Mediation of proliferating cell nuclear antigen (PCNA)-dependent DNA replication through a conserved p21(Cip1)-like PCNA-binding motif present in the third subunit of human DNA polymerase delta.

M Ducoux; S Urbach; G Baldacci; U Hübscher; S Koundrioukoff; Jesper Aagaard Christensen; P Hughes

doi:10.1074/jbc.M106990200

Mediation of proliferating cell nuclear antigen (PCNA)-dependent DNA replication through a conserved p21(Cip1)-like PCNA-binding motif present in the third subunit of human DNA polymerase delta.

M Ducoux, S Urbach, G Baldacci, U Hübscher, S Koundrioukoff, Jesper Aagaard Christensen, P Hughes

76 Citations (Scopus)

Abstract

The subunit that mediates binding of proliferating cell nuclear antigen (PCNA) to human DNA polymerase delta has not been clearly defined. We show that the third subunit of human DNA polymerase delta, p66, interacts with PCNA through a canonical PCNA-binding sequence located in its C terminus. Conversely, p66 interacts with the domain-interconnecting loop of PCNA, a region previously shown to be important for DNA polymerase delta activity and for binding of the cell cycle inhibitor p21(Cip1). In accordance with this, a peptide containing the PCNA-binding domain of p21(Cip1) inhibited p66 binding to PCNA and the activity of native three-subunit DNA polymerase delta. Furthermore, pull-down assays showed that DNA polymerase delta requires p66 for interaction with PCNA. More importantly, only reconstituted three-subunit DNA polymerase delta displayed PCNA-dependent DNA replication that could be inhibited by the PCNA-binding domain of p21(Cip1). Direct participation of p66 in PCNA-dependent DNA replication in vivo is demonstrated by co-localization of p66 with PCNA and DNA polymerase delta within DNA replication foci. Finally, in vitro phosphorylation of p66 by cyclin-dependent kinases suggests that p66 activity may be subject to cell cycle-dependent regulation. These results suggest that p66 is the chief mediator of PCNA-dependent DNA synthesis by DNA polymerase delta.

Original language	English
Journal	Journal of Biological Chemistry
Volume	276
Issue number	52
Pages (from-to)	49258-66
Number of pages	8
ISSN	0021-9258
DOIs	https://doi.org/10.1074/jbc.M106990200
Publication status	Published - 2001
Externally published	Yes

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Ducoux, M., Urbach, S., Baldacci, G., Hübscher, U., Koundrioukoff, S., Christensen, J. A., & Hughes, P. (2001). Mediation of proliferating cell nuclear antigen (PCNA)-dependent DNA replication through a conserved p21(Cip1)-like PCNA-binding motif present in the third subunit of human DNA polymerase delta. Journal of Biological Chemistry, 276(52), 49258-66. https://doi.org/10.1074/jbc.M106990200

Mediation of proliferating cell nuclear antigen (PCNA)-dependent DNA replication through a conserved p21(Cip1)-like PCNA-binding motif present in the third subunit of human DNA polymerase delta. / Ducoux, M; Urbach, S; Baldacci, G et al.

In: Journal of Biological Chemistry, Vol. 276, No. 52, 2001, p. 49258-66.

Research output: Contribution to journal › Journal article › Research › peer-review

Ducoux, M, Urbach, S, Baldacci, G, Hübscher, U, Koundrioukoff, S, Christensen, JA & Hughes, P 2001, 'Mediation of proliferating cell nuclear antigen (PCNA)-dependent DNA replication through a conserved p21(Cip1)-like PCNA-binding motif present in the third subunit of human DNA polymerase delta.', Journal of Biological Chemistry, vol. 276, no. 52, pp. 49258-66. https://doi.org/10.1074/jbc.M106990200

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title = "Mediation of proliferating cell nuclear antigen (PCNA)-dependent DNA replication through a conserved p21(Cip1)-like PCNA-binding motif present in the third subunit of human DNA polymerase delta.",

abstract = "The subunit that mediates binding of proliferating cell nuclear antigen (PCNA) to human DNA polymerase delta has not been clearly defined. We show that the third subunit of human DNA polymerase delta, p66, interacts with PCNA through a canonical PCNA-binding sequence located in its C terminus. Conversely, p66 interacts with the domain-interconnecting loop of PCNA, a region previously shown to be important for DNA polymerase delta activity and for binding of the cell cycle inhibitor p21(Cip1). In accordance with this, a peptide containing the PCNA-binding domain of p21(Cip1) inhibited p66 binding to PCNA and the activity of native three-subunit DNA polymerase delta. Furthermore, pull-down assays showed that DNA polymerase delta requires p66 for interaction with PCNA. More importantly, only reconstituted three-subunit DNA polymerase delta displayed PCNA-dependent DNA replication that could be inhibited by the PCNA-binding domain of p21(Cip1). Direct participation of p66 in PCNA-dependent DNA replication in vivo is demonstrated by co-localization of p66 with PCNA and DNA polymerase delta within DNA replication foci. Finally, in vitro phosphorylation of p66 by cyclin-dependent kinases suggests that p66 activity may be subject to cell cycle-dependent regulation. These results suggest that p66 is the chief mediator of PCNA-dependent DNA synthesis by DNA polymerase delta.",

author = "M Ducoux and S Urbach and G Baldacci and U H{\"u}bscher and S Koundrioukoff and Christensen, {Jesper Aagaard} and P Hughes",

note = "Keywords: Amino Acid Motifs; Amino Acid Sequence; Animals; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA Polymerase III; DNA Replication; Hela Cells; Humans; Immunohistochemistry; Molecular Sequence Data; Mutation; Peptide Fragments; Phosphorylation; Proliferating Cell Nuclear Antigen; Protein Binding; Protein Subunits; Recombinant Proteins; Sequence Alignment",

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T1 - Mediation of proliferating cell nuclear antigen (PCNA)-dependent DNA replication through a conserved p21(Cip1)-like PCNA-binding motif present in the third subunit of human DNA polymerase delta.

AU - Ducoux, M

AU - Urbach, S

AU - Baldacci, G

AU - Hübscher, U

AU - Koundrioukoff, S

AU - Christensen, Jesper Aagaard

AU - Hughes, P

N1 - Keywords: Amino Acid Motifs; Amino Acid Sequence; Animals; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA Polymerase III; DNA Replication; Hela Cells; Humans; Immunohistochemistry; Molecular Sequence Data; Mutation; Peptide Fragments; Phosphorylation; Proliferating Cell Nuclear Antigen; Protein Binding; Protein Subunits; Recombinant Proteins; Sequence Alignment

PY - 2001

Y1 - 2001

N2 - The subunit that mediates binding of proliferating cell nuclear antigen (PCNA) to human DNA polymerase delta has not been clearly defined. We show that the third subunit of human DNA polymerase delta, p66, interacts with PCNA through a canonical PCNA-binding sequence located in its C terminus. Conversely, p66 interacts with the domain-interconnecting loop of PCNA, a region previously shown to be important for DNA polymerase delta activity and for binding of the cell cycle inhibitor p21(Cip1). In accordance with this, a peptide containing the PCNA-binding domain of p21(Cip1) inhibited p66 binding to PCNA and the activity of native three-subunit DNA polymerase delta. Furthermore, pull-down assays showed that DNA polymerase delta requires p66 for interaction with PCNA. More importantly, only reconstituted three-subunit DNA polymerase delta displayed PCNA-dependent DNA replication that could be inhibited by the PCNA-binding domain of p21(Cip1). Direct participation of p66 in PCNA-dependent DNA replication in vivo is demonstrated by co-localization of p66 with PCNA and DNA polymerase delta within DNA replication foci. Finally, in vitro phosphorylation of p66 by cyclin-dependent kinases suggests that p66 activity may be subject to cell cycle-dependent regulation. These results suggest that p66 is the chief mediator of PCNA-dependent DNA synthesis by DNA polymerase delta.

AB - The subunit that mediates binding of proliferating cell nuclear antigen (PCNA) to human DNA polymerase delta has not been clearly defined. We show that the third subunit of human DNA polymerase delta, p66, interacts with PCNA through a canonical PCNA-binding sequence located in its C terminus. Conversely, p66 interacts with the domain-interconnecting loop of PCNA, a region previously shown to be important for DNA polymerase delta activity and for binding of the cell cycle inhibitor p21(Cip1). In accordance with this, a peptide containing the PCNA-binding domain of p21(Cip1) inhibited p66 binding to PCNA and the activity of native three-subunit DNA polymerase delta. Furthermore, pull-down assays showed that DNA polymerase delta requires p66 for interaction with PCNA. More importantly, only reconstituted three-subunit DNA polymerase delta displayed PCNA-dependent DNA replication that could be inhibited by the PCNA-binding domain of p21(Cip1). Direct participation of p66 in PCNA-dependent DNA replication in vivo is demonstrated by co-localization of p66 with PCNA and DNA polymerase delta within DNA replication foci. Finally, in vitro phosphorylation of p66 by cyclin-dependent kinases suggests that p66 activity may be subject to cell cycle-dependent regulation. These results suggest that p66 is the chief mediator of PCNA-dependent DNA synthesis by DNA polymerase delta.

U2 - 10.1074/jbc.M106990200

DO - 10.1074/jbc.M106990200

M3 - Journal article

C2 - 11595739

SN - 0021-9258

VL - 276

SP - 49258

EP - 49266

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 52

ER -

Mediation of proliferating cell nuclear antigen (PCNA)-dependent DNA replication through a conserved p21(Cip1)-like PCNA-binding motif present in the third subunit of human DNA polymerase delta.

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