Uncoupling of RAD51 focus formation and cell survival after replication fork stalling in RAD51D null CHO cells

• In vertebrate cells, the five RAD51 paralogs (XRCC2/3 and RAD51B/C/D) enhance the efficiency of homologous recombination repair (HRR). Stalling and breakage of DNA replication forks is a common event, especially in the large genomes of higher eukaryotes. When cells are exposed to agents that arrest DNA replication, such as hydroxyurea or aphidicolin, fork breakage can lead to chromosomal aberrations and cell killing. We assessed the contribution of the HRR protein RAD51D in resistance to killing by replication-associated DSBs. In response to hydroxyurea, the isogenic rad51d null CHO mutant fails to show any indication of HRR initiation, as assessed by induction RAD51 foci, as expected. Surprisingly, these cells have normal resistance to killing by replication inhibition from either hydroxyurea or aphidicolin, but show the expected sensitivity to camptothecin, which also generates replication-dependent DSBs. In contrast, we confirm that the V79 xrcc2 mutant does show increased sensitivity to hydroxyurea under some conditions, which was correlated to its attenuated RAD51 focus response. In response to the PARP1 inhibitor KU58684, rad51d cells, like other HRR mutants, show exquisite sensitivity (>1000-fold), which is also associated with defective RAD51 focus formation. Thus, rad51d cells are broadly deficient in RAD51 focus formation in response to various agents, but this defect is not invariably associated with increased sensitivity. Our results indicate that RAD51 paralogs do not contribute equally to cellular resistance of inhibitors of DNAreplication, and that the RAD51 foci associated with replication inhibition may not be a reliable indicator of cellular resistance to such agents.

Ämnesord

NATURVETENSKAP  -- Biologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences (hsv//eng)

Nyckelord

homologous recombination

RAD51 paralogs

RAD51 foci

hydroxyurea

DNA replication

Publikations- och innehållstyp

ref (ämneskategori)

art (ämneskategori)