Limited terminal transferase in human DNA polymerase mu defines the required balance between accuracy and efficiency in NHEJ

Abstract

DNA polymerase mu (Polmu) is a family X member implicated in DNA repair, with template-directed and terminal transferase (template-independent) activities. It has been proposed that the terminal transferase activity of Polmu can be specifically required during non-homologous end joining (NHEJ) to create or increase complementarity of DNA ends. By site-directed mutagenesis in human Polmu, we have identified a specific DNA ligand residue (Arg(387)) that is responsible for its limited terminal transferase activity compared to that of human TdT, its closest homologue (42% amino acid identity). Polmu mutant R387K (mimicking TdT) displayed a large increase in terminal transferase activity, but a weakened interaction with ssDNA. That paradox can be explained by the regulatory role of Arg(387) in the translocation of the primer from a non-productive E:DNA complex to a productive E:DNA:dNTP complex in the absence of a templating base, which is probably the rate limiting step during template-independent synthesis. Further, we show that the Polmu switch from terminal transferase to templated insertions in NHEJ reactions is triggered by recognition of a 5'-P at a second DNA end, whose 3'-protrusion could provide a templating base to facilitate such a special "pre-catalytic translocation step." These studies shed light on the mechanism by which a rate-limited terminal transferase activity in Polmu could regulate the balance between accuracy and necessary efficiency, providing some variability during NHEJ.

Fig. 1.

Specific residues involved in terminal transferase. (A) Crystal structures of TdT, either complexed with ssDNA (1KDH) or dNTP (1KEJ), and Polμ (1IHM). Residues selected for mutagenesis in Polμ, and their orthologues in TdT, are indicated in red sticks. DNA substrates are indicated in dark (primer strand) and light (template and downstream strand) blue. Incoming ddNTPs are indicated in magenta (non templated) or green (templated). (B) Amino acid sequence alignment of family X polymerases from different species (Hs, human; Mm, mouse; Sp, Schizosaccharomyces pombe; Sc, Saccharomyces cerevisiae; Li, Leishmania infantum; Taq, Thermus aquaticus; Bs, Bacillus subtilis; and ASFV, african swine fever virus) along the indicated regions. Residues selected for mutagenesis in Polμ are indicated with red dots. Invariant residues are depicted in white over black background, and conservative residues have gray background.

Fig. 2.

Arg³⁸⁷ regulates the rate of terminal transferase activity in human Polμ, while His³²⁹ is essential. (A) Polymerization reactions on a gapped-DNA substrate (see scheme) was carried out in the presence of 2.5 mM MgCl₂, the indicated amounts of dCTP, and 300 nM of either Polμ wt or the indicated mutants. After incubation for 30 min at 30 °C, extension of the 5′P-labeled primer (indicated with an asterisk) was analyzed by 8 M urea-20% PAGE and autoradiography, and the results quantitated by densitometry. (B) Terminal transferase activity on different substrates (ssDNA/polyT, blunt dsDNA and 3′-protruding (9T) dsDNA; 5 nM) was assayed for 30 min at 30 °C in the presence of 1 mM MnCl₂, 100 μM dTTP, and 600 nM of either Polμ wt or the indicated mutants, and analyzed by 8 M urea-20% PAGE and autoradiography.

Fig. 3.

Mutants at residue Arg³⁸⁷ have a reduced ssDNA binding affinity. The presence of dNTP reduces ssDNA binding capacity by Polμ. (A) A 5′-labeled ssDNA (polydT; 5 nM) was used as substrate for interaction with different concentrations of either Polμ wt or mutants R387K, R387A, and H329G. After incubation for 10 min at 22 °C, the protein/ssDNA complexes were retained in nitrocellulose filters, as described in Materials and Methods. (B) Schemes depicting two different complexes: non-productive (nucleotide absent), indicative of a E:DNA binary complex in which the 3′ end of primer is occupying the nucleotide pocket (white oval); and productive (nucleotide present), indicative of a E:DNA:dNTP ternary complex in which the primer has been relocated. Binding capacity of Polμ wt and R387K mutant (600 nM in each case) to ssDNA/polyT, either in the absence (left) or presence (right) of dTTP, was measured by nitrocellulose filter binding assay, as described in Materials and Methods.

Fig. 4.

Terminal transferase vs. NHEJ of incompatible ends. (A) Single nucleotide extension of a 3′-protruding (1T) dsDNA substrate (200 nM), either lacking (left panel) or having (right panel) a 5′-recessive P. The assay was performed as described in Materials and Methods, in the presence of 1 mM MnCl₂, 100 μM of the indicated ddNTP, and 200 nM of Polμ wt. After incubation for 30 min at 30 °C, +1 extension of the 5′P-labeled oligonucleotide was analyzed by 8 M urea-20% PAGE and autoradiography. (B) The observed nucleotide specificity can be the consequence of either untemplated polymerization (terminal transferase; scheme 1) or DNA-directed additions (dA insertion templated in trans), thus achieving precise joining of two DNA ends (scheme 2). Scheme 3 shows a hybrid situation in which the joining reaction is untemplated, as the 3′-protruding template base provided in trans is not in a proper register. The gray rectangle represents a cartoon of Polμ, in which the specific loop 1, involved in both terminal transferase and connectivity during NHEJ of incompatible ends, is highlighted. The orange area in Polμ represents the 8-kDa domain, specifically involved in 5′P recognition.

Fig. 5.

R387K has a low fidelity during NHEJ of incompatible ends. (A) Accurate NHEJ of minimally complementary ends, using a 5′-labeled 3′-protruding (GT) dsDNA substrate (5 nM), and a cold 3′-protruding (CA) dsDNA substrate (25 nM), both having a recessive 5′P. Mutant R387K displayed an accurate behavior comparable to wild-type Polμ. (B) Inaccurate NHEJ of incompatible ends, using a 5′-labeled 3′-protruding (G) dsDNA substrate (5 nM), having a recessive 5′P. Mutant R387K was more efficient but displayed an inaccurate behavior comparable to wild-type Polμ. (C) Accurate vs. inaccurate NHEJ of incompatible ends, using a 5′-labeled 3′-protruding (C) dsDNA substrate (5 nM), having a recessive 5′P. Mutant R387K displayed an error-prone behavior compared to the accuracy of wild-type Polμ. In all cases, assays were performed in the presence of 2.5 mM MgCl₂, 100 μM of the indicated ddNTP, and 200 nM of either Polμ wt or R387K mutant. After incubation for 30 min at 30 °C, +1 extension of the 5′P-labeled primer was analyzed by 8 M urea-20% PAGE and autoradiography.

Fig. 6.

Modeling the limiting step of Polμ's terminal transferase. (A) Stable/non-productive step (binary complex): Polμ (1IHM) was overimposed on the binary complex of TdT with ssDNA (1KDH). Only the partial structure of Polμ (wheat color) is shown for clarity. A 1nt 3′-protruding substrate (derived from the gapped substrate present in the Polμ crystal) was modeled (as in 1KDH) to reproduce the initial situation of a binary complex in which the primer (dark blue) occupies the NTP binding pocket. Residue His³²⁹ (as His³⁴² in TdT) is in “standby,” and Arg³⁸⁷ (modeled from Lys⁴⁰³ in TdT) strongly interacts with the primer strand. (B) Productive step for the terminal transferase (ternary complex in the absence of template): the primer (dark blue) has been relocated, and any incoming dNTP (magenta) sits in place with the assistance of His³²⁹ (rotated 180°). Arg³⁸⁷ stabilizes the new primer location by interacting with the template strand (nucleotide in yellow). (C) Productive step during NHEJ (ternary complex with a template provided in trans): a NHEJ reaction implying two 3′-protruding and incompatible ends (modeled from the gapped substrate present in the Polμ crystal) is shown. Primer relocation and Arg³⁸⁷ repositioning is facilitated by the template provided in trans (nucleotide in cyan) that allows selection of a complementary nucleotide (depicted in green).