The PCNA-associated protein PARI negatively regulates homologous recombination via the inhibition of DNA repair synthesis

Successful and accurate completion of the replication of damage-containing DNA requires mainly recombination and RAD18-dependent DNA damage tolerance pathways. RAD18 governs at least two distinct mechanisms: translesion synthesis (TLS) and template switching (TS)-dependent pathways. Whereas TS is mainly error-free, TLS can work in an error-prone manner and, as such, the regulation of these pathways requires tight control to prevent DNA errors and potentially oncogenic transformation and tumorigenesis. In humans, the PCNA-associated recombination inhibitor (PARI) protein has recently been shown to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HR in vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits the extent of HR and represents a new potential target for anticancer therapy

Mes-Mer-ising; Insights into Meiotic Recombination via the Mer2 and Mer3 complexes

Meiosis is a specialised form of cell division that results in the generation of haploid gametes. During meiosis I, it is essential that homologous chromosomes be linked in order that they be properly segregated. Linkages are generated through crossovers, arising from recombination mediated repair of double-stranded DNA breaks (DSBs). Meiotic DSBs are non-random, instead they arise through the control of the conserved topoisomerase Spo11 and its chromosome associated cofactors. In the budding yeast S. cerevisiae, Mer2 is a Spo11-associated factor. Using recombinant proteins and synthetic nucleosomes, combined with hybrid structural biology and biochemistry we extensively characterise Mer2. We discover several novel and unexpected interactions that provide exciting insights into the regulation of Spo11, and the formation of meiotic DSBs. Once DSBs are formed they are resected to ssDNA and enter into the homologous recombination (HR) pathway. In meiosis the homologous chromosome is used as a template. A number of factors bias the formation of crossovers from nascent HR repair intermediates. We discover that the helicase Mer3, protects “D-loop” repair intermediates by antagonising the anti-crossover factor Sgs1. Taken together, our in vitro efforts have provided much needed clarity on the initiation of meiotic recombination, its regulation, and on how the normally deleterious formation of crossovers is acilitated

Novel mechanistic insights into the role of Mer2 as the keystone of meiotic DNA break formation

In meiosis, DNA double-strand break (DSB) formation by Spo11 initiates recombination and enables chromosome segregation. Numerous factors are required for Spo11 activity, and couple the DSB machinery to the development of a meiosis-specific ‘axis-tethered loop’ chromosome organisation. Through in vitro reconstitution and budding yeast genetics, we here provide architectural insight into the DSB machinery by focussing on a foundational DSB factor, Mer2. We characterise the interaction of Mer2 with the histone reader Spp1, and show that Mer2 directly associates with nucleosomes, likely highlighting a contribution of Mer2 to tethering DSB factors to chromatin. We reveal the biochemical basis of Mer2 association with Hop1, a HORMA domain-containing chromosomal axis factor. Finally, we identify a conserved region within Mer2 crucial for DSB activity, and show that this region of Mer2 interacts with the DSB factor Mre11. In combination with previous work, we establish Mer2 as a keystone of the DSB machinery by bridging key protein complexes involved in the initiation of meiotic recombination

Mer3 helicase protects early crossover intermediates from STR complex disassembly during meiosis

During meiosis I it is necessary that homologous chromosomes are linked to one another so that they can be faithfully separated. S. cerevisiae Mer3 (HFM1 in mammals) is a SF2 helicase and member of the ZMM group of proteins, that facilitates the formation of class I crossovers during meiosis. Here we describe the structural organisation of Mer3 and, using AlphaFold modelling and XL-MS, we further characterise the previously described interaction with Mlh1-Mlh2. We find that Mer3 also forms a previously undescribed complex with the re- combination regulating factors Top3 and Rmi1 and that this interaction is competitive with Sgs1BLM helicase in a phosphodependent manner. Using in vitro reconstituted D-loop assays we show that Mer3 inhibits the anti-recombination activity of Sgs1/Top3/Rmi1 (STR) complex. Thus we provide a mechanism whereby Mer3 downregulates the anti-crossover activity of the STR complex, hence promoting the formation of crossovers during meiosis I

RECQ5 Helicase Cooperates with MUS81 Endonuclease in Processing Stalled Replication Forks at Common Fragile Sites during Mitosis

The MUS81-EME1 endonuclease cleaves late replication intermediates at common fragile sites (CFSs) during early mitosis to trigger DNA-repair synthesis that ensures faithful chromosome segregation. Here, we show that these DNA transactions are promoted by RECQ5 DNA helicase in a manner dependent on its Ser727 phosphorylation by CDK1. Upon replication stress, RECQ5 associates with CFSs in early mitosis through its physical interaction with MUS81 and promotes MUS81-dependent mitotic DNA synthesis. RECQ5 depletion or mutational inactivation of its ATP-binding site, RAD51-interacting domain, or phosphorylation site causes excessive binding of RAD51 to CFS loci and impairs CFS expression. This leads to defective chromosome segregation and accumulation of CFS-associated DNA damage in G1 cells. Biochemically, RECQ5 alleviates the inhibitory effect of RAD51 on 3'-flap DNA cleavage by MUS81-EME1 through its RAD51 filament disruption activity. These data suggest that RECQ5 removes RAD51 filaments stabilizing stalled replication forks at CFSs and hence facilitates CFS cleavage by MUS81-EME1

Silent recognition of flagellins from human gut commensal bacteria by Toll-like receptor 5

Flagellin, the protein unit of the bacterial flagellum, stimulates the innate immune receptor Toll-like receptor (TLR)5 following pattern recognition, or evades TLR5 through lack of recognition. This binary response fails to explain the weak agonism of flagellins from commensal bacteria, raising the question of how TLR5 response is tuned. Here, we describe a novel class of flagellin-TLR5 interaction, termed silent recognition. Silent flagellins are weak agonists despite high affinity binding to TLR5. This dynamic response is tuned by TLR5-flagellin interaction distal to the site of pattern recognition. Silent flagellins are produced primarily by the abundant gut bacteria Lachnospiraceae and are enriched in non-Western populations. These findings provide a mechanism for the innate immune system to tolerate commensal-derived flagellins