title: A genetic screen for replication initiation defective (rid) mutants in Schizosaccharomyces pombe.
abstract: In fission yeast the intra-S phase and DNA damage checkpoints are activated in response to inhibition of DNA replication or DNA damage, respectively. The intra-S phase checkpoint responds to stalled replication forks leading to the activation of the Cds1 kinase that both delays cell cycle progression and stabilizes DNA replication forks. The DNA damage checkpoint, that operates during the G2 phase of the cell cycle delays mitotic progression through activation of the checkpoint kinase, Chk1. Delay of the cell cycle is believed to be essential to allow time for either replication restart (in S phase) or DNA damage repair (in G2). Previously, our laboratory showed that fission yeast cells deleted for the N-terminal half of DNA polymerase ε (Cdc20) are delayed in S phase, but surprisingly require Chk1 rather than Cds1 to maintain cell viability. Several additional DNA replication mutants were then tested for their dependency on Chk1 or Cds1 when grown under semi-permissive temperatures. We discovered that mutants defective in DNA replication initiation are sensitive only to loss of Chk1, whilst mutations that inhibit DNA replication elongation are sensitive to loss of both Cds1 and Chk1. To confirm that the Chk1-sensitive, Cds1-insensitive phenotype (rid phenotype) is specific to mutants defective in DNA replication initiation, we completed a genetic screen for cell cycle mutants that require Chk1, but not Cds1 to maintain cell viability when grown at semi-permissive temperatures. Our screen identified two mutants, rid1-1 and rid2-1, that are defective in Orc1 and Mcm4, respectively. Both mutants show defects in DNA replication initiation consistent with our hypothesis that the rid phenotype is replication initiation specific. In the case of Mcm4, the mutation has been mapped to a highly conserved region of the protein that appears to be required for DNA replication initiation, but not elongation. Therefore, we conclude that the cellular response to inhibition of DNA replication initiation is distinct from blocking DNA replication elongation, and this difference can be exploited to identify mutants specifically defective in DNA replication initiation.
authors.affiliation: Department of Molecular and Cellular Phamacology, University of Miami School of Medicine PO Box 016189, Miami, FL 33140, USA. gdurso@miami.edu.
authors.forename: Alexandra M
authors.initials: AM
authors.lastname: Locovei
authors.forename: Ling
authors.initials: L
authors.lastname: Yin
authors.forename: Gennaro
authors.initials: G
authors.lastname: D'Urso
references.citation: Br J Cancer. 2006 Apr 24;94(8):1170-5
references.pmid: 16622441
references.citation: Nat Struct Biol. 2003 Mar;10(3):160-7
references.pmid: 12548282
references.citation: Genetics. 2007 Jul;176(3):1417-29
references.pmid: 17483413
references.citation: EMBO J. 2004 Jan 28;23(2):418-28
references.pmid: 14739927
references.citation: EMBO J. 1992 Apr;11(4):1343-50
references.pmid: 1563350
references.citation: J Clin Oncol. 2003 Dec 1;21(23):4306-13
references.pmid: 14645419
references.citation: Biol Cell. 2005 Oct;97(10):799-814
references.pmid: 15760303
references.citation: Genes Dev. 2003 Jul 15;17(14):1755-67
references.pmid: 12865299
references.citation: Nature. 2007 Jan 18;445(7125):281-5
references.pmid: 17167417
references.citation: EMBO J. 1986 Nov;5(11):2973-9
references.pmid: 16453724
references.citation: Br J Cancer. 2007 Apr 10;96(7):1107-11
references.pmid: 17342084
references.citation: Mol Cell. 2006 Jan 20;21(2):143-4
references.pmid: 16427002
references.citation: Methods Enzymol. 1991;194:795-823
references.pmid: 2005825
references.citation: Nat Chem Biol. 2008 Jun;4(6):357-65
references.pmid: 18469809
references.citation: Nat Cell Biol. 2006 Apr;8(4):358-66
references.pmid: 16531994
references.citation: Nat Genet. 2007 Jan;39(1):93-8
references.pmid: 17143284
references.citation: Mol Cell. 2006 Jan 6;21(1):29-39
references.pmid: 16387651
references.citation: Mol Genet Genomics. 2001 Aug;265(6):1039-49
references.pmid: 11523776
references.citation: Mol Cell Biol. 2004 Oct;24(19):8342-55
references.pmid: 15367656
references.citation: Genes Dev. 2008 Jul 1;22(13):1816-27
references.pmid: 18593882
references.citation: Mol Cell. 2008 Oct 10;32(1):106-17
references.pmid: 18851837
references.citation: Science. 2000 Dec 22;290(5500):2309-12
references.pmid: 11125146
references.citation: J Biol Chem. 2001 Jul 13;276(28):26189-96
references.pmid: 11344166
references.citation: Genetics. 1997 Nov;147(3):1025-41
references.pmid: 9383050
references.citation: Mol Cell. 2004 Oct 22;16(2):173-85
references.pmid: 15494305
references.citation: J Struct Biol. 2004 Apr-May;146(1-2):234-41
references.pmid: 15037254
references.citation: Nature. 2007 Jan 18;445(7125):328-32
references.pmid: 17167415
references.citation: J Biol Chem. 2004 Nov 26;279(48):49599-608
references.pmid: 15371427
references.citation: Mol Cell. 2008 Oct 24;32(2):166-8
references.pmid: 18951084
references.citation: Proc Natl Acad Sci U S A. 2004 Sep 28;101(39):14085-90
references.pmid: 15371597
references.citation: Mol Biol Cell. 2005 Nov;16(11):5269-82
references.pmid: 16148040
references.citation: Mol Cell Biol. 2004 Aug;24(16):7082-90
references.pmid: 15282308
references.citation: DNA Repair (Amst). 2007 Nov;6(11):1607-17
references.pmid: 17618841
references.citation: J Biol Chem. 2007 Feb 23;282(8):5346-55
references.pmid: 17189249
references.citation: Mol Biol Cell. 2007 Oct;18(10):3894-902
references.pmid: 17652453
references.citation: Genetics. 2007 Feb;175(2):553-66
references.pmid: 17151242
references.citation: J Cell Sci. 2000 Nov;113 ( Pt 22):3889-96
references.pmid: 11058076
references.citation: Curr Opin Genet Dev. 2001 Feb;11(1):64-70
references.pmid: 11163153
references.citation: J Biol Chem. 2004 Oct 29;279(44):45586-93
references.pmid: 15326181
references.citation: Curr Opin Genet Dev. 2001 Feb;11(1):78-82
references.pmid: 11163155
references.citation: Mol Cell Biol. 2003 Aug;23(16):5939-46
references.pmid: 12897162
references.citation: FEBS J. 2006 Mar;273(6):1224-39
references.pmid: 16519687
references.citation: Nature. 1998 Oct 1;395(6701):507-10
references.pmid: 9774107
references.citation: Mol Cell Biol. 2000 May;20(10):3667-76
references.pmid: 10779356
references.citation: Curr Biol. 2004 Sep 21;14(18):R778-86
references.pmid: 15380092
references.citation: Mol Cell. 2005 Sep 2;19(5):699-706
references.pmid: 16137625
references.citation: Mol Biol Cell. 2008 Oct;19(10):4374-82
references.pmid: 18667534
references.citation: Mol Cell Biol. 2001 Jul;21(14):4495-504
references.pmid: 11416129
references.citation: Biochemistry. 2008 Sep 23;47(38):9981-6
references.pmid: 18754676
references.citation: Nat Cell Biol. 2001 Nov;3(11):966-72
references.pmid: 11715017
references.citation: Science. 1998 May 8;280(5365):909-12
references.pmid: 9572736
references.citation: Cancer Epidemiol Biomarkers Prev. 2008 Oct;17(10):2855-64
references.pmid: 18843031
references.citation: Science. 2002 Aug 9;297(5583):1026-31
references.pmid: 12169736
references.citation: Nat Cell Biol. 2003 Feb;5(2):161-5
references.pmid: 12545175
references.citation: Cell. 2000 Sep 15;102(6):745-51
references.pmid: 11030618
references.citation: Eur J Cancer. 2010 Jan;46(1):33-40
references.pmid: 19815406
references.citation: EMBO J. 2004 Sep 15;23(18):3667-76
references.pmid: 15329670
references.citation: Nat Cell Biol. 2001 Nov;3(11):958-65
references.pmid: 11715016
references.citation: Curr Biol. 2001 Nov 27;11(23):R953-6
references.pmid: 11728320
references.citation: Mol Genet Genomics. 2007 Jun;277(6):685-99
references.pmid: 17323081
references.citation: Genes Cells. 2009 Jun;14(6):669-82
references.pmid: 19422421
references.citation: Mol Cell Biol. 1999 Jun;19(6):4262-9
references.pmid: 10330167
references.citation: Mol Cell. 2000 Oct;6(4):839-49
references.pmid: 11090622
references.citation: Cancer Res. 2004 Oct 1;64(19):7110-6
references.pmid: 15466207
references.citation: J Biol Chem. 2004 Apr 16;279(16):16144-53
references.pmid: 14766746
references.citation: J Biol Chem. 2009 Dec 4;284(49):34355-65
references.pmid: 19819872