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PMID: 8524220 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

p53 inhibits DNA replication in vitro in a DNA-binding-dependent manner.

Molecular and cellular biology ·Vol. 15 ·No. 12 ·1995-12-00 ·Pages 6554-60

Miller SD, Farmer G, Prives C

Abstract

The p53 tumor suppressor gene product is a sequence-specific DNA-binding protein that is necessary for the G1 arrest of many cell types. Consistent with its role as a cell cycle checkpoint factor, p53 has been shown to be capable of both transcriptional activation and repression. Here we show a new potential role for p53 as a DNA-binding-dependent regulator of DNA replication. Constructs containing multiple copies of the ribosomal gene cluster (RGC) p53 binding site cloned on the late side of the polyomavirus origin were used in in vitro replication assays. In the presence of p53, the replication of these constructs was strongly inhibited, while the replication of constructs containing a mutant version of the RGC site was not affected by p53. Several tumor-derived mutant p53 proteins were unable to inhibit replication of the construct with wild-type RGC sites. Additionally, the transactivator GAL4-VP16 was unable to inhibit replication of a construct containing GAL4 binding sites adjacent to the polyomavirus origin. We also show that the inhibition by p53 can occur from sites cloned as far as 600 bp from the origin. Preincubation experiments suggest that p53 inhibits replication at a step mediated by ATP, possibly by inhibiting the binding of polyomavirus T antigen to the core origin. The presence of an endogenous p53 binding site in the polyomavirus origin suggests potential mechanisms for the observed inhibition.

MeSH Terms
Amino Acid Sequence Animals Antigens, Polyomavirus Transforming/metabolism Base Sequence Binding Sites Cell Cycle Cell Line DNA Replication DNA-Binding Proteins/biosynthesis,metabolism Genes, p53 Kinetics Mice Molecular Sequence Data Mutagenesis, Site-Directed Recombinant Proteins/biosynthesis,metabolism Replication Origin Spodoptera Transcriptional Activation Transfection Tumor Cells, Cultured Tumor Suppressor Protein p53/biosynthesis,metabolism
Chemicals
Antigens, Polyomavirus Transforming DNA-Binding Proteins Recombinant Proteins Tumor Suppressor Protein p53
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Miller S D
Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
Farmer G
Prives C
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.15.12.6554
Published
1995-12-00
Pages
6554-60
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC230908
Subset
IM
Grants
NCI NIH HHS · CA26905 · United States
NCI NIH HHS · CA58316 · United States
External Links
PubMed source DOI: 10.1128/MCB.15.12.6554 PMC Full Text View journal details
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