DNA Elongation by the Human DNA Polymerase λ 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,Ulrich Hübscher

doi:10.1074/jbc.m411650200

Abstract

DNA polymerase lambda contains template-dependent (DNA polymerase) and template-independent (terminal transferase) activities. In this study we enzymologically characterized the terminal transferase activity of polymerase lambda (pol lambda-tdt). Pol lambda-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 lambda. Pol lambda-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 lambda 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 lambda.

Highlights

DNA polymerase1 ␭ is a recently described eukaryotic enzyme belonging to the pol X family, comprising other enzymes involved in DNA repair processes such as pol ␤, pol ␮, and terminal deoxynucleotidyltransferase (TdT) [1]
We aimed to investigate in details the biochemical properties of pol ␭-terminal transferase as well as the possible regulatory roles of the two auxiliary proteins, proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A)
The Terminal Transferase Activity of Pol ␭ Is Influenced by the 3Ј-End Terminal Sequence of the ssDNA Substrate—The activity of both cellular and viral terminal transferases have been shown to be influenced by the 3Ј-terminal sequence of the ssDNA substrate [26]

Summary

Introduction

DNA polymerase (pol)1 ␭ is a recently described eukaryotic enzyme belonging to the pol X family, comprising other enzymes involved in DNA repair processes such as pol ␤, pol ␮, and terminal deoxynucleotidyltransferase (TdT) [1]. Pol ␭ possesses multiple enzymatic activities, including DNA polymerase, terminal transferase, deoxyribose phosphate lyase, and polynucleotide synthetase, all localized in the C-terminal region containing the pol ␤-like core domain [2, 3]. On the basis of its biochemical properties, pol ␭ has been implicated in various DNA repair pathways such as abasic site translesion DNA synthesis, base excision repair, and the nonhomologous end joining type of double strand break repair even though no direct demonstration of its role in vivo has been demonstrated so far (6 –9). We aimed to investigate in details the biochemical properties of pol ␭-terminal transferase (pol ␭-tdt) as well as the possible regulatory roles of the two auxiliary proteins, PCNA and replication protein A (RP-A)

Objectives

Results

Conclusion