A specific loop in human DNA polymerase mu allows switching between creative and DNA-instructed synthesis

Abstract

Human DNA polymerase mu (Polmu) is a family X member that has terminal transferase activity but, in spite of a non-orthodox selection of the template information, displays its maximal catalytic efficiency in DNA-templated reactions. As terminal deoxynucleotidyl transferase (TdT), Polmu has a specific loop (loop1) that could provide this enzyme with its terminal transferase activity. When loop1 was deleted, human Polmu lacked TdT activity but improved DNA-binding and DNA template-dependent polymerization. Interestingly, when loop1 from TdT was inserted in Polmu (substituting its cognate loop1), the resulting chimaera displayed TdT activity, preferentially inserting dGTP residues, but had a strongly reduced template-dependent polymerization activity. Therefore, a specialized loop in Polmu, that could adopt alternative conformations, appears to provide this enzyme with a dual capacity: (i) template independency to create new DNA information, in which loop1 would have an active role by acting as a 'pseudotemplate'; (ii) template-dependent polymerization, in which loop1 must allow binding of the template strand. Recent in vivo and in vitro data suggest that such a dual capacity could be advantageous to resolve microhomology-mediated end-joining reactions.

Figure 1

TdT and Polμ have a specific loop at the palm subdomain. (A) Scheme of the multidomain structure of Polβ, Polμ and TdT. The N-terminal region containing a BRCT domain is only present in Polμ and TdT. Other domains, as 8 kDa, fingers (F), palm (P) and thumb (T) are shared by the three enzymes, and constitute an evolutionarily conserved Polβ polymerization core, present in most members of the DNA polymerase family X. The insert shows the position and flanking residues corresponding to loop1, exclusively present in the palm subdomain of TdT and Polμ. (B) Amino acid sequence alignment of human Polβ, mouse TdT and Polμ from different species (Hs, human; Mm, mouse; Rn, rat; Dr, trout) along the region containing loop1 in Polμ and TdT. Invariant residues acting as metal ligands are indicated with red dots. Polβ residues acting as DNA ligands are indicated with blue dots. Asterisks indicate putative residues involved in interactions of loop1 with the thumb subdomain. The region deleted in Δloop1 mutant is boxed in yellow. (C) Prediction of a mobile loop1 in Polμ as a switcher between creative and DNA-directed DNA synthesis. Some of the amino acid residues in TdT that are involved in the stabilization of loop1 (via connection to the thumb) are not conserved in Polμ. Particularly, the strong stacking interaction between the aromatic rings of TdT residues Phe385 (loop1) and His475 (thumb), is not conserved in Polμ (substituted by a cysteine (Cys370) residue). (D) By modelling human Polμ on the crystal structure of TdT (either as apoenzyme (PDB id: 1JMS), or complexed with dNTP(PDB id: 1KEJ) or ssDNA (PDB id: 1KDH), alternative conformations of loop1 can be predicted for Polμ. In this figure, represented by using the Swiss PDB Viewer program (), a gapped DNA has been also modelled based on a Polβ ternary complex (PDB id: 1BPY). Template strand is in magenta; the different conformations of loop1 are shown in red, yellow and green colours; subdomains in Polμ are light coloured in green (8 kDa), yellow (fingers), red (palm) and magenta (thumb), as shown in (A).

Figure 2

DNA-binding preferences of human Polμ. Formation of stable complexes between the wild-type Polμ and DNA was comparatively studied using the following DNA molecules, corresponding to different DNA repair intermediates: ssDNA, blunt-ended DNA (Blunt), template/primer (T/P) and 2 nt-gapped DNA (Gap). The assay was carried out using 4 nM of the corresponding labelled DNA primer, either alone or pre-hybridized to a DNA template (and to a downstream oligonucleotide, in the case of gap2-P), as described in Materials and Methods. Increasing concentrations (36, 72, 144, 432 and 864 nM) of Polμ wt were added to the reaction [50 mM Tris–HCl (pH 7.5), 1 mM DTT, 4% glycerol and 0.1 mg/ml BSA] and incubated 10 min at 30°C. Enzyme–DNA complexes (a and b) were identified by their retarded migration after 4% native PAGE and autoradiography. c, free hybridized DNA substrate; d, rest of non-hybridized primer.

Figure 3

Deletion of loop1 improves binding of Polμ to templated-DNA molecules. As shown in the figure for either a template/primer (A) or a 2 nt-gapped DNA substrate (B), elimination of loop1 in Polμ improves its DNA binding capacity (assessed by gel-shifting experiments as described in Figure 2), suggesting that loop1 would have some negative effect on DNA-directed DNA synthesis, probably interfering with the template binding region of human Polμ.

Figure 4

Deletion of loop1 does not affect template misalignment-mediated misincorporation and primer realignment by Polμ. (A) On a 2 nt-gapped DNA substrate (4 nM), deletion of loop1 did not destroy the capacity of human Polμ to catalyze a preferred insertion of dG in front of the second template base of the gap (dC). This ‘apparent misinsertion’ occurs as a consequence of Polμ's proneness to produce misalignement of the template dG, stabilized by the incoming dGTP nucleotide (‘dNTP selection mechanism’). (B) Loop1 is irrelevant to Polμ in order to realign a mismatched terminus to a distant complementary position, as it was described for the wild-type human Polμ (7). Insertion of dGTP in front of a distant dC template base is thus observed in both wild-type and Δloop1 mutant. In both cases (A and B) the reaction was carried out in the presence of 2 mM MgCl₂,1 µM of the indicated dNTP, and 600 nM of either Polμ wt or Δloop1 as indicated. After incubation for 30 min at 30°C, the extension of the 5′ P-labelled oligonucleotide was analysed by 8 M urea and 20% PAGE and autoradiography.

Figure 5

Loop 1 is critical for terminal transferase activity. (A) The assay was carried out using 4 nM of a labelled homopolymer (P15-T) as DNA primer. The reaction, carried out as described in Materials and Methods, was carried out in the presence of 1 mM MnCl₂, 10 µM of the indicated dNTP, and 600 nM of either Polμ wt, or Δloop1, or Ch-loop1, or commercial TdT (3 U). After incubation for 30 min at 30°C, the extension of the 5′-labelled oligonucleotide was analysed by 8 M urea and 20% PAGE and autoradiography. (B) The assay was identical to that described in part A, but providing Poly(dT)*/ PolidA as a template.