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

J. Biol. Chem., Vol. 280, Issue 3, 1971-1981, January 21, 2005

This Article Full Text Full Text (PDF) All Versions of this Article: 280/3/1971    most recent M411650200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map 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. Social Bookmarking                      What's this?

DNA Elongation by the Human DNA Polymerase Polymerase and Terminal Transferase Activities Are Differentially Coordinated by Proliferating Cell Nuclear Antigen and Replication Protein A^*

Giovanni Maga, Kristijan Ramadan¶, Giada. A. Locatelli, Igor Shevelev¶, Silvio Spadari, and Ulrich Hübscher¶

From the Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, via Abbiategrasso 207, I-27100 Pavia, Italy and the ^¶Institute of Veterinary Biochemistry and Molecular Biology, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland

DNA polymerase contains template-dependent (DNA polymerase) and template-independent (terminal transferase) activities. In this study we enzymologically characterized the terminal transferase activity of polymerase (pol -tdt). Pol -tdt activity was strongly influenced by the nature of the 3'-terminal sequence of the DNA substrate, and it required a single-stranded (ss) DNA 3'-overhang of about 9–12 nucleotides for optimal activity. The strong preference observed for pyrimidine versus purine nucleotide incorporation was found to be due, at least partially, to a steric block imposed by the residue Tyr-505 in the active site of pol . Pol -tdt was found to be able to elongate a 3'-ssDNA end by two alternative mechanisms: first, a template-independent one resulting in addition of 1 or 2 nucleotides, and second, a template-dependent one where a homopolymeric tract as short as 3 nucleotides at the 3'-end could be used as a template to direct DNA polymerization by a looping back mechanism. Furthermore repetitive cycles of DNA synthesis resulted in the expansion of such a short homopolymeric terminal sequence. Most importantly we found that the proliferating cell nuclear antigen was able to selectively block the looping back mechanism while stimulating the single terminal nucleotide addition. Finally replication protein A completely suppressed the transferase activity of pol while stimulating the polymerase activity, suggesting that proliferating cell nuclear antigen and replication protein A can coordinate the polymerase and the terminal transferase activities of pol .

Received for publication, October 13, 2004

^* This work was supported by European Union Project No. QLK3-CT-2002-02071 REBIOTECH (to G. M. and U. H.); by the Swiss National Science Foundation, the UBS "im Auftrage eines Kunden;" by the Wolfermann-Nägeli Stiftung and the University of Zürich; and by CARIPLO Foundation Project: "Oncogenetica e Proteomica della Replicazione" Grant 2003.1663/10.8441 (to G. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 39-0382546354; Fax: 39-0382422286; E-mail: maga{at}igm.cnr.it.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

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]

				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]

				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]