Nbs1 potentiates ATP-driven DNA unwinding and endonuclease cleavage by the Mre11/Rad50 complex.
The Nijmegen breakage syndrome gene product (Nbs1) was shown recently to associate in vivo with the Mre11 and Rad50 proteins, which play pivotal roles in eukaryotic DNA double-strand break repair, meiotic recombination, and telomere maintenance. We show in this work that the triple complex of recombinant Nbs1, Mre11, and Rad50 proteins binds cooperatively to DNA and forms a distinct protein-DNA species. The Mre11/Rad50/Nbs1 complex displays several enzymatic activities that are not seen without Nbs1, including partial unwinding of a DNA duplex and efficient cleavage of fully paired hairpins. Unwinding and hairpin cleavage are both increased by the presence of ATP. On nonhairpin DNA ends, ATP controls a switch in endonuclease specificity that allows Mre11/Rad50/Nbs1 to cleave a 3'-protruding strand at a double-/single-strand transition. Mutational analysis demonstrates that Rad50 is responsible for ATP binding by the complex, but the ATP-dependent activities are expressed only with Nbs1 present.
Paull
T T
TT
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0540, USA.
Gellert
M
M
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D034961:Antigens, Nuclear; D018797:Cell Cycle Proteins / Q000502:physiology*; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases / Q000502:physiology*; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004706:Endodeoxyribonucleases; D004720:Endonucleases / Q000378:metabolism*; D005090:Exodeoxyribonucleases; D005656:Fungal Proteins / Q000502:physiology*; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D000072200:Ku Autoantigen; D009687:Nuclear Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D013997:Time Factors
D034961:Antigens, Nuclear; D018797:Cell Cycle Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C112156:NBN protein, human; D009687:Nuclear Proteins; C086930:RAD50 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D004706:Endodeoxyribonucleases; D004720:Endonucleases; D005090:Exodeoxyribonucleases; C094191:MRE11 protein, S cerevisiae; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen