Proliferating human cells hypomorphic for origin recognition complex 2 and pre-replicative complex formation have a defect in p53 activation and Cdk2 kinase activation.
The Origin Recognition Complex (ORC) is a critical component of replication initiation. We have previously reported generation of an Orc2 hypomorph cell line (Delta/-) that expresses very low levels of Orc2 but is viable. We have shown here that Chk2 is phosphorylated, suggesting that DNA damage checkpoint pathways are activated. p53 was inactivated during the derivation of the Orc2 hypomorphic cell lines, accounting for their survival despite active Chk2. These cells also show a defect in the G1 to S-phase transition. Cdk2 kinase activation in G1 is decreased due to decreased Cyclin E levels, preventing progression into S-phase. Molecular combing of bromodeoxyuridine-labeled DNA revealed that once the Orc2 hypomorphic cells enter S-phase, fork density and fork progression are approximately comparable with wild type cells. Therefore, the low level of Orc2 hinders normal cell cycle progression by delaying the activation of G1 cyclin-dependent kinases. The results suggest that hypomorphic mutations in initiation factor genes may be particularly deleterious in cancers with mutant p53 or increased activity of Cyclin E/Cdk2.
Teer
Jamie K
JK
Biological and Biomedical Sciences Program, Harvard Medical School, Boston, Massachusetts 02115, USA.
Machida
Yuichi J
YJ
Labit
Helene
H
Novac
Olivia
O
Hyrien
Olivier
O
Marheineke
Kathrin
K
Zannis-Hadjopoulos
Maria
M
Dutta
Anindya
A
D002453:Cell Cycle / Q000235:genetics; D049108:Cell Enlargement; D049109:Cell Proliferation; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2 / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication / Q000235:genetics*; D004789:Enzyme Activation / Q000235:genetics; D006580:Genetic Carrier Screening; D045325:HCT116 Cells; D006801:Humans; D009154:Mutation; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D051738:Origin Recognition Complex / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D016196:S Phase / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
D019927:Cyclin E; D009363:Neoplasm Proteins; D051738:Origin Recognition Complex; D016159:Tumor Suppressor Protein p53; D051357:Cyclin-Dependent Kinase 2