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9, Radl 7, and RAD24 Are Required for S Phase Regulation in Saccharomyces Cerevisiae in Response to DNA Damage

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9, Radl 7, and RAD24 Are Required for S Phase Regulation in Saccharomyces Cerevisiae in Response to DNA Damage Copyright 0 1997 by the Genetics Society of America -9, RADl 7, and RAD24 Are Required for S Phase Regulation in Saccharomyces cerevisiae in Response to DNA Damage A. G. Paulovich, R. U. Margulies, B. M. Garvik and L. H. Hartwell Fred Hutchinson Cancer Research Center, Seattle, Washington 98104 Manuscript received July 30, 1996 Accepted for publication October 1, 1996 ABSTRACT We have previously shown that a checkpoint dependent on MECl and RAD53 slows the rate of S phase progression in Saccharomyces cereuisiae in response to alkylation damage. Whereas wild-type cells exhibit a slow S phase in response to damage,mal-1 and rad53 mutants replicate rapidly in the presence or absence of DNA damage. In this report, we show that other genes (RAD9, RADl 7, RAD24) involved in the DNA damage checkpoint pathway also play a role in regulating S phase in response to DNA damage. Furthermore,RAD9, RAD1 7, and RAD24 fall into two groups with respect to both sensitivity to alkylation and regulation of S phase. We also demonstrate that the more dramatic defect in S phase regulation in the mecl-l and rad53 mutants is epistatic to a less severe defect seen in rad9A, radl 7A, and rad24A. Furthermore, the triple rad9A radl 7A rad24A mutant also has a less severe defect than mcl-1 or rad53 mutants. Finally, we demonstrate the specificity of this phenotype by showing that the DNA repair and/or checkpoint mutants mgtlA, magla, apnlA, rev3A, radl8A, radl6A, dud-A100, sad4-1, tellA,rad26A, rad5lA, rad52-1, rad54A,radl4A, radlA, p0130-46, p0130-52, mad3A, pdslA/esp2A, pmlA, mlhlA, and mh2A are all proficient at S phase regulation, even though some of these mutations confersensitivity to alkylation. ANY types of cells havebeen shown to respond to ESP2 (YAMAMOTOet al. 1996a,b). How these same genes M DNA damage by regulating progression through participate in seemingly different checkpointsat differ- the ensuingmitotic cell cycle (HARTWELLand WEINERT ent cell cycle stages is still a mystery. However, recent 1989; CARR 1995; MURRAY1995). Regulation of cell cy- evidence suggests the possibility that checkpoint genes cle transitions in response to damageis a result of signal may encodeproteins involved directly in detection transduction pathways called “checkpoints” (HART- and/or processing of DNA lesions (LYDALLand WEIN- WELL and WEINERT1989). In Saccharomycescereuisim, ERT 1995, 1996). checkpoint pathways responding to DNA damage or to We recently demonstrated that in wild-type S. cereuis- inhibition of DNA replication regulate the entry into iae the rate of ongoing S phase is slowed, although not and progression through S phase and mitosis. Interest- blocked, when cells are subjected to alkylation damage ingly, many of the checkpoint genes that have been by exposure to sublethal doses of the monofunctional identified in yeast are necessary for controlling more alkylating agent methyl methanesulfonate (MMS) than one cell cycle transition. For example, the G1-S (PAULOVICHand HARTWELL 1995).Furthermore, we phase DNA damage checkpointis dependent onRAD9 showed that the slowing of S phase progression in re- (SIEDEet al. 1993, 1994), RAD24 (SIEDEet al. 1994), sponse to alkylation damage in yeast is dependent on and RAD53/MEC2/SPKl/SADl (ALLEN et al. 1994) (G1 the MECl and the RALl53 checkpoint genes (PAULOV- checkpoint status in mcl-1 and radl 7A has not been ICH and HARTWELL 1995); mcl-1or rad53mutants repli- determined), the SM checkpoint that inhibits mitosis cate damaged and undamaged DNA at comparable when cells are blocked in S phase is dependent on rates, ruling out a model in which lesions alone are MECl (WEINERTet al. 1994), RAD53 (ALLEN et al. 1994; able to slow replication and demonstratingthat the WEINERTet al. 1994), andPOLE (NAVASet al. 1995), and slowing of S phase is an active process. the G2-M DNA damage checkpoint is dependent on Inhibition of DNA replication in response to DNA damagehad previously beendemonstrated in Esche- RAD9 (WEINERTand HARTWELL 1988, 1990, 1993;), RADl 7 (WEINERTand HARTWELL1993; WEINERTet al. richia coli (CAIRNEs and DAVERN1966) as well asin mam- 1994), RAD24 (WEINERTet at. 1994), MECI/ESRI malian cells (PAINTERand YOUNG 1980; YOUNGand PAINTER1989; LARNER et aZ. 1994). This inhibition is (WEINERTet aZ. 1994), RAD53 (ALLEN et aZ. 1994; WEIN- due notonly to a decrease inthe initiation of replicons, ERT et al. 1994), MEc3 (WEINERTet al. 1994), andPDSI/ but also to a decrease in the rate of elongation of preex- isting nascentstrands (PAINTERand YOUNG 1980; Cmesponding authw; Lee Hartwell, Fred Hutchinson Cancer Re- search Center, 11124 Columbia St., Seattle, WA 98104. LARNER et al. 1994). Cells isolated from patients afflicted E-mail: [email protected] by the cancer-prone, neurodegenerative disorderataxia Genetics 145 45-62 (January, 1997) 46 A. G. Paulovich et al. TABLE 1 Yeast strains Strain Genotype 7830-2-4a MATa ura3 leu2 trpl his3 yMP10177 MATa ura3 leu2 trpl his3 rad9::LEU2 yMP10247 MATa ade2 ade3-130 leul-12 ura3-52 canl cyh2 SCR.:URA3 sap3 rad52-1 yMP10252 MATa ura3 leu2 trpl his3 mecl-l::HIS3 smll yMP10261 MATa ade2 ade3-130 ura3-52 canl cyh2 SCR.:URA3 sap3 trpl radlA yMP10318 MATa ura3 leu2 trpl his3 rad9A::LEU2 rad24A::TRPl yMP10333 MATa ade2 ade3-130 ura3 leu2 trpl cyh2 SCR:URA3 mgtlA::LEU2 yMP10359 MATa ura3 leu2 trpl his3 rad9A::HISjr rad24A::TRPl radl 7A::LEU2 yMP10365 MATa ura3 leu2 trpl his3 radl 7A::L.EU2 yMP10366 MATa ura3 leu2 trpl his3 rad24A::TRPl yMP10381 MATa ade2 ade3-I30 ura3 leu2 trpl cyh2 SCR:URA3 yMP10382 MATa ade2 ade3-130 ura3 leu2 trpl cyh2 SCR:URA3 rm3A::LEU2 yMP10425 MATa ade2 ade3-130 ura3 leu2 trpl qh2 SCR:URA3 radl8A::LEUZ yMP10428 MATa ura3 leu2 trpl his3 rad5lA::LEU2 yMP10447 MATa ura3 leu2 trpl his3 radl6A::URA3 yMP10464 MATa ura3 leu2 trpl his3 rad9A::LEU2 mec2-l::URA3 yMP10507 MATa ura3 leu2 trpl his3 rad24A::URA3 yMP10519 MATa his3 radl4A::HIS3 yMP10521 MATa ade2 ade3-I30 ura3 leu2 trpl cyh2 SCR.:URA3 mshZA::URA3 yMP10537 MATa ura3 leu2 trpl his3 rad9A::HIS3 radl 7A::LEU2 yMP10538 MATa ura3 leu2 trpl his3 rad24A::TRPl radl 7A::LEU2 yMP10590 MATa ade2 ade3-130 ura3 leu2 trpl cyh2 SCR:URA3 apnlA::LEU2 yMP10788 MATa ura3 leu2 trpl his3 rad53 yMP10789 MATa ura3 leu2 trpl his3 rad9A::TRPl yMP10796 MATa ura3 leu2 trpl his3 radl 7A::LEU2 rad53 yMP10798 MATa ura3 leu2 trpl his3 radl 7A::LEU2 rad53 yMP10801 MATa ura3 leu2 trpl his3 rad24A::TRPl rad53 yMP10844 MATa ura3 leu2 trpl his3 mecl-l smll yMP10845 MATa ura3 leu2 trpl his3 radl 7A::LEU2 smll yMP10847 MATa ura3 leu2 trpl his3 mecl-l smll yMP10848 MATa ura3 leu2 trpl his3 mecl-1 smll yMP10850 MATa ura3 leu2 trpl his3 radl 7A::LEU2 mecl-1 smll yMP10852 MATa ura3 leu2 trpl his3 radl 7A::LEUZ smll yMP10853 MATa ura3 leu2 trpl his3 radl 7A::LEU2 mecl-1 smll yMP10856 MATa ura3 leu2 trpI his3 radl 7A::LEUZ mecl-1 smll yMP10860 MATa ura3 leu2 trpl his3 smll yMP10863 MATa ura3 leu2 trpl his3 smll yMP10882 MATa ura3 leu2 trpl his3 rad9A::HIS3 mecl-1 smll yMP10884 MATa ura3 leu2 trpl his3 rad9A::HIS3 mecl-l smll yMP10886 MATa ura3 leu2 trpl his3 rad9A::HIS3 mecl-1 smll yMP10887 MATa ura3 leu2 trpl his3 rad9A::HIS3 smll yMP10889 MATa ura3 leu2 trpl his3 rad9A::HISjr smll yMP10903 MATa ura3 leu2 trpl his3 mecl-1 rad53 smll yMP10904 MATa ura3 leu2 trpl his3 mecl-1 rad53 smll yMP10910 MATa ade2 ade3-130 ura3 leu2 trpl cyh2 SCR::URA3 rad54A::LEU2 yMP10931 MATa ura3 leu2 trpl his3 mecl-1 rad24A::TRPl smll yMP10932 MATa ura3 leu2 trpl his3 rad24A::TRPl smll yMP10934 MATa ura3 leu2 trpl his3 rad24A::TRPl smll yMP10936 MATa ura3 leu2 trpl his3 mecl-1 rad24A::TRPl smll yMP10942 MATa ura3 leu2 trpl his3 mecl-l rad24A::TRPl smll yMP10943 MATa ura3 leu2 trpl his3 rad9A::HIS3 rad53 yMPlO944 MATa ura3 leu2 trpl his3 rad53 yMP10947 MATa ura3 leu2 trpl his3 rad9A::HIS3 rad53 yMP10949 MATa ura3 leu2 trpl his3 rad53 yMP10951 MATa ura3 leu2 trpl his3 rad53 yMP10952 MATa ura3 leu2 trpl his3 rad9A::HIS3 rad53 yMP10953 MATa ura3 leu2 trpl his3 radYA::HIS3 rad53 yMP10954 MATa ura3 leu2 trpl his3 rad9A::HIS3 rad53 yMP10955 MATa ura3 bu2 trbl his3 rad53 S Phase Regulation in S. cereuisiae 47 TABLE 1 Continued Strain Genotype yMP10956 MATa ura3 leu2 trpl his3 rad9A::HIS3 rad53 yMP10961 MATa ura3 leu2 trpl his3 rad24A::TRPl rad53 yMP10964 MATa ura3 leu2 trpl his3 rad24A::TRPl rad53 yMP10983 MATa ade2 ade3-I30 ura3 leu2 tql cyh2 SCfi:URA3 m1hlA::TRPl yMPllO58 MATa ura3 leu2 trpl mad3A::LEU2 yMP11069 MATa ura3 leu2 trpl his3 rad53 smll yMP11070 MATa ura3 leu2 trpl his3 rad53 smll yMP11071 MATa ura3 leu2 trpl his3 rad53 smll yMP11072 MATa ura3 leu2 trpl his3 rad53 smll yMPllO73 MATa ura3 leu2 trpl his3 rad53 smll yMP11074 MATa ura3 leu2 trpl his3 rad53 smll yMPllO82 MATa ade2ade3-130 ura3 leu2 trpl cyh2 SCfi:URA3pmlA::LEU2 Y202 MATa canl-100 ade2-1 hsi3-11,15 leu2-3,112 tql-1 ura3-IO0 Y286 MATa canl-100 ade2-l his3-11,15 leu2-3,112 trpl-I ura3-100 dunI-A100::HIS3 Y80 MATa ura3-1 his3-11,15 leu2-3,112 trpl-I ade2-1 cad-100 sad4 MATa ura3-l his3-11,15 leu2-3,112 trpl-I ade2-1 cad-100 sad4-1 TELl MATa ade2 his3 trpl ura3 leu2 tell MATa ade2ura3 his3 trpl leu2 PY38 MATa ura3-52 tqlA901 leu2-3,112 canl po130Al+pBL2II-POL30 p0130-46 MATa ura3-52 trplA901 leu2-3,112 canl po13OAl + p230-46 ~0130-52 MATa ura3-52 trplA 901 leu2-3,112 canl po130AI + p230-52 telangiectasia(AT) fail to inhibit both the initiation Yeast strains: Allyeast strains with the designation yMP (Table l),and also strain 7830-24a, are in the A364a back- and elongation of DNA replicons in response to DNA ground.
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