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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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Impact of mutations in a 3D framework. (A) Structural context of Loop1 and SD2 (green) as well as helix α14 (blue), just after the ALLGWTGS region, with respect to dNTP-binding site. SD1 is at the C-terminus of Loop1 and the beginning of the next β strand. (B) 3D representation of the effect of mutations. Each mutation described in Table 1 is colored according to its effect (blue = minimum; red = maximum).
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Figure 8: Impact of mutations in a 3D framework. (A) Structural context of Loop1 and SD2 (green) as well as helix α14 (blue), just after the ALLGWTGS region, with respect to dNTP-binding site. SD1 is at the C-terminus of Loop1 and the beginning of the next β strand. (B) 3D representation of the effect of mutations. Each mutation described in Table 1 is colored according to its effect (blue = minimum; red = maximum).

Mentions: The effect of systematic substitutions of SD2 residues was also studied, both with the DN => NS double mutant (as in pol μ) as well as with the three separate D473A, N474A and H475A single mutants (Figure 6A). When looking at the structure of Tdt, it is apparent that the SD1 and SD2 regions are close in space (Figure 8A).Figure 6.


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)

Impact of mutations in a 3D framework. (A) Structural context of Loop1 and SD2 (green) as well as helix α14 (blue), just after the ALLGWTGS region, with respect to dNTP-binding site. SD1 is at the C-terminus of Loop1 and the beginning of the next β strand. (B) 3D representation of the effect of mutations. Each mutation described in Table 1 is colored according to its effect (blue = minimum; red = maximum).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 8: Impact of mutations in a 3D framework. (A) Structural context of Loop1 and SD2 (green) as well as helix α14 (blue), just after the ALLGWTGS region, with respect to dNTP-binding site. SD1 is at the C-terminus of Loop1 and the beginning of the next β strand. (B) 3D representation of the effect of mutations. Each mutation described in Table 1 is colored according to its effect (blue = minimum; red = maximum).
Mentions: The effect of systematic substitutions of SD2 residues was also studied, both with the DN => NS double mutant (as in pol μ) as well as with the three separate D473A, N474A and H475A single mutants (Figure 6A). When looking at the structure of Tdt, it is apparent that the SD1 and SD2 regions are close in space (Figure 8A).Figure 6.

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