4 Zakharyevich, K. et al. Temporally and biochemically distinct activities of Exo1 during meiosis: double-strand break resection and resolution of double Holliday junctions.

32During prophase of the first meiotic division, cells deliberately break their DNA. 33These DNA breaks are repaired by homologous recombination, which facilitates 34proper chromosome segregation and enables reciprocal exchange of DNA seg-35 ments between homologous chromosomes, thus promoting genetic diversity in 36 the progeny 1 . A successful completion of meiotic recombination requires nucleo-37 lytic processing of recombination intermediates. Genetic and cellular data impli-38 cated a pathway dependent on the putative MLH1-MLH3 (MutLγ) nuclease in gen-39 erating crossovers, but mechanisms that lead to its activation were unclear 2-4 . 40Here, we have biochemically reconstituted key elements of this pro-crossover 41 pathway. First, we show that human MSH4-MSH5 (MutSγ), which was known to 42 support crossing over [5][6][7] , binds branched recombination intermediates and phys-43 ically associates with MutLγ. This helps stabilize the ensemble at joint molecule 44 structures and adjacent dsDNA. Second, we show that MutSγ directly stimulates 45 DNA cleavage by the MutLγ endonuclease, which demonstrates a novel and unex-46 pected function for MutSγ in triggering crossing-over. Third, we find that MutLγ 47 tion of yeast MutLγ is dependent on the integrity of the metal binding 75 DQHA(X)2E(X)4E motif within Mlh3, implicating the nuclease of Mlh3 in resolving 76 recombination intermediates 2,3,18,19,56 . Despite wealth of genetic and cellular data, 77 the mechanisms that control the MutLγ nuclease and lead to biased joint molecule 78 processing remained undefined. 79 80 81 82 83 84 4 Results 85 86 Human MutLγ is an ATP-stimulated endonuclease 87To study human MutLγ (hMLH1-hMLH3), we expressed and purified the hetero-88 dimer from insect cells ( Fig. 1a and Extended Data Fig. 1a,b). Similarly to the mis-89 match repair (MMR)-specific hMutLα (hMLH1-hPMS2) 20 , the hMLH1-hMLH3 90 complex non-specifically nicked double-stranded supercoiled DNA (scDNA) in the 91 presence of manganese without any other protein co-factor ( Fig. 1b,c, Extended 92 Data Fig. 1c), while almost no activity was observed with magnesium (Extended 93 Data Fig. 1d), which is believed to be the specific metal co-factor 20 . Mutations in 94 the conserved metal binding motif of hMLH3 abolished the endonuclease, indicat-95 ing that the DNA cleavage activity was intrinsic to the hMutLγ heterodimer ( Fig. 96 1d, see also Extended Data Fig. 1e). ATP promoted the nuclease activity >2-fold 97 ( Fig. 1d,e, Extended Data Fig. 1f-h). Experiments with various ATP analogs re-98 vealed that ATP hydrolysis by hMLH1-hMLH3 was required for the maximal stim-99 ulation of DNA cleavage (Fig. 1f, Extended Data Fig. 1h). The N-termini of both 100 hMLH1 and hMLH3 proteins contain conserved Walker motifs implicated in ATP 101 binding and hydrolysis 21 . To define whether the ATPase of hMLH1, hMLH3 or 102 both subunits of the heterodimer promotes its nucleolytic activity, we prepared 103 the respective hMutLγ variants with mutations in the conserved motifs of either 104 subu...

Activation of client protein kinases by the HSP90 molecular chaperone system is affected by phosphorylation at multiple sites on HSP90, the kinase-specific co-chaperone CDC37, and the kinase client itself. Removal of regulatory phosphorylation from client kinases and their release from the HSP90-CDC37 system depends on the Ser/Thr phosphatase PP5, which associates with HSP90 via its N-terminal TPR domain. Here, we present the cryoEM structure of the oncogenic protein kinase client BRAFV600E bound to HSP90-CDC37, showing how the V600E mutation favours BRAF association with HSP90-CDC37. Structures of HSP90-CDC37-BRAFV600E complexes with PP5 in autoinhibited and activated conformations, together with proteomic analysis of its phosphatase activity on BRAFV600E and CRAF, reveal how PP5 is activated by recruitment to HSP90 complexes. PP5 comprehensively dephosphorylates client proteins, removing interaction sites for regulatory partners such as 14-3-3 proteins and thus performing a ‘factory reset’ of the kinase prior to release.

GAGA is a Drosophila transcription factor that shows a high degree of post-translational modification. Here, we show that GAGA factor is acetylated in vivo. Lysine residues K325 and K373 on basic regions BR1 and BR3 of the DNA binding domain, respectively, are shown to be acetylated by PCAF. While BR1 is strictly required to stabilize DNA binding, BR3 is dispensable. However, acetylation of both lysine residues, either alone or in combination, weakens the binding to DNA. Despite the high degree of conservation of K325 and K373 in flies, their mutation to glutamine does not affect DNA binding. Molecular dynamics simulations, using acetylated K325 and a K325Q mutant of GAGA DNA binding domain in complex with DNA, are fully consistent with these results and provide a thermodynamic explanation for this observation. We propose that while K325 and K373 are not essential for DNA binding they have been largely conserved for regulatory purposes, thus highlighting a key regulatory system for GAGA factor in flies.