Alterations in DNA Replication and Histone Levels Promote Histone Gene Amplification in Saccharomyces cerevisiae
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1 Supporting Information Alterations in DNA Replication and Histone Levels Promote Histone Gene Amplification in Saccharomyces cerevisiae Diana E. Libuda and Fred Winston Copyright 2010 by the Genetics Society of America DOI: /genetics
2 2 SI D. E. Libuda and F. Winston FIGURE S1. HTA2-HTB2 amplification is not dependent upon the DNA replication checkpoint. DNA replication checkpoint deletions (mec1δ sml1δ, dun1δ, sml1δ) with (hta1-htb1)δ were analyzed for the HTA2-HTB2 amplification by CHEF gel Southern blot using a probe for the amplified region on chromosome II (amplification probe B; (Libuda and Winston 2006)). A representative CHEF gel Southern blot is shown for mec1δ sml1δ (hta1-htb1)δ strains. A summary of the tetrad and CHEF gel Southern analysis results for DNA replication checkpoint genes is in Table 2.
3 D. E. Libuda and F. Winston 3 SI TABLE S1 Sequences of oligonucleotides used in these studies name sequence use position (5 end of oligo) FO5008 CCTGCCCCAACAGATAAAAACAAGCAAGGGTCAACCG DPB3 deletion 12 bp 5 of DPB3 ORF TGAGATTGTACTGAGAGTGCAC FO5009 TTCTCATTGCGTGTATGTATATGTATAACATATTGCA DPB3 deletion 34 bp 3 of DPB3 ORF TCGCTGTGCGGTATTTCACACCG FO5010 GCGGCACAATAGCAGAAGCCACAGAATATTACCTAGA DPB4 deletion 102 bp 5 of DBP4 ORF GGAGATTGTACTGAGAGTGCAC FO5011 AGTGGTGGCAAGCACTACTAGACAGTTTCCATAGCGG DPB4 deletion 1 bp 3 of DPB4 ORF GGCTGTGCGGTATTTCACACCG FO5326 CGAGAGATTTGTTCTTATAAGACATCCCGATGTTCAG RRM3 deletion 12 bp 5 of RRM3 ORF GTCGCAGATTGTACTGAGAGTGCAC FO5327 CGCCGCATACGTAGGCTTAGCTAAATAGATGGCTTCT RRM3 deletion 152 bp 3 of RRM3 ORF AGACTGTGCGGTATTTCACACCG FO5006 GTTCACGCGCTTTGCCCACATTGGGATAGCGCCCACA CLB5 deletion 305 bp 5 of CLB5 ORF GCAGATTGTACTGAGAGTGCAC FO5007 CAGTTCAACCGTTTCCGGATGCATGTAGCGAGGGTA CLB5 deletion 418 bp 3 of CLB5 ORF GACCTGTGCGGTATTTCACACCG FO727 GGCGCGTGTAAGTTACAGACAAGCGATCCCTATTCCA HHT1-HHF1 deletion 100 bp 3 of HHT1 ORF TGAGATTGTACTGAGAGTGCAC
4 4 SI D. E. Libuda and F. Winston FO730 TAACCACATGGAAAGCCATAAATCTTGCCTCCTACAA HHT1-HHF1 deletion 250 bp 3 of HHF1 ORF GGCTGTGCGGTATTTCACACCG FO4596 GAGATGTTTGTATGATGTCCCCCCAGTCTAAATGCAT HHT2-HHF2 deletion 26 bp 3 of HHT2 ORF AGCTGTGCGGTATTTCACACCG FO4597 CACCGATTGTTTAACCACCGATTGTTTAACCACCGAA HHT2-HHF2 deletion 18 bp 3 of HHF2 ORF ACAGATTGTACTGAGAGTGCAC FO4363 AGGGGCTTAACATACAGTAAAAAAGGCAATTATAGTG DUN1 deletion 40 bp 5 of DUN1 ORF AAGCTGTGCGGTATTTCACACCG FO4364 CCAGATTCAAACAATGTTTTTGAAATAATGCTTCTCA DUN1 deletion 40 bp 3 of DUN1 ORF TGTAGATTGTACTGAGAGTGCAC FO4693 GTATCTAAGAGAAGAAAAGAACAGAACTAGTGGGAAA SML1 deletion 39 bp 5 of SML1 ORF TGAGATTGTACTGAGAGTGCAC FO4694 CTCACTAACCTCTCTTCAACTGCTCAATAATTTCCCG SML1 deletion 71 bp 3 of SML1 ORF CTCTGTGCGGTATTTCACACCG FO1075 AAGAACGACATACACCGCGTAAAGGCCCACAAGACTG MEC1 deletion 1 bp 5 of MEC1 ORF CCTGTGCGGTATTTCACACCG FO1076 ACATAATTGTTTCGATCACATTCATCAACGCTACTTC MEC1 deletion 6884 bp 3 of MEC1 ORF AAGATTGTACTGAGAGTGCAC FO6051 CCGTCTCAGAATTGGATCGT RNR3 Northern probe bp from RNR3 ATG FO6052 ATTGTTTCCGTTGGAACTGC RNR3 Northern probe bp from RNR3 ATG
5 D. E. Libuda and F. Winston 5 SI FO844 TGTATGTGTGTATGGTTTATTTGTG HTB1 Northern probe 162 bp 5 of HTB1 ORF FO907 GAGAACGATGGATTTAAAATCAAGAG HTB1 Northern probe 61 bp 5 of HTB1 ORF FO5415 CAAAGCAAATCAAACATTCTTGTC HTA2 Northern probe 20 bp 3 of HTA2 ORF FO5416 CACTGTCTTTAATAAAACGACGCTAT HTA2 Northern probe 134 bp 3 of HTA2 ORF FO5269 ACGGTTTCGGTGGTTAATTT HHF1 Northern probe +314 bp from HHF1 ATG FO5270 TCAAACTGTTTAAACCCAATTAAAA HHF1 Northern probe 175 bp 3 of HHF1 ORF FO6182 TTTTATATAGGACCACTGTTTTGTGA HHT2 Northern probe 91 bp 5 of HHT2 ORF FO6183 TGTGGAGTGTTTGCTTGGAT HHT2 Northern probe 1 bp 5 of HHT2 ORF FO5329 CAATCGGTGGTTAAACAATCGG HHF2 Northern probe +301 bp from HHF2 ATG FO5330 GGGAAGACTATCTAAGACAGTTCG HHF2 Northern probe 198 bp 3 of HHF2 ORF FO1408 TACCCGCCACGCGTTTTTTTCTTT ACT1 Northern probe -367 bp from ACT1 ATG FO1409 ATTGAAGAAGATTGAGCAGCGGTTTG ACT1 Northern probe bp from ACT1 ATG
6 6 SI D. E. Libuda and F. Winston TABLE S2 Results from synthetic genetic array analysis for regulators of (hta1-htb1)δ viability candidate gene d alternative gene names d systematic name d (hta1-htb1)δ growth e (SC-hisleu-arg+can b ) (hta1-htb1)δ yfg1δ a growth e (SC-his -leu -arg +can+g418 c ) gene ontology category d increases (hta1-htb1)δ viability ERG3 SYR1, PSO6 YLR056W ergosterol biosynthesis; endocytosis; ER IRA2 CCS1, GLC4 YOL081W ++ + mitochondria PRM4 YPL156C Mating SSE1 LPG3, MSI3 YPL106C HSP90 chaperone complex; telomere maintenance MSI1 CAC3 YBR195C ++ + CAF-1 Complex; chromatin, DNA repair, transcription DPB4 YDR121W DNA pol epsilon; ISW2 chromatin complex YDR431W YDR431W ++ + Dubious CIN8 KSL2, SDS15 YEL061C ++ + MT kinesin motor; Mitotic sister chromatid segregation RRM3 RTT104 YHR031C DNA helicase (rdna replication and Ty1 transposition) KKQ8 YKL168C putative kinase PAC10 GIM2, PFD3, RKS2 YGR078C tubulin; prefoldin complex YJL131C YJL131C putative protein; mitochondria localized BUD26 YDR241W dubious RPS6A YPL090C S ribosomal CLB5 YPR120C CDK in DNA replication HSV2 YGR223C phosphoinositide binding RPL19B YBL027W S ribosomal YBL081W YBL081W hypothetical protein AVT5 YBL089W ++ + putative transporter DST1 PPR2 YGL043W ++ + TFIIS; transcription elongation; meiotic recombination ayfg1δ indicates a gene deletion from the nonessential gene deletion set. Most of the deletion strains listed in this table have not been confirmed to have the correct deletion. bsc-his-leu-arg+can indicates selection for growth of (hta1-htb1)δ strains with or without yfg1δ (note: typical growth for (hta1-htb1)δ without yfg1δ on this medium is +/-). csc-his-leu-arg+can+g418 indicates selection for (hta1-htb1)δ yfg1δ. dinformation regarding gene names, systematic names, and gene ontology categories were obtained from the Saccharomyces Genome Database ( egrowth phenotype scores: ++, +++, and ++++ = a range of very strong growth; + = good growth; -/+ and +/- = intermediate growth; and - = no growth
7 D. E. Libuda and F. Winston 7 SI KEX2 SRB1, QDS1, VMA45 YNL238W ++ + proprotein convertase GCR2 YNL199C ++ + transcriptional activator (glycolysis) YNL198C YNL198C ++ + Dubious LSM1 SPB8 YJL124C degradation of cytoplasmic mrnas; RNA binding and catabolism FYV1 YDR024W ++ + Dubious YBR277C YBR277C Dubious NRG1 YDR043C Transcriptional Repressor; glucose repression RPS14A CRY1, RPL59 YCR031C S ribosomal; telomere maintenance PAT1 MRT1 YCR077C Topoisomerase II; chromosome segregation; mrna processing HHF1 YBR009C Histone H4 HHT1 YBR010W Histone H3 MBP1 YDL056W Transcription Factor; Repressor; DNA replication (complex with Swi6); Cell cycle RPP1A RPLA1 YDL081C p1 alpha ribosome SRB9 SSN2, NUT8, SCA1, UME2, RYE3, MED13 YDR443C Mediator complex; SRB8-11 complex; transcription; telomere maintenance SWI4 ART1 YER111C SBF complex (Swi4-6); DNA binding; transcription factor; cell cycle NRP1 YDL167C Unknown LSM7 YNL147W mrna catabolism; telomere maintenance MED1 YPR070W Mediator complex; transcription; telomere maintenance NUT1 MED5 YGL151W ++ + Mediator complex; transcription LSM6 YDR378C mrna catabolism YGL177W YGL177W ++ + Dubious PPZ1 YML016C ++ +/- Phosphatase; cell cycle progression YPL260W YPL260W ++ +/- Unknown YBR225W YBR225W ++ +/- Unknown MUP3 YHL036W ++ +/- Metionine permease VMR1 YHL035C ++ +/- potential Cdc28 substrate; vaculolar? SBP1 SSB1; SSBR1 YHL034C ++ +/- snorna binding; 35S transcript processing RPS25A RPS31A YGR027C ++ +/- 40S ribosomal YLL007C YLL007C ++ +/- Unknown YOL150C YOL150C ++ +/- Dubious
8 8 SI D. E. Libuda and F. Winston SRB8 SSN5; MED12; NUT6; GIG1; YCR080W YCR081W +++ +/- Mediator Complex; transcription; telomere maintenance HHT2 YNL031C +++ +/- Histone H3 BMH1 APR6 YER177W +++ +/- DNA binding; DNA damage checkpoint; signaling; vesical transport GIM3 PFD4 YNL153C +++ +/- prefoldin complex; chaperone complex CAN1 YEL063C + + arginine permease; deletion = can resistance MDM20 DEC1 YOLO076W + +/- NatB acetyl transferase complex; mitochondrial inheritance HAT2 YEL056W + +/- Hat1-Hat2 Acetyltransferase complex (Histone H3/H4); telomere silencing SRB10 SSN3, GIG2, NUT&, UME3, RYE5, CDK8 YPL042C + +/- CDK of Mediator complex; transcription; meiosis; telomere maintenance ATP5 OSC1 YDR298C + +/- ATP synthesis YDR336W YDR336W + +/- Unknown SRB2 MED20; HRS2 YHR041C + +/- Mediator complex; transcription factor; telomere maintenance decreases (hta1-htb1)δ viability RPS13A RPS9B; SUP36 YBR189W S ribosome YMR293C YMR293C + - Unknown; mitochondria YMR304C YMR304C-A + - Dubious CLA4 ERC10 YNL298W + - CDC42 activated kinase; cytokinesis; exit from mitosis; cell polarity VAM10 YOR068C + - Vaculoe fusion UAF30 YOR295W + - Upstream Activator Factor Complex (RNA pol I specific; contains Histone H3 and H4); transcription; rdna binding CPA1 YOR303W + - Citrulline biosynthesis (arginine precursor) SPE2 YOL052C + - spremidine/spermine biosynthesis RTG1 YOL067C + - transcriptional coactivator (bhlh); mitochondria YPL205C YPL205C + - Dubious POS5 YPL188W + - NADH kinase; mitochondria TGS1 YPL157W + - RNA methlytransferase; snrna capping; ribosome biogenesis YPL144W YPL144W + - Unknown; telomere maintenance
9 D. E. Libuda and F. Winston 9 SI UME1 WTM3 YPL139C + - transcription corepression; meiosis regulation MRP51 YPL118W + - mitochondria HIS7 YBR248C + - histidine biosynthesis DPB3 YBR278W + - DNA replication; DNA pol II; telomeric silencing TIM13 YGR181W + - mitochondria PDX1 YGR193C + - E3BP; mitochondria VAM3 PTH1 YOR106W + - Vacuole MCT1 YOR221C + - mitochondria YDR246C YDR246C + - Unknown; oxidoreductase MNN11 YER001W + - Golgi VPS35 GRD9; VPT7 YJL154C + - Endosome; Golgi VPS36 GRD12; VAC3; VPL11 YLR417W + - ESCRT-II complex; telomere maintenance YLR235C YLR235C + - Dubious KAP114 YGL241W + - Imports Spt15, Histone H2A and H2B, and NAP1 SNF8 VPS22 YPL002C + - ESCRT-II complex; transcription regulated by glucose; telomere maintenance VPS66 YPR139C + - Vacuole SEM1 DSS1; HOD1 YDR363W-A S proteasome; telomere maintenance YNL171C YNL171C + - Dubious DOA1 UFD3; ZZZ4 YKL213C + - promotes NHEJ in stationary phase; DSB DNA repair; Ubiquitin BSC2 YDR275W + - Unknown RPS7A RPS30 YOR096W S ribosome COG8 DOR1 YML071C + - Golgi complex YML090W YML090W + - Dubious VPS9 VPL31; VPT9 YML097C + - vacuole; telomere maintenance MYO5 YMR109W + - Myosin PKR1 YMR123W + - V-ATPase assembly factor in ER BRR1 YPR057W + - snrnp protein; RNA binding SPT10 CRE1; SUD1 YJL127C + - Putative histone acetyltransferase; Activates Histone genes; transcription; chromatin ARG3 YJL088W + - Arginine biosynthesis THR1 YHR025W + - threonine metabolism PPA1 VMA16 YHR026W + - Vacuole VPS25 VPT25 YJR102C + - ESRT-II complex; Ubiquitin; Telomere maintenance KCS1 YDR017C + - Vacuole PMP1 YCR024C-A + - enzyme regulator VPS53 YJL029C + - Golgi HIT1 YJR055W + - Unknown; telomere maintenance ERI1 RIN1 YPL096C-A + - Er protein
10 10 SI D. E. Libuda and F. Winston HDA1 YNL021W + - Histone deacetylase complex; deletion increases H2B, H3, and H4 acetylation; chromatin; transcription COG6 COD2; SEC37 YNL041C + - Golgi VPS27 GRD11; SSV17; VPL23; VPL27; VPT27; DID7 YNR006W + - ER protein UBP1 YDL122W + - Ubiquitin; ER FAR11 YNL127W + - pheromone cell cycle arrest HIS5 YIL116W + - histidine biosynthesis FLX1 YIL134W + - FAD transport; mitochondria RRB1 YMR131C + - ribosome LAT1 ODP2, PDA2 YNL071W + - pyruvate dehydrogenase; mitochondria MSK1 YNL073W + - lysine-trna ligase; mitochondria NNF2 YGR089W + - chromosome segregation; genetic and physical interactions w/ Rpb8 (RNA pol I, II, III) YDL062W YDR062W + - Dubious YDR455C YDR455C + - Dubious TOM1 YDR457W + - E3 ubiquitin ligase; mrna export; regulation of transcriptional coactivators VPS60 MOS10; CHM5 YDR486C + - vacuole QRI8 UBC7 YMR022W + -/+ ubiguitin; ER; chromatin assembly/disassembly HSC82 HSP90 YMR186W + -/+ chaperone of Hsp90; protein folding; telomere maintenance MDJ2 YNL328C + -/+ Mitochondria RPS6B LPG18; RPS101; RPS102 YBR181C + -/+ 40S ribosome KIN1 YDR122W + -/+ serine/threonine kinase LEU3 YLR451W + -/+ leucine biosynthesis HXK2 HEX1; HKB; SCI2 YGL253W + -/+ glucose metabolism AEP3 YPL005W + -/+ mrna metabolism; mitochondria YNL190W YNL190W + -/+ Unknown PEX1 PAS1 YKL197C + -/+ ATPase; peroxisome HIS6 YIL020C + -/+ histidine biosynthesis RPL34B YIL052C + -/+ 60S ribosome
11 D. E. Libuda and F. Winston 11 SI VPS20 CHM6; VPT20; VPL10 YMR077C + -/+ ESCRT-III complex; ubiquitin; vacuole IES3 YLR052W + -/+ INO80 chromatin remodeling complex NAB6 YML117W + -/+ putative RNA binding protein SLM5 YCR024C + -/+ Mitochondria YCR049C YCR049C + -/+ Dubious CSM2 YIL132C + -/+ meiotic chromosome segregation; DNA repair RPL16A RPL13 YIL133C + -/+ 60S ribosome VPS3 PEP6; VPL3; VPT17 YDR495C + -/+ Vacuole PUF6 YDR496C + -/+ mrna binding; transcription; mating; ribosome biogenesis YGL149W YGL149W + -/+ Dubious CIK1 YMR198W - - microtubule motor; spindle; meiosis; sister chromatid segregation GSG1 TRS85; MUM1 YDR108W - - ER; Golgi; meiosis; TRAPP complex BFR1 YOR198C - - mrnp complex; mrna metabolism; meiosis; mitosis regulation RPB4 CTF15 YJL140W - - RNA pol II; transcription; telomere maintenance; mrna export VPS66 YPR139C - - Vacuole RPS8A YBL072C S ribosome PER1 COS16 YCR044C - - ER STP22 VPS23 YCL008C -/+ - ESCRT-1 complex; ubiquitin; telomere maintenance SAT4 HAL4 YCR008W -/+ - mitotic cell cycle; kinase YGL218W YGL218W -/+ - Dubious RTG2 YGL252C -/+ - mitochondria; SLIK complex; transcription VMA7 YGR020C -/+ - Vacuole RPL34B YIL052C -/+ - 60S ribosome; telomere maintenance RIM13 CPL1 YMR154C -/+ - protease SLM5 YCR024C -/+ - Mitochondria FEN2 YCR028C -/+ - plasma membrane FKH2 YNL068C -/+ - transcription factor; cell cycle; silencing RPS21B YJL136C -/+ - 40S ribosome SEL1 UBX2 YML013W +/- - ubiquitin; ER YML013C-A YML012C-A YML013C-A +/- - Dubious (overlaps with SEL1) YAP1 PAR1; SNQ3 YML007W +/- - bzip transcription factor MRPL3 YMR024W +/- - mitochondria
12 12 SI D. E. Libuda and F. Winston ADA1 HFI1; SUP110; SRM12; GAN1 YPL254W +/- - SAGA; transcription; chromatin; histone acetylation; telomere maintenance HTA2 H2A2 YBL003C +/- - Histone H2A
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