Effect of amino acid substitutions in the rad50 ATP binding domain on DNA double strand break repair in yeast.
The Saccharomyces cerevisiae Rad50-Mre11-Xrs2 complex plays a central role in the cellular response to DNA double strand breaks. Rad50 has a globular ATPase head domain with a long coiled-coil tail. DNA binding by Rad50 is ATP-dependent and the Rad50-Mre11-Xrs2 complex possesses DNA unwinding and endonuclease activities that are regulated by ATP. Here we have examined the role of the Rad50 Walker type A ATP binding motif in DNA double strand break repair by a combination of genetic and biochemical approaches. Replacement of the conserved lysine residue within the Walker A motif with alanine, glutamate, or arginine results in the same DNA damage sensitivity and homologous recombination defect as the rad50 deletion mutation. The Walker A mutations also cause a deficiency in non-homologous end-joining. As expected, complexes containing the rad50 Walker A mutant proteins are defective in ATPase, ATP-dependent DNA unwinding, and ATP-stimulated endonuclease activities. Although the DNA end-bridging activity of the Rad50-Mre11-Xrs2 complex is ATP-independent, the end-bridging activity of complexes containing the rad50 Walker A mutant proteins is salt-sensitive. These results provide a molecular explanation for the observed in vivo defects of the rad50 Walker mutant strains and reveal a novel ATP-independent function for Rad50 in DNA end-bridging.
Chen
Ling
L
Department of Molecular Medicine, Institute of Biotechnology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245, USA.
Trujillo
Kelly M
KM
Van Komen
Stephen
S
Roh
Dong Hyun
DH
Krejci
Lumir
L
Lewis
L Kevin
LK
Resnick
Michael A
MA
Sung
Patrick
P
Tomkinson
Alan E
AE
D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000409:Alanine / Q000737:chemistry; D020816:Amino Acid Motifs; D001120:Arginine / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000502:physiology*; D004307:Dose-Response Relationship, Radiation; D004720:Endonucleases / Q000378:metabolism; D005720:Gamma Rays; D005816:Genetic Complementation Test; D018698:Glutamic Acid / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000502:physiology*; D013997:Time Factors
D004268:DNA-Binding Proteins; C086930:RAD50 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D004247:DNA; D001120:Arginine; D004720:Endonucleases; D000251:Adenosine Triphosphatases; D008239:Lysine; D000409:Alanine