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Conferring a template-dependent polymerase activity to terminal deoxynucleotidyltransferase by mutations in the Loop1 region.

Romain F, Barbosa I, Gouge J, Rougeon F, Delarue M - Nucleic Acids Res. (2009)

Bottom Line: First we describe the effect of mutations on six different positions chosen to destabilize Tdt Loop1 structure, either by alanine substitution or by deletion; they result at most in a reduction of Tdt activity, but adding Co(++) restores most of this Tdt activity.Among them, the single-point mutant F401A displays a sequence-specific replicative polymerase phenotype that is stable upon Co(++) addition.These results are discussed in light of the available crystal structures.

View Article: PubMed Central - PubMed

Affiliation: Unité de Dynamique Structurale des Macromolécules and URA 2581 du C.N.R.S., Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France.

ABSTRACT
Terminal deoxynucleotidyltransferase (Tdt) and DNA polymerase mu (pol mu) are two eukaryotic highly similar proteins involved in DNA processing and repair. Despite their high sequence identity, they differ widely in their activity: pol mu has a templated polymerase activity, whereas Tdt has a non-templated one. Loop1, first described when the Tdt structure was solved, has been invoked as the major structural determinant of this difference. Here we describe attempts to transform Tdt into pol mu with the minimal number of mutations in and around Loop1. First we describe the effect of mutations on six different positions chosen to destabilize Tdt Loop1 structure, either by alanine substitution or by deletion; they result at most in a reduction of Tdt activity, but adding Co(++) restores most of this Tdt activity. However, a deletion of the entire Loop1 as in pol lambda does confer a limited template-dependent polymerase behavior to Tdt while a chimera bearing an extended pol mu Loop1 reproduces pol mu behavior. Finally, 16 additional substitutions are reported, targeted at the two so-called 'sequence determinant' regions located just after Loop1 or underneath. Among them, the single-point mutant F401A displays a sequence-specific replicative polymerase phenotype that is stable upon Co(++) addition. These results are discussed in light of the available crystal structures.

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SD1 region mutants (QKC). (A) The activity without CoCl2 but in the presence of 4 mM MgCl2 of wild-type Tdt, Q402E, Q402A, K403R, K403A and C404A mutants is shown for each dNTP and a mixture of all dNTPs. (B) The activity without CoCl2 but in the presence of 4 mM MgCl2 of Tdt wild-type, along with the triple mutant in SD1 switching QKC into ERS (as in pol μ), D399A, H400A, F401A and F405A mutants is shown for each dNTP and a mixture of all dNTPs. The effect of Co++ addition (1 mM) to the reaction buffer is shown in Supplementary Figure S2.
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Figure 5: SD1 region mutants (QKC). (A) The activity without CoCl2 but in the presence of 4 mM MgCl2 of wild-type Tdt, Q402E, Q402A, K403R, K403A and C404A mutants is shown for each dNTP and a mixture of all dNTPs. (B) The activity without CoCl2 but in the presence of 4 mM MgCl2 of Tdt wild-type, along with the triple mutant in SD1 switching QKC into ERS (as in pol μ), D399A, H400A, F401A and F405A mutants is shown for each dNTP and a mixture of all dNTPs. The effect of Co++ addition (1 mM) to the reaction buffer is shown in Supplementary Figure S2.

Mentions: The multialignement of all known pol μ sequences and all known Tdt sequences reveals two blocks of sequences completely conserved in each sub-family but not identical to each other [Figure 2 of (22)], which we call here the two ‘sequence determinant’ regions SD1 and SD2 (Figure 1). The SD1 contains the QKC tripeptide, just outside of Loop1. As it is part of our grafted sequence that confers pol μ behavior to Tdt, we decided to mutate it separately (Figure 5A). The triple mutant QKC→ERS does incorporate the correct nucleotide (G) when in presence of dGTP, and does not incorporate efficiently any of the three other dNTPs. When in presence of all four dNTPs it adds only one nucleotide, but then stops completely. Therefore we characterize this mutant not as a pol μ, but rather as a ‘stuck’ polymerase; to explain this effect, we speculate that one of the products of the reaction (elongated primer or PPi) may be an inhibitor or cannot be released.Figure 5.


Conferring a template-dependent polymerase activity to terminal deoxynucleotidyltransferase by mutations in the Loop1 region.

Romain F, Barbosa I, Gouge J, Rougeon F, Delarue M - Nucleic Acids Res. (2009)

SD1 region mutants (QKC). (A) The activity without CoCl2 but in the presence of 4 mM MgCl2 of wild-type Tdt, Q402E, Q402A, K403R, K403A and C404A mutants is shown for each dNTP and a mixture of all dNTPs. (B) The activity without CoCl2 but in the presence of 4 mM MgCl2 of Tdt wild-type, along with the triple mutant in SD1 switching QKC into ERS (as in pol μ), D399A, H400A, F401A and F405A mutants is shown for each dNTP and a mixture of all dNTPs. The effect of Co++ addition (1 mM) to the reaction buffer is shown in Supplementary Figure S2.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2724280&req=5

Figure 5: SD1 region mutants (QKC). (A) The activity without CoCl2 but in the presence of 4 mM MgCl2 of wild-type Tdt, Q402E, Q402A, K403R, K403A and C404A mutants is shown for each dNTP and a mixture of all dNTPs. (B) The activity without CoCl2 but in the presence of 4 mM MgCl2 of Tdt wild-type, along with the triple mutant in SD1 switching QKC into ERS (as in pol μ), D399A, H400A, F401A and F405A mutants is shown for each dNTP and a mixture of all dNTPs. The effect of Co++ addition (1 mM) to the reaction buffer is shown in Supplementary Figure S2.
Mentions: The multialignement of all known pol μ sequences and all known Tdt sequences reveals two blocks of sequences completely conserved in each sub-family but not identical to each other [Figure 2 of (22)], which we call here the two ‘sequence determinant’ regions SD1 and SD2 (Figure 1). The SD1 contains the QKC tripeptide, just outside of Loop1. As it is part of our grafted sequence that confers pol μ behavior to Tdt, we decided to mutate it separately (Figure 5A). The triple mutant QKC→ERS does incorporate the correct nucleotide (G) when in presence of dGTP, and does not incorporate efficiently any of the three other dNTPs. When in presence of all four dNTPs it adds only one nucleotide, but then stops completely. Therefore we characterize this mutant not as a pol μ, but rather as a ‘stuck’ polymerase; to explain this effect, we speculate that one of the products of the reaction (elongated primer or PPi) may be an inhibitor or cannot be released.Figure 5.

Bottom Line: First we describe the effect of mutations on six different positions chosen to destabilize Tdt Loop1 structure, either by alanine substitution or by deletion; they result at most in a reduction of Tdt activity, but adding Co(++) restores most of this Tdt activity.Among them, the single-point mutant F401A displays a sequence-specific replicative polymerase phenotype that is stable upon Co(++) addition.These results are discussed in light of the available crystal structures.

View Article: PubMed Central - PubMed

Affiliation: Unité de Dynamique Structurale des Macromolécules and URA 2581 du C.N.R.S., Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France.

ABSTRACT
Terminal deoxynucleotidyltransferase (Tdt) and DNA polymerase mu (pol mu) are two eukaryotic highly similar proteins involved in DNA processing and repair. Despite their high sequence identity, they differ widely in their activity: pol mu has a templated polymerase activity, whereas Tdt has a non-templated one. Loop1, first described when the Tdt structure was solved, has been invoked as the major structural determinant of this difference. Here we describe attempts to transform Tdt into pol mu with the minimal number of mutations in and around Loop1. First we describe the effect of mutations on six different positions chosen to destabilize Tdt Loop1 structure, either by alanine substitution or by deletion; they result at most in a reduction of Tdt activity, but adding Co(++) restores most of this Tdt activity. However, a deletion of the entire Loop1 as in pol lambda does confer a limited template-dependent polymerase behavior to Tdt while a chimera bearing an extended pol mu Loop1 reproduces pol mu behavior. Finally, 16 additional substitutions are reported, targeted at the two so-called 'sequence determinant' regions located just after Loop1 or underneath. Among them, the single-point mutant F401A displays a sequence-specific replicative polymerase phenotype that is stable upon Co(++) addition. These results are discussed in light of the available crystal structures.

Show MeSH
Related in: MedlinePlus