Abstract
Homologous recombination (HR) has an intimate relationship with genome replication, both during repair of DNA lesions that might prevent DNA synthesis and in tackling stalls to the replication fork. Recent studies led us to ask if HR might have a more central role in replicating the genome of Leishmania, a eukaryotic parasite. Conflicting evidence has emerged regarding whether or not HR genes are essential, and genome-wide mapping has provided evidence for an unorthodox organisation of DNA replication initiation sites, termed origins. To answer this question, we have employed a combined CRISPR/Cas9 and DiCre approach to rapidly generate and assess the effect of conditional ablation of RAD51 and three RAD51-related proteins in Leishmania major. Using this approach, we demonstrate that loss of any of these HR factors is not immediately lethal but in each case growth slows with time and leads to DNA damage and accumulation of cells with aberrant DNA content. Despite these similarities, we show that only loss of RAD51 or RAD51-3 impairs DNA synthesis and causes elevated levels of genome-wide mutation. Furthermore, we show that these two HR factors act in distinct ways, since ablation of RAD51, but not RAD51-3, has a profound effect on DNA replication, causing loss of initiation at the major origins and increased DNA synthesis at subtelomeres. Our work clarifies questions regarding the importance of HR to survival of Leishmania and reveals an unanticipated, central role for RAD51 in the programme of genome replication in a microbial eukaryote.
Highlights
Homologous recombination (HR) has critical roles in the genome maintenance of all organisms, mainly through repair of double stranded DNA breaks [1]
PCR showed this approach to be very efficient for RAD51 and the three RAD51 paralogues, since selection using only puromycin resulted in all wild type (WT) copies of each gene being replaced by floxed and tagged versions after a single transformation (S1 Fig)
Because RAD51 and the RAD51 paralogue mutants may generate similar phenotypes [44, 46], since each contributes to HR [29], we used the same approach to modify the L. major gene encoding the orthologue of T. brucei PIF6
Summary
Homologous recombination (HR) has critical roles in the genome maintenance of all organisms, mainly through repair of double stranded DNA breaks [1]. HR is a multistep repair process initiated by resection of the ends of double-stranded DNA breaks to generate single stranded DNA overhangs. This processing provides access to a key player in HR: the Rad recombinase (RecA in bacteria, RadA in archaea)[2], which catalyses invasion of the singlestranded DNA into intact homologous duplex DNA, allowing template-directed repair of the broken DNA site. An even more intimate association between HR and DNA replication has been described in bacteria and archaea, where RecA [14,15,16,17] and RadA [18] can mediate DNA replication when origins (the genome sites where DNA synthesis begins during replication) have been removed
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