Identification of residues important for DNA binding in the full-length human Rad52 protein.
Human Rad52 (HsRad52) is a DNA-binding protein (418 residues) that promotes the catalysis of DNA double strand break repair by the Rad51 recombinase. HsRad52 self-associates to form ring-shaped oligomers as well as higher order complexes of these rings. Analysis of the structural and functional organization of protein domains suggests that many of the determinants of DNA binding lie within the N-terminal 85 residues. Crystal structures of two truncation mutants, HsRad52(1-212) and HsRad52(1-209) support the idea that this region makes up an important part of the DNA binding domain. Here, we report the results of saturating alanine scanning mutagenesis of the N-terminal domain of full-length HsRad52 in which we identify residues that are likely involved in direct contact with single-stranded DNA (ssDNA). Our results largely agree with the position of side-chains seen in the crystal structures but also suggest that certain DNA binding and cross-subunit interactions differ between the 11 subunit ring in the crystal structures of the truncation mutant proteins versus the seven subunit ring formed by full-length HsRad52.
Lloyd
Janice A
JA
Department of Biochemistry and Molecular Pharmacology, Aaron Lazare Research Building, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605-2324, USA.
McGrew
Dharia A
DA
Knight
Kendall L
KL
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D002384:Catalysis; D002461:Cell Line, Transformed; D064447:Checkpoint Kinase 2; D002850:Chromatography, Gel; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry; D004249:DNA Damage; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051135:Rad51 Recombinase; D051721:Rad52 DNA Repair and Recombination Protein; D011995:Recombination, Genetic; D017386:Sequence Homology, Amino Acid
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C497194:RAD52 protein, human; D051721:Rad52 DNA Repair and Recombination Protein; D004247:DNA; D001120:Arginine; D064447:Checkpoint Kinase 2; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D000409:Alanine