A genetic screen for replication initiation defective (rid) mutants in Schizosaccharomyces pombe.
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.
Locovei
Alexandra M
AM
Department of Molecular and Cellular Phamacology, University of Miami School of Medicine PO Box 016189, Miami, FL 33140, USA. gdurso@miami.edu.
Yin
Ling
L
D'Urso
Gennaro
G
Br J Cancer. 2006 Apr 24;94(8):1170-5
16622441
Nat Struct Biol. 2003 Mar;10(3):160-7
12548282
Genetics. 2007 Jul;176(3):1417-29
17483413
EMBO J. 2004 Jan 28;23(2):418-28
14739927
EMBO J. 1992 Apr;11(4):1343-50
1563350
J Clin Oncol. 2003 Dec 1;21(23):4306-13
14645419
Biol Cell. 2005 Oct;97(10):799-814
15760303
Genes Dev. 2003 Jul 15;17(14):1755-67
12865299
Nature. 2007 Jan 18;445(7125):281-5
17167417
EMBO J. 1986 Nov;5(11):2973-9
16453724
Br J Cancer. 2007 Apr 10;96(7):1107-11
17342084
Mol Cell. 2006 Jan 20;21(2):143-4
16427002
Methods Enzymol. 1991;194:795-823
2005825
Nat Chem Biol. 2008 Jun;4(6):357-65
18469809
Nat Cell Biol. 2006 Apr;8(4):358-66
16531994
Nat Genet. 2007 Jan;39(1):93-8
17143284
Mol Cell. 2006 Jan 6;21(1):29-39
16387651
Mol Genet Genomics. 2001 Aug;265(6):1039-49
11523776
Mol Cell Biol. 2004 Oct;24(19):8342-55
15367656
Genes Dev. 2008 Jul 1;22(13):1816-27
18593882
Mol Cell. 2008 Oct 10;32(1):106-17
18851837
Science. 2000 Dec 22;290(5500):2309-12
11125146
J Biol Chem. 2001 Jul 13;276(28):26189-96
11344166
Genetics. 1997 Nov;147(3):1025-41
9383050
Mol Cell. 2004 Oct 22;16(2):173-85
15494305
J Struct Biol. 2004 Apr-May;146(1-2):234-41
15037254
Nature. 2007 Jan 18;445(7125):328-32
17167415
J Biol Chem. 2004 Nov 26;279(48):49599-608
15371427
Mol Cell. 2008 Oct 24;32(2):166-8
18951084
Proc Natl Acad Sci U S A. 2004 Sep 28;101(39):14085-90
15371597
Mol Biol Cell. 2005 Nov;16(11):5269-82
16148040
Mol Cell Biol. 2004 Aug;24(16):7082-90
15282308
DNA Repair (Amst). 2007 Nov;6(11):1607-17
17618841
J Biol Chem. 2007 Feb 23;282(8):5346-55
17189249
Mol Biol Cell. 2007 Oct;18(10):3894-902
17652453
Genetics. 2007 Feb;175(2):553-66
17151242
J Cell Sci. 2000 Nov;113 ( Pt 22):3889-96
11058076
Curr Opin Genet Dev. 2001 Feb;11(1):64-70
11163153
J Biol Chem. 2004 Oct 29;279(44):45586-93
15326181
Curr Opin Genet Dev. 2001 Feb;11(1):78-82
11163155
Mol Cell Biol. 2003 Aug;23(16):5939-46
12897162
FEBS J. 2006 Mar;273(6):1224-39
16519687
Nature. 1998 Oct 1;395(6701):507-10
9774107
Mol Cell Biol. 2000 May;20(10):3667-76
10779356
Curr Biol. 2004 Sep 21;14(18):R778-86
15380092
Mol Cell. 2005 Sep 2;19(5):699-706
16137625
Mol Biol Cell. 2008 Oct;19(10):4374-82
18667534
Mol Cell Biol. 2001 Jul;21(14):4495-504
11416129
Biochemistry. 2008 Sep 23;47(38):9981-6
18754676
Nat Cell Biol. 2001 Nov;3(11):966-72
11715017
Science. 1998 May 8;280(5365):909-12
9572736
Cancer Epidemiol Biomarkers Prev. 2008 Oct;17(10):2855-64
18843031
Science. 2002 Aug 9;297(5583):1026-31
12169736
Nat Cell Biol. 2003 Feb;5(2):161-5
12545175
Cell. 2000 Sep 15;102(6):745-51
11030618
Eur J Cancer. 2010 Jan;46(1):33-40
19815406
EMBO J. 2004 Sep 15;23(18):3667-76
15329670
Nat Cell Biol. 2001 Nov;3(11):958-65
11715016
Curr Biol. 2001 Nov 27;11(23):R953-6
11728320
Mol Genet Genomics. 2007 Jun;277(6):685-99
17323081
Genes Cells. 2009 Jun;14(6):669-82
19422421
Mol Cell Biol. 1999 Jun;19(6):4262-9
10330167
Mol Cell. 2000 Oct;6(4):839-49
11090622
Cancer Res. 2004 Oct 1;64(19):7110-6
15466207
J Biol Chem. 2004 Apr 16;279(16):16144-53
14766746
J Biol Chem. 2009 Dec 4;284(49):34355-65
19819872