Abstract
SummaryGenome stability requires coordination of DNA replication origin activation and replication fork progression. RTEL1 is a regulator of homologous recombination (HR) implicated in meiotic cross-over control and DNA repair in C. elegans. Through a genome-wide synthetic lethal screen, we uncovered an essential genetic interaction between RTEL1 and DNA polymerase (Pol) epsilon. Loss of POLE4, an accessory subunit of Pol epsilon, has no overt phenotype in worms. In contrast, the combined loss of POLE-4 and RTEL-1 results in embryonic lethality, accumulation of HR intermediates, genome instability, and cessation of DNA replication. Similarly, loss of Rtel1 in Pole4−/− mouse cells inhibits cellular proliferation, which is associated with persistent HR intermediates and incomplete DNA replication. We propose that RTEL1 facilitates genome-wide fork progression through its ability to metabolize DNA secondary structures that form during DNA replication. Loss of this function becomes incompatible with cell survival under conditions of reduced origin activation, such as Pol epsilon hypomorphy.
Highlights
DNA replication origins are established at thousands of sites throughout the genome through a combination of structural and functional chromatin determinants that promote loading of inactive MCM2-7 double hexamers around DNA replication origins (Fragkos et al, 2015)
Genetic analysis revealed that rtel-1 is synthetic lethal when combined with mutations in dog-1/FANCJ, mus-81, him-6/BLM, and rcq-5, all of which are homologs of genes involved in human genetic diseases and the maintenance of genome stability at replication forks (Barber et al, 2008)
Rtel-1 Is Synthetic Lethal with pole-4 in C. elegans To confirm our observations with RNAi, we obtained a genetic deletion of pole-4, tm4613, which removed the majority of the coding region of the gene, apart from the first 35 nucleotides of exon 1; this likely represents a bona fide null allele (Figure S2A). pole-4(tm4613) mutants appeared superficially wild type, and the loss of pole-4 did not result in any significant loss of viability. rtel-1 mutants show greater than 90% viability as previously described (Barber et al, 2008)
Summary
DNA replication origins are established at thousands of sites throughout the genome through a combination of structural and functional chromatin determinants that promote loading of inactive MCM2-7 double hexamers around DNA replication origins (Fragkos et al, 2015). The bulk of DNA replication at active replication forks is performed by the conserved DNA polymerase complexes Pol delta and Pol epsilon, which act on the lagging and leading strand, respectively (Burgers and Kunkel, 2017) The latter is considered to be a stable component of the replisome and is required for efficient CMG formation in budding yeast (Bell and Labib, 2016). Genetic analysis revealed that rtel-1 is synthetic lethal when combined with mutations in dog-1/FANCJ, mus-81, him-6/BLM, and rcq-5, all of which are homologs of genes involved in human genetic diseases and the maintenance of genome stability at replication forks (Barber et al, 2008) Mutants of these genes when combined with rtel-1 displayed persistent RAD-51 foci in the germline and embryonic lethality, indicating that RTEL-1 is essential in their absence. Subsequent studies showed that RTEL1 facilitates efficient telomere and genome-wide replication in vertebrates (Vannier et al, 2013), but its precise function during DNA replication remains unclear
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