fide Rad51 proteins that may contribute, in a different manner, to homologous recombination, and that they might replace ScRad51 in a hypothetical yeast protein complex inactivating different functions required for recombinational repair.
Ayora
Silvia
S
Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, CSIC, Cantoblanco, Campus Universidad Autónoma de Madrid, 28049, Spain.
Piruat
José I
JI
Luna
Rosa
R
Reiss
Bernd
B
Russo
Vincenzo E A
VE
Aguilera
Andrés
A
Alonso
Juan C
JC
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D019068:Bryopsida / Q000201:enzymology* / Q000235:genetics; D002846:Chromatography, Affinity; D023902:Chromosome Pairing / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005816:Genetic Complementation Test; D008851:Micropore Filters; D008969:Molecular Sequence Data; D011485:Protein Binding; D051135:Rad51 Recombinase; D011995:Recombination, Genetic / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017385:Sequence Homology
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D004247:DNA; C495151:RAD51 protein, S cerevisiae; D051135:Rad51 Recombinase; D000251:Adenosine Triphosphatases