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RECMITMEI Report Summary

Project ID: 268639
Funded under: FP7-IDEAS-ERC
Country: United Kingdom

Final Report Summary - RECMITMEI (Regulating recombination in mitotic and meiotic cells)

My lab previously discovered the Fe-S cluster helicase RTEL1 as a key regulator of homologous recombination. We hypothesized that RTEL1 could metabolise D-loop intermediates to influence the outcome of recombination reactions in various different cellular contexts, including meiotic recombination, DNA replication and at telomeres. RTEL1 was first described in 2004 by the Lansdorp lab as a factor that controls telomere length differences between different strains of mice. It was shown that telomeres are frequently lost from the chromosome end in the absence of RTEL1, but a molecular explanation for this phenomenon was lacking. We postulated that the telomere dysfunction of RTEL1 deficient mouse cells might reflect a failure to regulate HR or metabolise an HR intermediate formed at telomeres. Indeed, as part of this project we proceeded to show that RTEL1 uses its D-loop disruption activity to disassemble telomere (t)-loop structures to prevent unscheduled and catastrophic telomere processing by the SLX1/4 nuclease complex, which provided an explanation for the telomere loss observed in RTEL1 deficient cell. He also showed that RTEL1 performs a genetically distinct function in dismantling telomeric G4-DNA structures to avert telomere fragility during replication of the chromosome end. We also established how the two distinct functions of RTEL1 are controlled at telomeres. Through a proteomic approach he discovered that RTEL1 interacts with PCNA, an essential replication protein, via a PIP box motif. Analysis of knock-in mice and cells harboring a point mutation in the RTEL1 PIP box revealed a critical role in unwinding telomeric G4-DNA structures and facilitating global genome replication. Ageing studies of these knock-in mice also established that RTEL1 acts as tumour suppressor gene. The clinical importance of our work was reinforced by subsequent GWAS studies, which identified RTEL1 as a susceptibility locus for astrocytomas and high-grade gliomas. We also discovered that the Shelterin protein, TRF2, recruits RTEL1 to telomeres in S-phase to promote t-loop unwinding, which required a previously uncharacterized metal-coordinating C4C4 motif in RTEL1 and a unique interaction surface within the TRFH domain of TRF2.
Our genetic studies in mouse cells revealed that the PIP-box and C4C4 motifs in RTEL1 convey distinct and separable functions, which impact on the targeting of RTEL1 to replication forks and telomeres, respectively. Importantly, we also demonstrated that the TRF2-RTEL1 interaction is abolished by mutations in RTEL1 that are causal for Hoyeraal-Hreidarsson syndrome (HHS), a severe form of Dyskeratosis congenita. We showed that the RTEL1 pR1264H mutation, which is causal for HHS and is present at a carrier frequency of 1% within the Ashkenazi Orthodox Jewish population and 0.45% in the general Ashkenazi Jewish population, specifically disrupts the TRF2-RTEL1 interaction. Our findings therefore have major implications for the understanding of human telomere dysfunction disorders. Our proteomic analysis of RTEL1 also identified an interaction with MMS19, which was first discovered in a yeast screen for methylmethane sulfonate sensitive mutants over 30 years ago and also associates with Rad3/XPD. But how MMS19 functions in cells was completely unclear. Since XPD and RTEL1 are both Fe-S cluster helicases, we hypothesized that MMS19 could be involved in Fe-S cluster biogenesis. Indeed, we showed that MMS19 also forms a complex with the cytoplasmic Fe-S assembly (CIA) proteins CIAO1, IOP1, and MIP18, which together facilitate the incorporation of Fe-S clusters into client apo-proteins, including RTEL1, XPD and many other essential replication and repair proteins. Our work provided a molecular explanation for the pleiotropic phenotypes associated with MMS19 deficiency and why defects in mitochondrial Fe-S cluster biogenesis confer genome instability. Genetic screens in C. elegans also uncovered a synthetic lethal interaction between the novel helicase helq-1 and the Rad51 paralog rfs-1, which resulted from a block to meiotic DSB repair at a post-strand invasion step of HR. While RAD-51-ssDNA filaments assemble at processed meiotic DSBs with normal kinetics in helq-1 rfs-1 double mutants, persistence of RAD-51 foci and genetic interactions with rtel-1 suggested a failure in the disassembly of RAD-51 from stable strand-invasion intermediates. Indeed, biochemical analysis revealed that HELQ-1 promotes the disassembly of RAD-51 nucleoprotein filaments formed on double-stranded, but not single-stranded DNA. We proposed that HELQ-1 and RFS-1 promote post-synaptic RAD-51 filament disassembly, which is essential for completion of meiotic DSB repair. We proceeded to demonstrate that HelQ helicase deficient mice phenotypically resemble those with FA, exhibiting subfertility, germ cell attrition, DNA damage sensitivity and tumour predisposition. Proteomic analysis revealed that HELQ is physically associated with the RAD51 paralog sub-complex: RAD51B, RAD51C, RAD51D and XRCC2 (BCDX2). Loss of HELQ in cells was found to result in hyper-activation of the FA pathway and persistence of recombination intermediates at damaged replication fork.
Our findings provided an explanation for the prevalence of non-synonymous variants in HELQ, which are significantly associated with upper aerodigestive tract cancers, particularly amongst smokers; and variants in HELQ associated with early menopause, which may reflect the germ cell defects and ovarian dysgenesis observed in HELQ deficient mice. The mechanism by which Rad51 paralogs directly stimulate the recombinase activity of Rad51 had remained enigmatic for many years. We previously discovered a Rad51 paralog complex, RFS-1/RIP-1, from C. elegans, which is essential for HR and RAD-51 focus formation specifically at replication blocking lesions in vivo. By exploiting the biochemical tractability of this complex, we demonstrated that Rad51 paralogs bind to and structurally remodel the pre-synaptic RAD-51-ssDNA filament to a stabilized, “open”, flexible conformation, which facilitates strand exchange with the template duplex. Our results therefore defined the underlying mechanism of HR stimulation by Rad51 paralogs and established a new paradigm for HR mediator action. Over the last decade, 53BP1 has emerged as a key factor controlling DSB repair pathway choice during the cell cycle. 53BP1 promotes NHEJ by acting as a barrier to resection of DSBs in G1 but how this is achieved was unclear. We discovered that Rif1 deficient mice are immune-compromised and defective for 53BP1-mediated fusion of dysfunctional telomeres and for toxic NHEJ in BRCA1-deficient cells. We established that RIF1 physically interacts with 53BP1 and is essential for suppressing resection at DSBs. Our discovery that RIF1 is critical for 53BP1-dependent NHEJ has important ramifications for understanding DSB pathway choice and the tumour suppressive functions of BRCA1. Overall, our work during this project has provided a mechanistic understanding of various factors that regulate homologous recombination during DNA repair, DNA replication, meiosis and/or at telomeres. Importantly, our discoveries have helped to illuminate the molecular basis of several human genetic disorders highlighting the clinical impact of our work.

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United Kingdom
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