Abstract
Efficient repair of chromosomal double-strand breaks (DSBs) by homologous recombination relies on the formation of a Rad51 recombinase filament that forms on single-stranded DNA (ssDNA) created at DSB ends. This filament facilitates the search for a homologous donor sequence and promotes strand invasion. Recently caffeine treatment has been shown to prevent gene targeting in mammalian cells by increasing non-productive Rad51 interactions between the DSB and random regions of the genome. Here we show that caffeine treatment prevents gene conversion in yeast, independently of its inhibition of the Mec1ATR/Tel1ATM-dependent DNA damage response or caffeine's inhibition of 5′ to 3′ resection of DSB ends. Caffeine treatment results in a dosage-dependent eviction of Rad51 from ssDNA. Gene conversion is impaired even at low concentrations of caffeine, where there is no discernible dismantling of the Rad51 filament. Loss of the Rad51 filament integrity is independent of Srs2's Rad51 filament dismantling activity or Rad51's ATPase activity and does not depend on non-specific Rad51 binding to undamaged double-stranded DNA. Caffeine treatment had similar effects on irradiated HeLa cells, promoting loss of previously assembled Rad51 foci. We conclude that caffeine treatment can disrupt gene conversion by disrupting Rad51 filaments.
Highlights
Repair of DNA double-strand breaks (DSBs) is highly conserved within eukaryotic cells
In an accompanying paper we show that caffeine impairs resection, which precedes Rad51 loading, by causing the rapid loss of the Sae2 and Dna2 resection proteins inhibiting repair by single-strand annealing (SSA)
Caffeine’s effect cannot be explained by interfering with either of the known pathways that regulate Rad51 filament assembly on singlestranded DNA (ssDNA), the filament dissociating activity of Srs2 or the removal of Rad51 non- bound on double-stranded DNA (dsDNA), by Rad54, Rdh54 or Uls1
Summary
Repair of DNA double-strand breaks (DSBs) is highly conserved within eukaryotic cells. Cdk activation facilitates the 5 to 3 resection of the broken ends, leaving 3 singlestranded DNA (ssDNA) tails that are first coated by replication protein A (RPA). Rad is recruited to RPA-coated ssDNA and facilitates the formation of a filament of the Rad recombination protein [5,6,7,8]. After the Rad filament forms, it facilitates a search for homology throughout the genome and promotes strand invasion between the ssDNA and homologous double-stranded DNA (dsDNA). Recombination can occur when the DSB is flanked by homologous sequences In this process, termed single-strand annealing (SSA), resection exposes complementary strands of the two flanking homologies that can anneal in a Rad52-dependent, but Rad51-independent manner, leading to a deletion
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