Abstract
Damage tolerance mechanisms mediating damage-bypass and gap-filling are crucial for genome integrity. A major damage tolerance pathway involves recombination and is referred to as template switch. Template switch intermediates were visualized by 2D gel electrophoresis in the proximity of replication forks as X-shaped structures involving sister chromatid junctions. The homologous recombination factor Rad51 is required for the formation/stabilization of these intermediates, but its mode of action remains to be investigated. By using a combination of genetic and physical approaches, we show that the homologous recombination factors Rad55 and Rad57, but not Rad59, are required for the formation of template switch intermediates. The replication-proficient but recombination-defective rfa1-t11 mutant is normal in triggering a checkpoint response following DNA damage but is impaired in X-structure formation. The Exo1 nuclease also has stimulatory roles in this process. The checkpoint kinase, Rad53, is required for X-molecule formation and phosphorylates Rad55 robustly in response to DNA damage. Although Rad55 phosphorylation is thought to activate recombinational repair under conditions of genotoxic stress, we find that Rad55 phosphomutants do not affect the efficiency of X-molecule formation. We also examined the DNA polymerase implicated in the DNA synthesis step of template switch. Deficiencies in translesion synthesis polymerases do not affect X-molecule formation, whereas DNA polymerase δ, required also for bulk DNA synthesis, plays an important role. Our data indicate that a subset of homologous recombination factors, together with DNA polymerase δ, promote the formation of template switch intermediates that are then preferentially dissolved by the action of the Sgs1 helicase in association with the Top3 topoisomerase rather than resolved by Holliday Junction nucleases. Our results allow us to propose the choreography through which different players contribute to template switch in response to DNA damage and to distinguish this process from other recombination-mediated processes promoting DNA repair.
Highlights
Proliferating cells are constantly exposed to DNA damage from both endogenous and exogenous sources
One DNA damage tolerance mechanism involving recombination factors, template switch, uses the information on the newly synthesized sister chromatid to fill in the gaps arising during replication under damaging conditions
This process leads to the formation of repair structures involving sister chromatid junctions in the proximity of replication forks
Summary
Proliferating cells are constantly exposed to DNA damage from both endogenous and exogenous sources. These DNA lesions can cause replication fork collapse and cell cycle arrest thereby posing a serious threat to genome integrity. To avoid the catastrophic consequences associated with fork demise, cells have evolved multiple mechanisms by which arrested or stalled replication forks can be rescued. These mechanisms are collectively referred to as DNA damage tolerance (DDT) mechanisms and involve factors belonging to two main repair pathways: the RAD52 homologous recombination (HR) and the RAD6/RAD18 post-replication repair (PRR) pathways [1,2]. The other DDT mechanism copies the information from undamaged segments of the genome, usually in an error-free manner and is referred to as template switch [2,5,6,7]
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