Terminal Deoxynucleotidyl Transferase Indiscriminately Incorporates Ribonucleotides and Deoxyribonucleotides

Abstract

Terminal deoxynucleotidyl transferase (TdT) catalyzes the condensation of deoxyribonucleotides on 3-hydroxyl ends of DNA strands in a template-independent manner and adds N-regions to gene segment junctions during V(D)J recombination. Although TdT is able to incorporate a few ribonucleotides in vitro, TdT discrimination between ribo-and deoxyribonucleotides has never been studied. We found that TdT shows only a minor preference for incorporation of deoxyribonucleotides over ribonucleotides on DNA strands. However, incorporation of ribonucleotides alone or in the presence of deoxyribonucleotides generally leads to premature chain termination, reflecting an impeded accommodation of ribo-or mixed ribo/deoxyribonucleic acid substrates by TdT. An essential catalytic aspartate in TdT was identified, which is a first step toward understanding the apparent lack of sugar discrimination by TdT.A traditional classification of polymerases, as DNA or RNA polymerases, relies on their ability to use dNTPs 1 or rNTPs as nucleotide substrates (1, 2). Sugar selectivity is extremely high, several orders of magnitude (3-7), allowing nucleic acid polymerases to accomplish specialized tasks in the presence of competing nucleotide substrates. Terminal deoxynucleotidyl transferase (TdT, EC2.7.7.31) is a DNA polymerase, which contributes to the diversification of antigen receptors by adding nucleotides, called N-regions, to gene segment junctions during V(D)J recombination (8,9). In vitro, TdT most efficiently polymerizes deoxynucleotides on 3Ј-hydroxyl ends of DNA strands in a template-independent manner (10, 11). However, shortly after the isolation of TdT, it was shown that TdT could add a few ribonucleotides to oligodeoxynucleotide initiators and incorporate ribonucleotides into growing DNA chains (12, 13). The methodology at that time allowed neither accurate determination of the reaction kinetic parameters nor calculation of the TdT selectivity factor for deoxyribonucleotides over ribonucleotides. Having recently developed a new method for producing large quantities of pure and unproteolyzed recombinant TdT in bacteria (14), we have been able to compare the activity of the two TdT isoforms found in the mouse (15), to undertake three-dimensional structural analyses (16), and to further improve the enzymatic characterization of the protein.Here, we present our analysis of the sugar selectivity of TdT, and our investigation of its enzymatic behavior in vitro in the presence of both ribo-and deoxyribonucleotides. We report that the short murine TdT, which is the isoform conserved across the vertebrate phylum (17), has only minor preference for the incorporation of deoxyribo-over ribonucleotides on DNA strands in vitro and that under conditions that reproduce the in vivo ribo/deoxyribonucleotide pool imbalance (18), incorporation of ribonucleotides by TdT leads to premature termination of chain elongation. As a first step toward analyzing the mechanisms underlying the apparent lack of sugar discrimination by TdT, unique among nucleic a...

Biochemical Properties of Saccharomyces cerevisiae DNA Polymerase IV

“…In contrast with pol and pol ␤, which favor incorporation of dATP over rATP by factors approaching 10,000-fold, pol IV incorporates rATP and rCTP only 46-fold and 11-fold less efficiently than it incorporates dATP and dCTP, respectively (Table I). This limited discrimination against rNTP incorporation is similar to that reported previously for pol and TdT (15,24). Upon insertion of a rNTP, further extension by pol IV in the presence of all four rNTPs is inefficient (data not shown).…”

Molecular modeling and functional characterization of the monomeric primase–polymerase domain from the Sulfolobus solfataricus plasmid pIT3

“…The latter two enzymes are functionally malleable to the point of carrying out various nucleic acid synthesis reactions on a wide range of substrates [31][32][33]. Furthermore, like the TdT enzyme [34], the Rep245 protein can incorporate ribo-and deoxynucleotides in vitro. A noteworthy finding is that this functional equivalence is matched by structural relationships between the catalytic subunit of archaeal primases and the active site of the X family of polymerases [23].…”

DNA polymerases in nonhomologous end joining: Are there any benefits to standing out from the crowd?

“…Pol4 Gonzalez-Barrera et al, 2005], terminal deoxynucleotidyl transferase (TdT) [Kato et al, 1967;Boule et al, 2001], and polymerase l (Pol l) [Nick McElhinny and Ramsden, 2003;Ruiz et al, 2003] do not efficiently exclude ribonucleotide triphosphates, and thus extend DNA primers with deoxynucleotides or ribonucleotides with similar efficiency (low-sugar selectivity). Low-sugar selectivity is also observed in members of the archaeo-eukaryotic primase family implicated in synthesis during bacterial NHEJ [Della et al, 2004;Pitcher et al, 2007;Zhu and Shuman, 2008], suggesting that it might be generally important for NHEJ function.…”

“…4). Indeed, TdT is generally promiscuous in synthesis; it can extend primers with deoxynucleotides, ribonucleotides [Kato et al, 1967;Boule et al, 2001], and even non-nucleotide triphosphates (where the base is substituted with aromatic rings) with comparable efficiency [Arzumanov et al, 2000].…”