Limits...
Expanding the repertoire of DNA polymerase substrates: template-instructed incorporation of non-nucleoside triphosphate analogues by DNA polymerases beta and lambda.

Crespan E, Alexandrova L, Khandazhinskaya A, Jasko M, Kukhanova M, Villani G, Hübscher U, Spadari S, Maga G - Nucleic Acids Res. (2006)

Bottom Line: We have recently shown that neither the base nor the sugar moieties of a nucleotide is an essential feature for its incorporation by DNA polymerases (pols) lambda and beta.This compound represents the first example of a substrate lacking both nucleobase and ribose residue, showing distinct base-pairing properties with normal bases.Therefore, this NNTP analog can be considered as the prototype of an entirely novel class of DNA pol substrates.

View Article: PubMed Central - PubMed

Affiliation: Istituto di Genetica Molecolare, IGM-CNR, via Abbiategrasso 207, I-27100 Pavia, Italy.

ABSTRACT
We have recently shown that neither the base nor the sugar moieties of a nucleotide is an essential feature for its incorporation by DNA polymerases (pols) lambda and beta. Here we present the identification of novel non-nucleoside triphosphate (NNTP) derivatives belonging to three classes: (i) non-substrate-specific inhibitors of DNA pol lambda; (ii) substrate inhibitors which could preferentially be incorporated by either DNA pol lambda wild type or its Y505A mutant and (iii) the substrate inhibitor N-(Biphenylcarbonyl)-4-oxobutyl triphosphate which could be incorporated exclusively by DNA pol beta in a Mg2+-dependent manner, and preferentially pairs with A on the template. This compound represents the first example of a substrate lacking both nucleobase and ribose residue, showing distinct base-pairing properties with normal bases. Therefore, this NNTP analog can be considered as the prototype of an entirely novel class of DNA pol substrates.

Show MeSH

Related in: MedlinePlus

Incorporation of non-nucleoside triphosphate analogues (NNTPs) by DNA polymerase λ wt and the Y505A mutant. Reactions were carried out as detailed in Materials and Methods. The different migration of the +1 products (indicated by arrows) are due to different electrophoretic mobilities of the incorporated NNTPs. The corresponding incorporation products are indicated with arrows. The different structures resulted in different electrophoretic mobility among the NNTPs. (A) The various NNTPs (10 μM) were tested for incorporation opposite to an abasic site (lanes 1–10) or on an intact template (lanes 11–20) and in the presence of Mn2+. Lane 21, reaction was carried out in the presence of 100 μM of all four dNTPs. (B) As in (A) but in the presence of the DNA pol λ Y505A mutant. Lane 16, control reaction in the absence of added nucleotides.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1761426&req=5

fig2: Incorporation of non-nucleoside triphosphate analogues (NNTPs) by DNA polymerase λ wt and the Y505A mutant. Reactions were carried out as detailed in Materials and Methods. The different migration of the +1 products (indicated by arrows) are due to different electrophoretic mobilities of the incorporated NNTPs. The corresponding incorporation products are indicated with arrows. The different structures resulted in different electrophoretic mobility among the NNTPs. (A) The various NNTPs (10 μM) were tested for incorporation opposite to an abasic site (lanes 1–10) or on an intact template (lanes 11–20) and in the presence of Mn2+. Lane 21, reaction was carried out in the presence of 100 μM of all four dNTPs. (B) As in (A) but in the presence of the DNA pol λ Y505A mutant. Lane 16, control reaction in the absence of added nucleotides.

Mentions: DNA polymerase λ and its mutant Y505A show different abilities of incorporating NNTPs in the presence of an abasic site on the template strand. The different NNTPs shown in Figure 1 were evaluated for their ability to be incorporated by DNA pol λ wild type and the Y505A mutant on a DNA template either undamaged or bearing an abasic (AP) site at position +1. As shown in Figure 2A, DNA pol λ wild type on the undamaged template was able to incorporate only compound IIb, whereas in the presence of an AP site, also compounds Ia and IIa, together with IIb, could be incorporated. When the mutant Y505A was tested, some striking differences emerged. As shown in Figure 2B, compound IIb was now incorporated opposite to an AP site only. Moreover, compound Ib, which was not a substrate for the wild-type enzyme, was incorporated on both templates, with a higher efficiency on the intact one. Another interesting difference was that compound Ia was now incorporated on both templates by the mutant enzyme. No incorporation could be detected for both enzymes when Mg2+ replaced Mn2+. Thus, it appears that both the presence/absence of tyrosine 505 in the nucleotide binding site, and the nature of the template (either undamaged or carrying an AP site) differentially affected the ability of various NNTPs to act as substrates for DNA pol λ. As apparent by the accumulation of only +1 products, all the compounds acted as chain terminators. This was expected, due to the lack of available 3′-hydroxyl groups for subsequent elongation. In order to investigate in more details the observed differences in NNTPs incorporation by DNA pol λ and the Y505A mutant, the kinetic parameters (Km, kcat, kcat/Km) for the incorporation reaction were determined and the calculated values are summarized in Table 2. As controls, kinetic parameters for the incorporation of dCTP or dATP, depending on the template used, were also determined. Inspection of these data revealed several differences, discussed in detail in the following sections.


Expanding the repertoire of DNA polymerase substrates: template-instructed incorporation of non-nucleoside triphosphate analogues by DNA polymerases beta and lambda.

Crespan E, Alexandrova L, Khandazhinskaya A, Jasko M, Kukhanova M, Villani G, Hübscher U, Spadari S, Maga G - Nucleic Acids Res. (2006)

Incorporation of non-nucleoside triphosphate analogues (NNTPs) by DNA polymerase λ wt and the Y505A mutant. Reactions were carried out as detailed in Materials and Methods. The different migration of the +1 products (indicated by arrows) are due to different electrophoretic mobilities of the incorporated NNTPs. The corresponding incorporation products are indicated with arrows. The different structures resulted in different electrophoretic mobility among the NNTPs. (A) The various NNTPs (10 μM) were tested for incorporation opposite to an abasic site (lanes 1–10) or on an intact template (lanes 11–20) and in the presence of Mn2+. Lane 21, reaction was carried out in the presence of 100 μM of all four dNTPs. (B) As in (A) but in the presence of the DNA pol λ Y505A mutant. Lane 16, control reaction in the absence of added nucleotides.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1761426&req=5

fig2: Incorporation of non-nucleoside triphosphate analogues (NNTPs) by DNA polymerase λ wt and the Y505A mutant. Reactions were carried out as detailed in Materials and Methods. The different migration of the +1 products (indicated by arrows) are due to different electrophoretic mobilities of the incorporated NNTPs. The corresponding incorporation products are indicated with arrows. The different structures resulted in different electrophoretic mobility among the NNTPs. (A) The various NNTPs (10 μM) were tested for incorporation opposite to an abasic site (lanes 1–10) or on an intact template (lanes 11–20) and in the presence of Mn2+. Lane 21, reaction was carried out in the presence of 100 μM of all four dNTPs. (B) As in (A) but in the presence of the DNA pol λ Y505A mutant. Lane 16, control reaction in the absence of added nucleotides.
Mentions: DNA polymerase λ and its mutant Y505A show different abilities of incorporating NNTPs in the presence of an abasic site on the template strand. The different NNTPs shown in Figure 1 were evaluated for their ability to be incorporated by DNA pol λ wild type and the Y505A mutant on a DNA template either undamaged or bearing an abasic (AP) site at position +1. As shown in Figure 2A, DNA pol λ wild type on the undamaged template was able to incorporate only compound IIb, whereas in the presence of an AP site, also compounds Ia and IIa, together with IIb, could be incorporated. When the mutant Y505A was tested, some striking differences emerged. As shown in Figure 2B, compound IIb was now incorporated opposite to an AP site only. Moreover, compound Ib, which was not a substrate for the wild-type enzyme, was incorporated on both templates, with a higher efficiency on the intact one. Another interesting difference was that compound Ia was now incorporated on both templates by the mutant enzyme. No incorporation could be detected for both enzymes when Mg2+ replaced Mn2+. Thus, it appears that both the presence/absence of tyrosine 505 in the nucleotide binding site, and the nature of the template (either undamaged or carrying an AP site) differentially affected the ability of various NNTPs to act as substrates for DNA pol λ. As apparent by the accumulation of only +1 products, all the compounds acted as chain terminators. This was expected, due to the lack of available 3′-hydroxyl groups for subsequent elongation. In order to investigate in more details the observed differences in NNTPs incorporation by DNA pol λ and the Y505A mutant, the kinetic parameters (Km, kcat, kcat/Km) for the incorporation reaction were determined and the calculated values are summarized in Table 2. As controls, kinetic parameters for the incorporation of dCTP or dATP, depending on the template used, were also determined. Inspection of these data revealed several differences, discussed in detail in the following sections.

Bottom Line: We have recently shown that neither the base nor the sugar moieties of a nucleotide is an essential feature for its incorporation by DNA polymerases (pols) lambda and beta.This compound represents the first example of a substrate lacking both nucleobase and ribose residue, showing distinct base-pairing properties with normal bases.Therefore, this NNTP analog can be considered as the prototype of an entirely novel class of DNA pol substrates.

View Article: PubMed Central - PubMed

Affiliation: Istituto di Genetica Molecolare, IGM-CNR, via Abbiategrasso 207, I-27100 Pavia, Italy.

ABSTRACT
We have recently shown that neither the base nor the sugar moieties of a nucleotide is an essential feature for its incorporation by DNA polymerases (pols) lambda and beta. Here we present the identification of novel non-nucleoside triphosphate (NNTP) derivatives belonging to three classes: (i) non-substrate-specific inhibitors of DNA pol lambda; (ii) substrate inhibitors which could preferentially be incorporated by either DNA pol lambda wild type or its Y505A mutant and (iii) the substrate inhibitor N-(Biphenylcarbonyl)-4-oxobutyl triphosphate which could be incorporated exclusively by DNA pol beta in a Mg2+-dependent manner, and preferentially pairs with A on the template. This compound represents the first example of a substrate lacking both nucleobase and ribose residue, showing distinct base-pairing properties with normal bases. Therefore, this NNTP analog can be considered as the prototype of an entirely novel class of DNA pol substrates.

Show MeSH
Related in: MedlinePlus