Conditional knockout of RAD51-related genes in Leishmania major reveals a critical role for homologous recombination during genome replication


Autoři: Jeziel D. Damasceno aff001;  João Reis-Cunha aff002;  Kathryn Crouch aff001;  Dario Beraldi aff001;  Craig Lapsley aff001;  Luiz R. O. Tosi aff003;  Daniella Bartholomeu aff002;  Richard McCulloch aff001
Působiště autorů: The Wellcome Centre for Integrative Parasitology, University of Glasgow, Institute of Infection, Immunity and Inflammation, Sir Graeme Davies Building, 120 University Place, Glasgow, United Kingdom aff001;  Laboratório de Imunologia e Genômica de Parasitos, Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brasil aff002;  Department of Cell and Molecular Biology, Ribeirão Preto Medical School, University of São Paulo; Ribeirão Preto, SP, Brazil aff003
Vyšlo v časopise: Conditional knockout of RAD51-related genes in Leishmania major reveals a critical role for homologous recombination during genome replication. PLoS Genet 16(7): e32767. doi:10.1371/journal.pgen.1008828
Kategorie: Research Article
doi: 10.1371/journal.pgen.1008828

Souhrn

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.

Klíčová slova:

Cell cycle and cell division – DNA – DNA extraction – DNA replication – DNA synthesis – Homologous recombination – Leishmania – Polymerase chain reaction


Zdroje

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2020 Číslo 7

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