RAD51 mutants cause replication defects and chromosomal instability.
RAD51 is important for restarting stalled replication forks and for repairing DNA double-strand breaks (DSBs) through a pathway called homology-directed repair (HDR). However, analysis of the consequences of specific RAD51 mutants has been difficult since they are toxic. Here we report on the dominant effects of two human RAD51 mutants defective for ATP binding (K133A) or ATP hydrolysis (K133R) expressed in mouse embryonic stem (ES) cells that also expressed normal mouse RAD51 from the other chromosome. These cells were defective for restarting stalled replication forks and repairing breaks. They were also hypersensitive to camptothecin, a genotoxin that generates breaks specifically at the replication fork. In addition, these cells exhibited a wide range of structural chromosomal changes that included multiple breakpoints within the same chromosome. Thus, ATP binding and hydrolysis are essential for chromosomal maintenance. Fusion of RAD51 to a fluorescent tag (enhanced green fluorescent protein [eGFP]) allowed visualization of these proteins at sites of replication and repair. We found very low levels of mutant protein present at these sites compared to normal protein, suggesting that low levels of mutant protein were sufficient for disruption of RAD51 activity and generation of chromosomal rearrangements.
Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center, San Antonio, Texas, USA.
Tae Moon
TM
Kim
Jun Ho
JH
Ko
Lingchuan
L
Hu
Sung-A
SA
Kim
Alexander J R
AJ
Bishop
Jan
J
Vijg
Cristina
C
Montagna
Paul
P
Hasty
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002166:Camptothecin / Q000494:pharmacology; D002460:Cell Line; D043171:Chromosomal Instability; D002869:Chromosome Aberrations; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D053595:Embryonic Stem Cells / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D051379:Mice; D011401:Promoter Regions, Genetic / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering; D051135:Rad51 Recombinase / Q000235:genetics* / Q000378:metabolism*
D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; C495154:Rad51 protein, mouse; D002166:Camptothecin