GEMC1 is a TopBP1-interacting protein required for chromosomal DNA replication.
Many of the factors required for chromosomal DNA replication have been identified in unicellular eukaryotes. However, DNA replication is poorly understood in multicellular organisms. Here, we report the identification of GEMC1 (geminin coiled-coil containing protein 1), a novel vertebrate protein required for chromosomal DNA replication. GEMC1 is highly conserved in vertebrates and is preferentially expressed in proliferating cells. Using Xenopus laevis egg extract we show that Xenopus GEMC1 (xGEMC1) binds to the checkpoint and replication factor TopBP1, which promotes binding of xGEMC1 to chromatin during pre-replication complex (pre-RC) formation. We demonstrate that xGEMC1 interacts directly with replication factors such as Cdc45 and the kinase Cdk2-CyclinE, through which it is heavily phosphorylated. Phosphorylated xGEMC1 stimulates initiation of DNA replication, whereas depletion of xGEMC1 prevents the onset of DNA replication owing to the impairment of Cdc45 loading onto chromatin. Similarly, inhibition of GEMC1 expression with morpholino and siRNA oligos prevents DNA replication in embryonic and somatic vertebrate cells. These data suggest that GEMC1 promotes initiation of chromosomal DNA replication in multicellular organisms by mediating TopBP1- and Cdk2-dependent recruitment of Cdc45 onto replication origins.
Balestrini
Alessia
A
Genome Stability, London Research Institute, Hertfordshire, UK.
Cosentino
Claudia
C
Errico
Alessia
A
Garner
Elizabeth
E
Costanzo
Vincenzo
V
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000378:metabolism; D002875:Chromosomes; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins; D008969:Molecular Sequence Data; D010063:Ovum; D011485:Protein Binding; D018741:Replication Origin / Q000235:genetics; D029867:Xenopus Proteins / Q000378:metabolism; D014982:Xenopus laevis
D002352:Carrier Proteins; C502211:Cdc45 protein, Xenopus; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C551026:GMNC protein, Xenopus; C509993:TopBP1 protein, Xenopus; D029867:Xenopus Proteins; C495891:Cdk2 protein, Xenopus; D051357:Cyclin-Dependent Kinase 2
Dev Dyn. 2002 Dec;225(4):499-510
12454926
EMBO J. 2001 Apr 17;20(8):2097-107
11296242
Mol Cell. 2002 Feb;9(2):233-40
11864598
Cell. 1988 Jul 29;54(3):423-31
3293802
Cell. 1987 Jan 30;48(2):205-17
3026635
Mol Biol Cell. 2002 May;13(5):1462-72
12006645
J Cell Biol. 1993 Dec;123(6 Pt 1):1321-31
8253833
EMBO J. 1998 Oct 1;17(19):5699-707
9755170
Genes Dev. 2003 May 1;17(9):1141-52
12730133
Cell. 2005 Jun 17;121(6):887-98
15960976
Nature. 2007 Jan 18;445(7125):281-5
17167417
Cell Div. 2007 Jun 05;2:16
17547773
EMBO J. 2003 May 15;22(10):2526-35
12743046
Nature. 2007 Jan 18;445(7125):328-32
17167415
Nat Cell Biol. 2004 Jul;6(7):648-55
15220931
Cell. 1998 Jun 12;93(6):1043-53
9635433
EMBO J. 2008 Mar 19;27(6):876-85
18309293
Mol Cell. 2004 Jul 23;15(2):245-58
15260975
J Cell Biol. 2002 Nov 25;159(4):541-7
12438414
Mol Cell. 2000 Apr;5(4):617-27
10882098
EMBO J. 2008 Mar 5;27(5):758-69
18256689
J Cell Sci. 2004 Dec 1;117(Pt 25):6019-30
15536124
Nature. 2004 Oct 28;431(7012):1118-23
15496876
Curr Biol. 2004 Sep 21;14(18):R778-86
15380092
Cell. 1986 Nov 21;47(4):577-87
3779837
Methods Mol Biol. 2004;241:255-67
14970660
Proc Natl Acad Sci U S A. 1998 Sep 1;95(18):10453-8
9724724
EMBO J. 1998 Sep 1;17(17):5182-91
9724654
Curr Opin Cell Biol. 2000 Dec;12(6):690-6
11063933
Mol Cell Biol. 2006 Jul;26(13):4843-52
16782873