RAD51 mutants cause replication defects and chromosomal instability

Tae Moon Kim, Jun Ho Ko, Lingchuan Hu, Sung A. Kim, Alexander J R Bishop, Jan Vijg, Cristina Montagna, Paul Hasty

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)3663-3680
Number of pages18
JournalMolecular and Cellular Biology
Volume32
Issue number18
DOIs
StatePublished - Sep 2012

Fingerprint

Chromosomal Instability
Adenosine Triphosphate
Mutant Proteins
Hydrolysis
Chromosomes
Camptothecin
Double-Stranded DNA Breaks
Poisons
Mutagens
Proteins
Maintenance

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

Cite this

Kim, T. M., Ko, J. H., Hu, L., Kim, S. A., Bishop, A. J. R., Vijg, J., ... Hasty, P. (2012). RAD51 mutants cause replication defects and chromosomal instability. Molecular and Cellular Biology, 32(18), 3663-3680. https://doi.org/10.1128/MCB.00406-12

RAD51 mutants cause replication defects and chromosomal instability. / Kim, Tae Moon; Ko, Jun Ho; Hu, Lingchuan; Kim, Sung A.; Bishop, Alexander J R; Vijg, Jan; Montagna, Cristina; Hasty, Paul.

In: Molecular and Cellular Biology, Vol. 32, No. 18, 09.2012, p. 3663-3680.

Research output: Contribution to journalArticle

Kim, Tae Moon ; Ko, Jun Ho ; Hu, Lingchuan ; Kim, Sung A. ; Bishop, Alexander J R ; Vijg, Jan ; Montagna, Cristina ; Hasty, Paul. / RAD51 mutants cause replication defects and chromosomal instability. In: Molecular and Cellular Biology. 2012 ; Vol. 32, No. 18. pp. 3663-3680.
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