p12(DOC-1), a growth suppressor, associates with DNA polymerase alpha/primase.
p12(DOC-1) is a growth suppressor identified and isolated from normal keratinocytes. Ectopic expression of p12(DOC-1) in squamous carcinoma cells led to the reversion of in vitro transformation phenotypes including anchorage independence, doubling time, and morphology. Here we report that p12(DOC-1) associates with DNA polymerase alpha/primase (pol-alpha:primase) in vitro and in cells. The pol-alpha:primase binding domain in p12(DOC-1) is mapped to the amino-terminal six amino acid (MSYKPN). The biological effect of p12(DOC-1) on pol-alpha:primase was examined using in vitro DNA replication assays. Using the SV40 DNA replication assay, p12(DOC-1) suppresses DNA replication, leveling at approximately 50%. Similar results were obtained using the M13 single-stranded DNA synthesis assay. Analysis of the DNA replication products revealed that p12(DOC-1) affects the initiation step, not the elongation phase. The p12(DOC-1) suppression of DNA replication is likely to be mediated either by a direct inhibitory effect on pol-alpha:primase or by its effect on cyclin-dependent kinase 2 (CDK2), a recently identified p12(DOC-1)-associated protein known to stimulate DNA replication by phosphorylating pol-alpha:primase. p12(DOC-1) suppresses CDK2-mediated phosphorylation of pol-alpha:primase. These data support a role of p12(DOC-1) as a regulator of DNA replication by direct inhibition of pol-alpha:primase or by negatively regulating the CDK2-mediated phosphorylation of pol-alpha:primase.
Matsuo
K
K
Laboratory of Molecular Pathology, Division of Oral Pathology, and. Harvard University, School of Dental Medicine, Boston, Massachusetts 02115, USA.
Shintani
S
S
Tsuji
T
T
Nagata
E
E
Lerman
M
M
McBride
J
J
Nakahara
Y
Y
Ohyama
H
H
Todd
R
R
Wong
D T
DT
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D042846:CDC2-CDC28 Kinases; D002460:Cell Line; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004256:DNA Polymerase I / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019915:DNA Primase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006131:Growth Inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D025521:Tumor Suppressor Proteins
C096151:CDK2AP1 protein, human; D004791:Enzyme Inhibitors; D006131:Growth Inhibitors; D011506:Proteins; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D019915:DNA Primase; C116932:DNA polymerase alpha-primase; D004256:DNA Polymerase I