HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 18/14/1743 04-2268fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by MAGA, G. Articles by HÜBSCHER, U. Search for Related Content PubMed PubMed Citation Articles by MAGA, G. Articles by HÜBSCHER, U.

The human DNA polymerase interacts with PCNA through a domain important for DNA primer binding and the interaction is inhibited by p21^/WAF1/CIP1

GIOVANNI MAGA^*,1, GIUSEPPINA BLANCA^*,, IGOR SHEVELEV, ISABELLE FROUIN, KRISTIJAN RAMADAN, SILVIO SPADARI^*, GIUSEPPE VILLANI and ULRICH HÜBSCHER

^* Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, Pavia, Italy;
Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique, Toulouse, France; and
Institute of Veterinary Biochemistry and Molecular Biology, University of Zürich, Zürich, Switzerland

¹Correspondence: E-mail: maga{at}igm.cnr.it

SPECIFIC AIMS

In this work we aimed to investigate 1) which structural domain of pol was important for primer binding and/or for PCNA interaction; 2) which domain of PCNA could mediate the interaction with pol ; and 3) whether the tumor suppressor p21^/WAF1/CIP1 could affect the interaction between pol and PCNA.

PRINCIPAL FINDINGS

1. Using co-immunoprecipitation experiments, we demonstrated that DNA polymerase (pol ) interacts with proliferating cell nuclear antigen (PCNA) in vivo in human cells

2. Enzymological characterization of selected pol mutants revealed that the residues located in an helix-hairpin-helix motif are important for the interaction of pol with the primer/template DNA
By using pull-down assays, we have shown that these residues are also involved in the pol interaction with PCNA (Fig. 1 A, B).

View larger version (29K): [in this window] [in a new window]   Figure 1. The F338A mutation abolishes the physical interaction of pol with the front side of PCNA. A) Recombinant human PCNA (5 µg) was incubated with recombinant his-tagged human pol either wt (lane 4: 3 µg), or the E330A (lane 1: 5 µg), V334A (lane 2: 5 µg) and F338A (lane 3: 4.5 µg) mutants. In a control reaction, PCNA was incubated in the presence of 5 µg of BSA (lane 5). NiNTA-agarose beads were added to each reaction. Pulled-down fractions were analyzed with antibodies specific to pol and PCNA. The position of the proteins reacting with each antibody is indicated on the left side of the panel. B) Recombinant his-tagged human pol either wt (lane 1: 3 µg) or the V334A (lane 2: 5 µg) and F338A (lane 3: 4.5 µg) mutants bound to NiNTA-agarose beads were incubated with HeLa cells total extract (500 µl, 6 mg/mL). As negative control, 5 µg of BSA were incubated in the presence of the beads and the extract (lane 4). Samples were analyzed with antibodies specific to pol and PCNA. The position of the proteins reacting with each antibody is indicated on the left side of the panel. C,upper panel) Recombinant human PCNA, either wild-type (filled circles), or the LAPK251 (squares), QLGI125 (open circles), and SHV43 mutants (triangles), were titrated in the presence of 25 nM pol and 50 nM (3'-OH ends) of the homopolymeric DNA template poly(dA)/oligo(dT). Total amounts of incorporated radioactive dTTP were plotted as a function of the PCNA concentrations (expressed as trimers). C, lower panel) Pol (25 nM) was tested in the presence of increasing concentrations of poly(dA)/oligo(dT). The kinetic parameters Km, kcat, and kcat/Km were calculated in the absence or presence of increasing amounts of PCNA, either wt (circles) or the QLGI125 mutant (triangles). The increase in the kcat/Km values calculated for each set of reactions with respect to the control without PCNA, was plotted as a function of the PCNA concentrations. D) Co-immunoprecipitation of pol and PCNA with polyclonal anti-pol antibodies. Human recombinant pol (500 ng) was incubated in the presence of PCNA (2 µg) wild-type (lane 1) or the SHV43 (lane 2) and the LAPK251 (lane 3) mutants, in PBS. Anti-pol antibodies coupled to Protein G-Sepharose beads were then added. As a negative control, PCNA wild-type was subjected to immunoprecipitation with anti-pol IgG-covered beads but in the absence of pol (lane 4). Beads were washed and bound immunocomplexes were transferred to a membrane and probed with either anti-pol antibodies (upper panel) or with anti-PCNA antibodies (lower panel).

3. The major sites of interaction between PCNA and many of its partners are the interdomain connecting loop (ID loop) of PCNA (aa 121 to 132) and the facing hydrophobic pocket (aa 42 to 46)
Using recombinant PCNA proteins carrying mutations in each of these domains, we have shown that residues 43–45 of the hydrophobic pocket are essential for interaction with pol (Fig. 1C, D ). Residues 125–128 of the ID loop, also play an important role in stabilizing this interaction (Fig. 1C ).

4. The tumor suppressor protein p21^/WAF1/CIP1 can efficiently compete in pull-down assays in vitro with pol for binding to PCNA (Fig. 2 A, B) and can completely abolish the PCNA-dependent stimulation of pol activity (Fig. 2 C, D)

View larger version (30K): [in this window] [in a new window]   Figure 2. The p21 protein competes with pol for binding to PCNA. A) Pull-down assays were performed by incubating GST-p21 (500 ng) with either PCNA alone (1 µg, lanes 1, 4), pol alone (0.75 µg, lane 2), or both PCNA and pol at different PCNA/pol molar ratios (1/1, lane 3; 1/2 lane 5; 2/1, lane 6). Gluthatione-Sepharose beads were then added, washed, and the bound proteins were visualized by immunoblotting with antibodies against pol (upper panel), p21 (middle panel), or PCNA (lower panel). Lane 7: negative control with pol and PCNA incubated together with the beads in the absence of GST-p21. Quantification of the blots is shown at the bottom of the panel as relative (%) amounts of recovered PCNA with respect to the controls. B) Pull-down assays were performed by incubating his-tagged recombinant human pol (500 ng) with wt PCNA (500 ng) either alone (lane 2) or in combination with 200 ng (lane 3) or 400 ng (lane 4) of p21. Ni-NTA beads specific for his-tag binding, were then added, washed, and bound proteins were visualized by immunoblotting with antibodies against pol (upper panel), or PCNA (lower panel). Lane 1: negative control with wt PCNA incubated together with the beads in the absence of pol . Quantification of the blots is shown at the bottom of the panel as relative (%) amounts of recovered PCNA with respect to the controls. C) Pol reactions were carried out in the presence of the 5'-end labeled DNA oligonucleotide primer/template 18/73-mer and in the absence (lane 2) or presence of increasing amounts of wt PCNA (20 nM, lane 3; 50 nM, lane 4) either alone or in combination with increasing amounts of p21 (150 nM, lane 5; 300 nM, lane 6). Products were resolved on a 7M urea/20% PAA gel. D) Recombinant human wt PCNA was titrated in the presence of 20 nM pol and 50 nM (3'-OH ends) of the homopolymeric DNA template poly(dA)/oligo(dT) in either the absence (squares) or presence of 200 nM p21 (triangles) or 400 nM p21 (circles). Total amounts of incorporated radioactive dTTP in the absence or presence of PCNA were determined to calculate the stimulation of pol activity by PCNA. The resulting stimulation (fold) was then plotted as a function of the PCNA concentrations (expressed as trimers).

CONCLUSIONS AND SIGNIFICANCE

The interplay among pol , pol , PCNA, and p21, based on our findings, is presented in Fig. 3 .

View larger version (43K): [in this window] [in a new window]   Figure 3. Schematic diagram.

The discovery that pol binds to PCNA at the same site as many other partners, can have implications for the regulation of such interaction. Based on our previous observations, we have suggested that PCNA could promote bypass of abasic sites during DNA replication. Abasic sites are frequent and are estimated to occur 10,000 times per cell, per day. A mechanism for the bypass of these DNA lesions in eukaryotic cells has been previously proposed, involving pol and pol . Our data, showing that pol interacts with PCNA at the same site as pol , suggest the possibility of a "switching" between the replicative pol and the translesion pol , through direct competition for PCNA, yielding to elongation of the nascent DNA strand past abasic sites.

The p21^/WAF1/CIP1protein is induced upon DNA damage, senescence, or differentiation of cells and blocks progression the cell cycle through its interaction CDKs and PCNA. Indeed, p21 can form a complex with PCNA, preventing interaction with replication factor C and pol . In this paper, we present evidence that p21 can efficiently compete with pol for binding to PCNA (Fig. 2) . This is the first time that p21 has been shown to prevent the interaction of PCNA with a pol other than pol and pol . Further in vivo studies are needed to investigate the physiological significance of this interaction, however these findings raise the possibility that, in principle, the interaction of pol with PCNA might be subjected to cell cycle and/or checkpoint control. Recent genetic data suggest that pol is particularly subjected to –1 frameshift errors. Thus, accurate regulation of its lesion bypass activity by PCNA- and p21-concerted action might be important for preventing genetic instability.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2268fje;

This article has been cited by other articles:

				E. Crespan, U. Hubscher, and G. Maga Error-free bypass of 2-hydroxyadenine by human DNA polymerase {lambda} with Proliferating Cell Nuclear Antigen and Replication Protein A in different sequence contexts Nucleic Acids Res., August 1, 2007; 35(15): 5173 - 5181. [Abstract] [Full Text] [PDF]

				E. Crespan, L. Alexandrova, A. Khandazhinskaya, M. Jasko, M. Kukhanova, G. Villani, U. Hubscher, S. Spadari, and G. Maga Expanding the repertoire of DNA polymerase substrates: template-instructed incorporation of non-nucleoside triphosphate analogues by DNA polymerases {beta} and {lambda} Nucleic Acids Res., January 12, 2007; 35(1): 45 - 57. [Abstract] [Full Text] [PDF]

				F. Vandermoere, I. E. Yazidi-Belkoura, Y. Demont, C. Slomianny, J. Antol, J. Lemoine, and H. Hondermarck Proteomics Exploration Reveals That Actin Is a Signaling Target of the Kinase Akt Mol. Cell. Proteomics, January 1, 2007; 6(1): 114 - 124. [Abstract] [Full Text] [PDF]

				K. K.L. Chan, Q.-M. Zhang, and G. L. Dianov Base excision repair fidelity in normal and cancer cells Mutagenesis, May 1, 2006; 21(3): 173 - 178. [Abstract] [Full Text] [PDF]

				G. Maga, I. Shevelev, G. Villani, S. Spadari, and U. Hubscher Human replication protein A can suppress the intrinsic in vitro mutator phenotype of human DNA polymerase {lambda} Nucleic Acids Res., March 6, 2006; 34(5): 1405 - 1415. [Abstract] [Full Text] [PDF]

				I. Frouin, M. Toueille, E. Ferrari, I. Shevelev, and U. Hubscher Phosphorylation of human DNA polymerase {lambda} by the cyclin-dependent kinase Cdk2/cyclin A complex is modulated by its association with proliferating cell nuclear antigen Nucleic Acids Res., September 20, 2005; 33(16): 5354 - 5361. [Abstract] [Full Text] [PDF]

				G. A. Locatelli, R. Di Santo, E. Crespan, R. Costi, A. Roux, U. Hubscher, I. Shevelev, G. Blanca, G. Villani, S. Spadari, et al. Diketo Hexenoic Acid Derivatives Are Novel Selective Non-Nucleoside Inhibitors of Mammalian Terminal Deoxynucleotidyl Transferases, with Potent Cytotoxic Effect against Leukemic Cells Mol. Pharmacol., August 1, 2005; 68(2): 538 - 550. [Abstract] [Full Text] [PDF]

				E. Crespan, S. Zanoli, A. Khandazhinskaya, I. Shevelev, M. Jasko, L. Alexandrova, M. Kukhanova, G. Blanca, G. Villani, U. Hubscher, et al. Incorporation of non-nucleoside triphosphate analogues opposite to an abasic site by human DNA polymerases {beta} and {lambda} Nucleic Acids Res., July 25, 2005; 33(13): 4117 - 4127. [Abstract] [Full Text] [PDF]

				G. Maga, K. Ramadan, Giada. A. Locatelli, I. Shevelev, S. Spadari, and U. Hubscher DNA Elongation by the Human DNA Polymerase {lambda} Polymerase and Terminal Transferase Activities Are Differentially Coordinated by Proliferating Cell Nuclear Antigen and Replication Protein A J. Biol. Chem., January 21, 2005; 280(3): 1971 - 1981. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 18/14/1743 04-2268fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by MAGA, G. Articles by HÜBSCHER, U. Search for Related Content PubMed PubMed Citation Articles by MAGA, G. Articles by HÜBSCHER, U.