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Critical amino acids in Escherichia coli UmuC responsible for sugar discrimination and base-substitution fidelity.

Vaisman A, Kuban W, McDonald JP, Karata K, Yang W, Goodman MF, Woodgate R - Nucleic Acids Res. (2012)

Bottom Line: While the Y11F substitution has a minimal effect on sugar selectivity, it results in an increase in spontaneous mutagenesis.In comparison, an F10L substitution increases sugar selectivity and the overall fidelity of pol V Mut.Molecular modeling analysis reveals that the branched side-chain of L10 impinges on the benzene ring of Y11 so as to constrict its movement and as a consequence, firmly closes the steric gate, which in wild-type enzyme fails to guard against ribonucleoside triphosphates incorporation with sufficient stringency.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-3371, USA.

ABSTRACT
The active form of Escherichia coli DNA polymerase V responsible for damage-induced mutagenesis is a multiprotein complex (UmuD'(2)C-RecA-ATP), called pol V Mut. Optimal activity of pol V Mut in vitro is observed on an SSB-coated single-stranded circular DNA template in the presence of the β/γ complex and a transactivated RecA nucleoprotein filament, RecA*. Remarkably, under these conditions, wild-type pol V Mut efficiently incorporates ribonucleotides into DNA. A Y11A substitution in the 'steric gate' of UmuC further reduces pol V sugar selectivity and converts pol V Mut into a primer-dependent RNA polymerase that is capable of synthesizing long RNAs with a processivity comparable to that of DNA synthesis. Despite such properties, Y11A only promotes low levels of spontaneous mutagenesis in vivo. While the Y11F substitution has a minimal effect on sugar selectivity, it results in an increase in spontaneous mutagenesis. In comparison, an F10L substitution increases sugar selectivity and the overall fidelity of pol V Mut. Molecular modeling analysis reveals that the branched side-chain of L10 impinges on the benzene ring of Y11 so as to constrict its movement and as a consequence, firmly closes the steric gate, which in wild-type enzyme fails to guard against ribonucleoside triphosphates incorporation with sufficient stringency.

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The inhibitory effect of cytosine arabinoside incorporation on DNA synthesis by wild-type pol V (A) and the Y11A variant (B). Insertions of dCTP (lanes 2, 4, 6) versus ara-C (lanes 3, 5, 7) with, or without, dGTP alone (4, 5), or in combination with dATP and dTTP (6, 7) were analyzed using a substrate containing five consecutive Gs in the template. Reactions were carried out in the presence of 100 µM nucleotides for 5 min; all reactions contained 1 mM ATP. The identity of the nucleotide incorporated is shown below each track. The extended sequence of the template is indicated to the right of the gel. The reaction shown in lane 0 contained no polymerase and the reactions in lane 1 contained no other nucleotides except the 1 mM ATP. The well and primer locations are indicated by arrows at the top and bottom of the gel, respectively.
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gks233-F5: The inhibitory effect of cytosine arabinoside incorporation on DNA synthesis by wild-type pol V (A) and the Y11A variant (B). Insertions of dCTP (lanes 2, 4, 6) versus ara-C (lanes 3, 5, 7) with, or without, dGTP alone (4, 5), or in combination with dATP and dTTP (6, 7) were analyzed using a substrate containing five consecutive Gs in the template. Reactions were carried out in the presence of 100 µM nucleotides for 5 min; all reactions contained 1 mM ATP. The identity of the nucleotide incorporated is shown below each track. The extended sequence of the template is indicated to the right of the gel. The reaction shown in lane 0 contained no polymerase and the reactions in lane 1 contained no other nucleotides except the 1 mM ATP. The well and primer locations are indicated by arrows at the top and bottom of the gel, respectively.

Mentions: To better understand the steric interactions between Y11A and an incoming rNTP, we compared reactions in the presence of dCTP and a steric isomer of CTP, cytosine-1-β-d-arabinofuranoside (Ara-C), which also has a 2′-hydroxyl group, only in a different conformation (Figure 5). Ara-C was incorporated as efficiently as dCTP by both wild-type pol V and Y11A, similarly outcompeting ATP and incorrect dNTPs incorporation opposite template G and equally interfering with further primer extension. No products beyond the +5 position are seen even in the presence of correct downstream nucleotides. Efficient incorporation of Ara-C is not unusual even for DNA polymerases with strict sugar selectivity, since a ribose with a 2′-OH group on the opposite side of the plane of the sugar (compared to rNTP) avoids the collision with the steric gate residue. However, extension of the resulting primer ends for these polymerases, as well as for wild-type pol V and Y11A, is largely inhibited despite the presence of a 3′-hydroxyl group on the sugar moiety of the terminal nucleotide. Therefore, even though the Y11A mutation greatly facilitates accommodation of a nucleotide with a 2′-OH group in the polymerase active site, it does not improve primer extension when the sugar pucker at its terminus is in a 2′-endo conformation. These experiments also indicated that dG and/or dC misincorporations occur more often when catalyzed by wild-type pol V than by Y11A [e.g. wild-type pol V in the presence of dGTP and dCTP synthesizes ∼5 nt longer product than Y11A (compare lanes 4 in Figure 5A and B), and the resulting 16-mer requires elongation of a primer with five consecutive C/A and C/T and/or G/A and G/T mispairs]. These data support our assumption that Y11A could actually be more accurate while incorporating dNTPs than the wild-type enzyme.Figure 5.


Critical amino acids in Escherichia coli UmuC responsible for sugar discrimination and base-substitution fidelity.

Vaisman A, Kuban W, McDonald JP, Karata K, Yang W, Goodman MF, Woodgate R - Nucleic Acids Res. (2012)

The inhibitory effect of cytosine arabinoside incorporation on DNA synthesis by wild-type pol V (A) and the Y11A variant (B). Insertions of dCTP (lanes 2, 4, 6) versus ara-C (lanes 3, 5, 7) with, or without, dGTP alone (4, 5), or in combination with dATP and dTTP (6, 7) were analyzed using a substrate containing five consecutive Gs in the template. Reactions were carried out in the presence of 100 µM nucleotides for 5 min; all reactions contained 1 mM ATP. The identity of the nucleotide incorporated is shown below each track. The extended sequence of the template is indicated to the right of the gel. The reaction shown in lane 0 contained no polymerase and the reactions in lane 1 contained no other nucleotides except the 1 mM ATP. The well and primer locations are indicated by arrows at the top and bottom of the gel, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3401427&req=5

gks233-F5: The inhibitory effect of cytosine arabinoside incorporation on DNA synthesis by wild-type pol V (A) and the Y11A variant (B). Insertions of dCTP (lanes 2, 4, 6) versus ara-C (lanes 3, 5, 7) with, or without, dGTP alone (4, 5), or in combination with dATP and dTTP (6, 7) were analyzed using a substrate containing five consecutive Gs in the template. Reactions were carried out in the presence of 100 µM nucleotides for 5 min; all reactions contained 1 mM ATP. The identity of the nucleotide incorporated is shown below each track. The extended sequence of the template is indicated to the right of the gel. The reaction shown in lane 0 contained no polymerase and the reactions in lane 1 contained no other nucleotides except the 1 mM ATP. The well and primer locations are indicated by arrows at the top and bottom of the gel, respectively.
Mentions: To better understand the steric interactions between Y11A and an incoming rNTP, we compared reactions in the presence of dCTP and a steric isomer of CTP, cytosine-1-β-d-arabinofuranoside (Ara-C), which also has a 2′-hydroxyl group, only in a different conformation (Figure 5). Ara-C was incorporated as efficiently as dCTP by both wild-type pol V and Y11A, similarly outcompeting ATP and incorrect dNTPs incorporation opposite template G and equally interfering with further primer extension. No products beyond the +5 position are seen even in the presence of correct downstream nucleotides. Efficient incorporation of Ara-C is not unusual even for DNA polymerases with strict sugar selectivity, since a ribose with a 2′-OH group on the opposite side of the plane of the sugar (compared to rNTP) avoids the collision with the steric gate residue. However, extension of the resulting primer ends for these polymerases, as well as for wild-type pol V and Y11A, is largely inhibited despite the presence of a 3′-hydroxyl group on the sugar moiety of the terminal nucleotide. Therefore, even though the Y11A mutation greatly facilitates accommodation of a nucleotide with a 2′-OH group in the polymerase active site, it does not improve primer extension when the sugar pucker at its terminus is in a 2′-endo conformation. These experiments also indicated that dG and/or dC misincorporations occur more often when catalyzed by wild-type pol V than by Y11A [e.g. wild-type pol V in the presence of dGTP and dCTP synthesizes ∼5 nt longer product than Y11A (compare lanes 4 in Figure 5A and B), and the resulting 16-mer requires elongation of a primer with five consecutive C/A and C/T and/or G/A and G/T mispairs]. These data support our assumption that Y11A could actually be more accurate while incorporating dNTPs than the wild-type enzyme.Figure 5.

Bottom Line: While the Y11F substitution has a minimal effect on sugar selectivity, it results in an increase in spontaneous mutagenesis.In comparison, an F10L substitution increases sugar selectivity and the overall fidelity of pol V Mut.Molecular modeling analysis reveals that the branched side-chain of L10 impinges on the benzene ring of Y11 so as to constrict its movement and as a consequence, firmly closes the steric gate, which in wild-type enzyme fails to guard against ribonucleoside triphosphates incorporation with sufficient stringency.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-3371, USA.

ABSTRACT
The active form of Escherichia coli DNA polymerase V responsible for damage-induced mutagenesis is a multiprotein complex (UmuD'(2)C-RecA-ATP), called pol V Mut. Optimal activity of pol V Mut in vitro is observed on an SSB-coated single-stranded circular DNA template in the presence of the β/γ complex and a transactivated RecA nucleoprotein filament, RecA*. Remarkably, under these conditions, wild-type pol V Mut efficiently incorporates ribonucleotides into DNA. A Y11A substitution in the 'steric gate' of UmuC further reduces pol V sugar selectivity and converts pol V Mut into a primer-dependent RNA polymerase that is capable of synthesizing long RNAs with a processivity comparable to that of DNA synthesis. Despite such properties, Y11A only promotes low levels of spontaneous mutagenesis in vivo. While the Y11F substitution has a minimal effect on sugar selectivity, it results in an increase in spontaneous mutagenesis. In comparison, an F10L substitution increases sugar selectivity and the overall fidelity of pol V Mut. Molecular modeling analysis reveals that the branched side-chain of L10 impinges on the benzene ring of Y11 so as to constrict its movement and as a consequence, firmly closes the steric gate, which in wild-type enzyme fails to guard against ribonucleoside triphosphates incorporation with sufficient stringency.

Show MeSH
Related in: MedlinePlus