Synthetic viability genomic screening defines Sae2 function in DNA repair

• DOI: 10.15252/embj.201590973
• Publication Date: 2015-04-22T02:41:17Z
• Authors: Fabio Puddu; Tobias Oelschlaegel; Ilaria Guerini; Nicola J Geisler; Hengyao Niu; Mareike Herzog; Israel Salguero; Bernardo Ochoa‐Mo...; Emmanuelle Viré; Patrick Sung; David J Adams; Thomas M Keane; Stephen P Jackson
• Keywords: Haven

Article21 April 2015Open Access Synthetic viability genomic screening defines Sae2 function in DNA repair Fabio Puddu The Gurdon Institute and Department of Biochemistry, University Cambridge, UK Search for more papers by this author Tobias Oelschlaegel Ilaria Guerini Nicola J Geisler Hengyao Niu Molecular Biophysics Yale School Medicine, New Haven, CT, USA Mareike Herzog Wellcome Trust Sanger Institute, Hinxton, Israel Salguero Bernardo Ochoa-Montaño Emmanuelle Viré Patrick Sung David Adams Thomas M Keane Stephen P Jackson Corresponding Author Information Puddu1,‡, Oelschlaegel1,‡, Guerini1, Geisler1, Niu3, Herzog1,2, Salguero1, Ochoa-Montaño1, Viré1, Sung3, Adams2, Keane2 1,2 1The 2The 3Molecular ‡These authors contributed equally to work *Corresponding author. Tel: [phone]; E-mail: [email protected] EMBO Journal (2015)34:1509-1522https://doi.org/10.15252/embj.201590973 PDFDownload PDF article text main figures. Peer ReviewDownload a summary the editorial decision process including letters, reviewer comments responses feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract double-strand break (DSB) homologous recombination (HR) requires 3′ single-stranded (ssDNA) generation 5′ DNA-end resection. During meiosis, yeast cooperates with nuclease Mre11 remove covalently bound Spo11 from DSB termini, allowing resection HR ensue. Mitotic roles have remained enigmatic, however, since cells lacking these display modest defects but marked damage hypersensitivities. By combining classic genetic suppressor high-throughput sequencing, we identify mutations that strongly suppress sensitivities sae2∆ cells. assessing impacts at cellular, biochemical structural levels, propose that, addition promoting resection, crucial role activity mitotic is facilitate removal ssDNA associated ends. Thus, without or activity, partly processed DSBs impairs strand invasion HR. Synopsis nucleases cooperate their interplay still less well understood. A synthetic approach offers new insights into structure roles. Classic combined sequencing bioinformatics provides widely applicable non-null mutations. Specific MRE11 Sae2. Cellular, effects resolve paradoxes related promote removing toxic Mre11-bound intermediates. Introduction most cytotoxic form damage, ineffective leading mutations, chromosomal rearrangements genome instability can yield cancer, neurodegenerative disease, immunodeficiency and/or infertility (Jackson Bartek, 2009). arise ionising radiation radiomimetic drugs are generated when replication forks encounter breaks other lesions, alkylation adducts sites abortive topoisomerase activity. also physiological intermediates meiotic recombination, being introduced during prophase I II-type enzyme becomes linked end each side (Keeney et al, 1997). two pathways non-homologous end-joining (NHEJ) (Lisby 2004; Symington Gautier, 2011). In NHEJ, ends need little no processing before ligated (Daley 2005). contrast, involving degradation break, yielding tails mediate via pairing invading sister chromatid, which template. Reflecting above requirements, defective components hypersensitivity various DNA-damaging agents. This illustrated Saccharomyces cerevisiae harbouring Mre11–Rad50–Xrs2 (MRX) complex, binds juxtaposes (Williams 2008) and, through catalytic functions, activities involved (Furuse 1998; Williams 2008; Stracker Petrini, Once clean, partially resected has been generated, enzymes Exo1 Sgs1/Dna2 then thought act, generating extensive regions needed effective (Mimitou Symington, Zhu 2008). Notably, while essential meiosis cells, only somewhat delayed absence almost unaffected mre11-nd (nuclease-dead) (Ivanov 1994; Moreau 1999), indicating existence MRX-nuclease-independent routes generation. Another protein S. Sae2, functional homolog human CtIP (Sartori 2007; You Despite obvious domains, reported endonuclease vitro (Lengsfeld Makharashvili 2014; Wang 2014), functions tightly regulated cell cycle- damage-dependent phosphorylations (Baroni Huertas Jackson, 2009; Barton 2014). many ways, appears together MRX repair. For instance, as mre11S rad50S hypomorphic alleles phenocopy SAE2 deletion (sae2Δ) unprocessed Spo11–DNA complexes Kleckner, 1995; Nairz Klein, 1997; Prinz Furthermore, recent findings indicated stimulates particularly protein-bound (Cannavo Cejka, Also, both cause towards anti-cancer drug camptothecin (Deng 2005), yields repaired Nevertheless, key differences between exist, sae2Δ leads persistence 2004) hyperactivation MRX-associated Tel1 kinase (Usui 2001), ATM, inactivation abrogates (Fukunaga These findings, displaying mild (Clerici highlight how cannot be readily explained it simply cooperating enhance As below, mutants whole-genome determine genotype, led model resolves apparent regarding namely fact either causes agents, defect strains negligible compared mre11∆ lack an increase rather than loss. Our invokes Mre11/MRX critical step proceed effectively on Results SVGS identifies suppressors To gain why hypersensitive performed (SVGS; Fig 1A). do this, took cultures strain (bearing full locus) plated them YPD plates supplemented camptothecin, stabilises cleavage replication-dependent Sae2-dependent 2005) (Fig isolated 48 surviving treatment spontaneously arose population analysed. verifying all indeed contained gene yet were resistant, subsequent analyses revealed 10 clones largely fully suppressed DNA-alkylating agent methyl methanesulphonate (MMS), inhibitor hydroxyurea (HU), DSB-generating phleomycin ultraviolet light (Supplementary S1). Figure 1. suppressing Outline was used (CPT) hypersensitivity. Validation suppression phenotypes; subset (sup25–sup30) recovered shown along identified clone. Summary results (SVGS) ORF type mutation number times found mutated putatively driving resistance. Download figure PowerPoint causing phenotypes, analysed next-generation Illumina sequencing. We tools (see 4) altering open reading frames within reference 24 resistance retaining agents possessed TOP1 1B C), thereby providing proof-of-principle methodology (TOP1 non-essential encodes I, target). Strikingly, remaining clones, one different single codon, resulting amino acid residue His37 replaced Arg Tyr (mre11-H37R mre11-H37Y, respectively; C Supplementary S1; note mutually exclusive). While some additional potential worthy further examination, resistant camptothecin. Because broader phenotypes undefined mechanism action, focused characterising (mre11SUPsae2∆) alleles. mre11SUPsae2∆ lies functionally structurally evolutionarily conserved α-helical region, among quite divergent fungal species 2A). anticipated previous studies, deleting did not hypersensitivities revealing mre11-H37R mre11-H37Y behaving null (unpublished observation). line destabilise Mre11, producing proteins expressed equivalent levels wild-type 2B). expression resensitised lesser extent MMS 2C), recessive respectively. established sae2∆mre11SUPsae2∆ upon treatment. Importantly, independent introduction confirmed conferred 2D). caused prevent its Mec1/Tel1-dependent (sae2-MT) CDK-dependent (sae2-S267A) phosphorylation 2E F). observed mutating Ala (mre11-H37A; 2G), suggesting mediated abrogation specific likely reflected alteration introducing bulky chains. 2. suppresses CPT A. Alignment region containing H37 species; secondary prediction above. B. Western blot anti-Mre11 antibody extracts prepared shows alter (* indicate cross-reacting proteins). C. sup28 sup29 rescued expressing (wt) Mre11. D. E, F. sae2MT (sae2-2,5,6,8,9) sae2-S267A CPT, camptothecin; Phleo, phleomycin. G. mre11-H37A does sae2∆. heightened signalling measured Rad53 hyperphosphorylation 2006). had read-out inactivity 3A). sporulation impaired deficiency, aberrant accumulation 5′-bound THR4 hot spot (Goldway 1993; 3B; S2A, itself present). etoposide, produces bearing Top2 S2B; ERG6 permeability plasma membrane etoposide), significant must exist etoposide-induced DSBs. 3. checkpoint hyperactivation. rescue defect. single-strand annealing (SSA). SSA strains. E. sae2∆-dependent cycle arrest induction. F, Ku required mre11-H37R-mediated H. xrs2∆ I. Next, examined (SSA), using system wherein locus contains HO site flanked direct sequence repeats. system, induction until complementary sequences become exposed anneal, deletes repeats (Fishman-Lobell 1992; Vaze 2002; 3C). 2006), SSA-mediated resume progression after fast agreement published alleviate 3D E). Finally, effect telomere-associated complex It simultaneous SGS1 lethality/sickness, possibly due excessive telomere shortening Hardy test whether phenotype, crossed sae2∆mre11-H37R sgs1∆ strain. S2C, unable recover neither sgs1∆sae2∆ nor sgs1∆sae2∆mre11-H37R implying phenotype. conclusion, negatively affect Mre11-dependent maintenance demonstrated Southern analysis S2D). Together, data requiring Mre11-mediated intermediates, attributed Sae2-mediated bridging Subsequent channelling NHEJ because factor Yku70 sensitivity 3F). addition, previously sae2∆-mediated loss 2010; Foster 2011) considerably mre11-H37Y. require Exo1, contrast 2010), reliant 3G). Further characterisations, mre11-H37R, 3H), allele, phenocopies somehow preventing Sae2–MRX interactions stimulation Hopfner 2000; Cannavo 3I). H37R binding explore might operate, over-expressed purified Mre11H37R Mre11H37A 4A S2F) subjected analyses. All similar fractionated profiles, grossly stability. Since promotes initially speculated would having intrinsically high, Sae2-independent Surprisingly, case, actually exhibiting lower 4B). electrophoretic mobility shift assays, reduced double-stranded (dsDNA; 4C) abrogated 4D). Conversely, alanine, result supsae2∆ dsDNA-binding 4. biochemically, level homogeneity cultures. exonuclease assay release labelled nucleotide, indicated. C, Electrophoretic assays dsDNA (C) (D). Quantification Overnight grown diluted medium doses CPT. Colony growth scored 3–6 days later. Averages standard deviations point. Intragenic (mre11-H125N) mre11-H37R. treated (E). Dotted lines represent dotted Taken minor suggested suppressive weakened idea, same polypeptide, substantially active His125 Asn (Moreau 2001; mre11-H125N; 4E S2F G), prevents blocked 1999). Even mre11-H37R,H125N MMS, Mre11-nuclease-mediated H37R-dependent suppression, Sae2-deficient setting 4G S2G). sensitive mre11-H125N strains, corresponding carrying allele comparable (compare curves 1 2 3 4 4F) sae2∆-induced nuclease-independent Identifying interface mediating operate relate data, screened could propagated plasmid mutagenic coli strain, libraries plasmids mre11 sae2∆mre11∆ transformants capable presence 5A). Through retrieval, verification, 12 suppressors, nine point three double S3A). One mutant initial arising identified. Among mre11-P110L mre11-L89V, located domains II III, strong predictions 5B). Two another presumably responsible phenotype (because acts own), whereas third mre11-Q70R mre11-G193S. site-directed mutagenesis, mre11-Q70R, alters highly 5C). Ensuing comparisons S3B) extents mre11-L89V marginally weaker 5D). 5. mutagenesis LOF: loss-of-function SUP: shaded boxes blue shapes phosphoesterase motifs structures, Fungal alignment Q70. Structural residues 1–414, obtained homology modelling pombe structures. water-accessible surface monomers shades blue. Red: whose