Adenomatous polyposis coli protein regulates the cellular response to DNA replication stress

Mariana G. Brocardo, James A. Borowiec, Beric R. Henderson

Research output: Contribution to journalArticleResearchpeer-review

Abstract

The adenomatous polyposis coli (APC) tumor suppressor traffics between nucleus and cytoplasm to perform distinct functions. Here we identify a specific role for APC in the DNA replication stress response. The silencing of APC caused an accumulation of asynchronous cells in early S phase and delayed S phase progression in cells released from hydroxyurea-mediated replication arrest. Immunoprecipitation assays revealed a selective binding of APC to replication protein A 32 kDa subunit (RPA32), and the APC-RPA32 complex increased at chromatin after hydroxyurea treatment. Interestingly, APC knock-down prevented accumulation at chromatin of the stress-induced S33- and S29-phosphorylated forms of RPA32, and reduced the expression of ATR-phosphorylated forms of S317-phospho-Chk1 and γ-H2AX. Using RPA32-inducible cells we showed that reconstitution of RPA32 diminished the S-phase delay caused by loss of APC. In contrast to full-length APC, the truncated APC mutant protein expressed in SW480 colon cancer cells was impaired in its binding and regulation of RPA32, and failed to regulate cell cycle after replication stress. We propose that APC associates with RPA at stalled DNA replication forks and promotes the ATR-dependent phosphorylation of RPA32, Chk1 and γ-H2AX in response to DNA replication stress, thereby influencing the rate of re-entry into the cell cycle.

LanguageEnglish
Pages1354-1364
Number of pages11
JournalInternational Journal of Biochemistry and Cell Biology
Volume43
Issue number9
DOIs
Publication statusPublished - Sep 2011
Externally publishedYes

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Adenomatous Polyposis Coli Protein
Replication Protein A
Adenomatous Polyposis Coli
DNA Replication
DNA
Cells
S Phase
Hydroxyurea
Chromatin
Cell Cycle
Phosphorylation
Reentry
Mutant Proteins
Tumors
Immunoprecipitation
Assays
Colonic Neoplasms
Cytoplasm

Cite this

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title = "Adenomatous polyposis coli protein regulates the cellular response to DNA replication stress",
abstract = "The adenomatous polyposis coli (APC) tumor suppressor traffics between nucleus and cytoplasm to perform distinct functions. Here we identify a specific role for APC in the DNA replication stress response. The silencing of APC caused an accumulation of asynchronous cells in early S phase and delayed S phase progression in cells released from hydroxyurea-mediated replication arrest. Immunoprecipitation assays revealed a selective binding of APC to replication protein A 32 kDa subunit (RPA32), and the APC-RPA32 complex increased at chromatin after hydroxyurea treatment. Interestingly, APC knock-down prevented accumulation at chromatin of the stress-induced S33- and S29-phosphorylated forms of RPA32, and reduced the expression of ATR-phosphorylated forms of S317-phospho-Chk1 and γ-H2AX. Using RPA32-inducible cells we showed that reconstitution of RPA32 diminished the S-phase delay caused by loss of APC. In contrast to full-length APC, the truncated APC mutant protein expressed in SW480 colon cancer cells was impaired in its binding and regulation of RPA32, and failed to regulate cell cycle after replication stress. We propose that APC associates with RPA at stalled DNA replication forks and promotes the ATR-dependent phosphorylation of RPA32, Chk1 and γ-H2AX in response to DNA replication stress, thereby influencing the rate of re-entry into the cell cycle.",
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Adenomatous polyposis coli protein regulates the cellular response to DNA replication stress. / Brocardo, Mariana G.; Borowiec, James A.; Henderson, Beric R.

In: International Journal of Biochemistry and Cell Biology, Vol. 43, No. 9, 09.2011, p. 1354-1364.

Research output: Contribution to journalArticleResearchpeer-review

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