Terminal deoxynucleotidyl transferase: the story of a misguided DNA polymerase

Abstract

Nearly every DNA polymerase characterized to date exclusively catalyzes the incorporation of mononucleotides into a growing primer using a DNA or RNA template as a guide to direct each incorporation event. There is, however, one unique DNA polymerase designated terminal deoxynucleotidyl transferase that performs DNA synthesis using only single-stranded DNA as the nucleic acid substrate. In this chapter, we review the biological role of this enigmatic DNA polymerase and the biochemical mechanism for its ability to perform DNA synthesis in the absence of a templating strand. We compare and contrast the molecular events for template-independent DNA synthesis catalyzed by terminal deoxynucleotidyl transferase with other well-characterized DNA polymerases that perform template-dependent synthesis. This includes a quantitative inspection of how terminal deoxynucleotidyl transferase binds DNA and dNTP substrates, the possible involvement of a conformational change that precedes phosphoryl transfer, and kinetic steps that are associated with the release of products. These enzymatic steps are discussed within the context of the available structures of terminal deoxynucleotidyl transferase in the presence of DNA or nucleotide substrate. In addition, we discuss the ability of proteins involved in replication and recombination to regulate the activity of the terminal deoxynucleotidyl transferase. Finally, the biomedical role of this specialized DNA polymerase is discussed focusing on its involvement in cancer development and its use in biomedical applications such as labeling DNA for detecting apoptosis.

Figure 1

Simplified models for template-dependent and template-independent DNA polymerase activity. (A) Most DNA polymerases require double-stranded DNA as a substrate, where the 5′→3′ strand is used as a primer and the complementary strand 3′→5′ is used as a template. (B) Terminal deoxynucleotidyl transferase is unique in its ability to catalyze phosphoryl transfer in the absence of a template that can not be accommodated in its active site.

Figure 2

Overview of the V(D)J recombination process that generates functional Ig heavy chain from the inactive gene segments in developing B- or T-lymphocytes. Shown is the V(D)J recombination process for the formation of a functional IgH gene where DJ assembly occurs prior to the combination with the V segment. See text for further details.

Figure 3

A simplified model for (A) the RAG-cleavage phase generating double-strand breaks and (B) DNA repair through non-homologous end-joining pathway during DJ gene segment assembly of the V(D)J recombination mechanism.

Figure 4

Simplified overview of the enzymatic steps and the role of terminal deoxynucleotidyl transferase in lymphocyte gene rearrangement. The variability of the recombined gene segments is increased through the random addition of non-templated (N) nucleotides catalyzed by the terminal deoxynucleotidyl transferase prior to complementary pairing and extension by template-dependent DNA polymerases.

Figure 5

The catalytic mechanism model for the nucleotidyl transfer reaction catalyzed by terminal deoxynucleotidyl transferase. See text for details.

Figure 6

Chemical structures of various 5-substituted indolyl nucleotides used to probe the activity of TdT.

Figure 7

Schematic representations of the different domains found in the four X-family DNA polymerases. Each domain is labeled and colored for clarity. NLS represents the nuclear localization signal motif, and BRCT indicates the BRCA1 carboxy terminus domain.

Figure 8

Crystal structure of terminal deoxynucleotidyl transferase showing the finger, thumb, palm and index finger (8kDa) subdomains that work synergistically to catalyze nucleotide incorporation. Inset 1 shows the hand-like morphology of most DNA polymerases. Inset 2 shows a surface model for the ring-like structure of TdT in which the primer strand is located perpendicular to the hole where dNTPs presumably diffuse to enter the active site. The ternary complex structure was prepared using the available binary crystal structures of murine TdT (PDB ID codes 1KEJ (TdT•ddATP) and 1KDH (TdT•ssDNA) [116]). MOE (www.chemcomp.com) was used for all structural modeling.

Figure 9

The active site of TdT (PDB ID code: 1KEJ) as defined by amino acids that exist within 6 Å of the bound nucleotide substrate, ddATP. The incoming nucleotide is shown in ball-stick representation in CPK color scheme. The two cobalt ions are colored as cyan. This figure was prepared using the UCSF Chimera package (http://www.cgl.ucsf.edu/chimera).

Figure 10

Comparing the crystal structures of template-independent TdT enzyme (PDB code: 1KEJ) and template-dependent DNA polymerase, pol λ (PDB code: 1XSN). (A) Superimposed structures of TdT and pol λ. The superimposed structures show an incredibly high degree of similarity between the two polymerases of the family X despite the fact that they catalyze different modes of DNA polymerization (template-independent versus template-dependent). (B) The ribbon structure of TdT (left) with the putative model of primer-template duplex derived from the human pol λ ternary complex (right) to show the “lariat-like” loop (in magenta) that prevents the ability of TdT to accommodate a templating strand. (C) Molecular surface model of TdT (left) with the putative model of primer-template duplex (shown in ribbon) derived from the human pol λ ternary complex (right). The dNTP is shown in ball and stick model in CPK color scheme. The models were prepared using the MOE package (www.chemcomp.com).

Figure 11

Chemical structures for different inhibitors of TdT. (A) Cordycepin. (B) RDS 2119.