Reconstitution of the strand invasion step of double-strand break repair using human Rad51 Rad52 and RPA proteins.
The human Rad51 recombinase is essential for the repair of double-strand breaks in DNA that occur in somatic cells after exposure to ionising irradiation, or in germ line cells undergoing meiotic recombination. The initiation of double-strand break repair is thought to involve resection of the double-strand break to produce 3'-ended single-stranded (ss) tails that invade homologous duplex DNA. Here, we have used purified proteins to set up a defined in vitro system for the initial strand invasion step of double-strand break repair. We show that (i) hRad51 binds to the ssDNA of tailed duplex DNA molecules, and (ii) hRad51 catalyses the invasion of tailed duplex DNA into homologous covalently closed DNA. Invasion is stimulated by the single-strand DNA binding protein RPA, and by the hRad52 protein. Strikingly, hRad51 forms terminal nucleoprotein filaments on either 3' or 5'-ssDNA tails and promotes strand invasion without regard for the polarity of the tail. Taken together, these results show that hRad51 is recruited to regions of ssDNA occurring at resected double-strand breaks, and that hRad51 shows no intrinsic polarity preference at the strand invasion step that initiates double-strand break repair.
McIlwraith
M J
MJ
Clare Hall Laboratories, Imperial Cancer Research Fund, South Mimms, Hertfordshire, EN6 3LD, UK.
Van Dyck
E
E
Masson
J Y
JY
Stasiak
A Z
AZ
Stasiak
A
A
West
S C
SC
D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000648:ultrastructure; D004260:DNA Repair / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D004278:DNA, Superhelical / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D008854:Microscopy, Electron; D008957:Models, Genetic; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D051135:Rad51 Recombinase; D011938:Rec A Recombinases / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics; D051716:Replication Protein A; D012689:Sequence Homology, Nucleic Acid
D004277:DNA, Single-Stranded; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; D004247:DNA; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D011938:Rec A Recombinases