Rad51 contribution to DNA tolerance mechanisms post UV-C

MARÍA BELÉN VALLERGA; SABRINA FLORENCIA MANSILLA; MARÍA BELÉN FEDERICO; MARTÍN HABIF; MARINA A. GONZÁLEZ BESTEIRO; AGOSTINA BERTOLÍN; VANESA GOTTIFREDI

Buenos Aires

Congreso; Ubiquitin & UBLs. At the crossroads from chromatin to protein; 2014

EMBO

Exposure of cells to genotoxic stimuli causes replication fork stalling at DNA lesions which might lead to fork collapse and subsequent cell death. To preserve viability, cells activate tolerance mechanisms to replicate across damaged DNA. The main known mechanism underlying UVC tolerance is Translesion DNA Synthesis (TLS), with polymerase eta as one of its key players. Template Switching (TS) is another DNA tolerance pathway involving at least some of the classic homologous recombination factors. TS relies on the recombinogenic protein Rad51 which facilitates homology searching for an alternative undamaged template for lesion bypass. Protection of fork degradation has been reported after hydroxyurea treatment as a recombinogenic independent function of Rad51. The contribution of either functions of Rad51 to UV DNA damage tolerance is poorly understood. In our present work we seek to assess the impact of Rad51 on replication fork progression. Using the DNA fiber assay we show that Rad51 prevents massive UV-triggered and mirin sensitive degradation of newly synthesized DNA. DNA degradation is limited to nucleotid incorporated before UV irradiation and is independent of PAR inhibition, a factor required for fork reversal. The Fiber assay also allowed us to observe that TLS (polymerase eta) and TS factors facilitate progression of ongoing replication forks, independently of each other, with no overlapping functions. DNA elongation after UV irradiation was largely affected by PAR inhibition, but this effect was not further modulated when Rad51 was depleted. We also found that Rad51 protects cell viability as its depletion caused a persistent increase of 53BP1 foci formation (a replication stress marker) and nuclear fragmentation (cell death marker). Thus in this work we demonstrate that Rad51 is required for cell survival after UV irradiation being this effect potentially associated with a Rad51-dependent effect on the quality and the extent of fork elongation after UV irradiation.