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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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Specificity of nucleotide incorporation by mutant pol Vs. Reactions catalyzed by F10L (A–D), Y11A (E–H) or Y11F (I–L) in the presence 100 µM of each individual dNTP (A, B, E, F, I, J) or rNTP (C, D, G, H, K, L) were carried out for 5 min. All reactions contained 1 mM ATP. The identity of the nucleotide incorporated is shown below each lane and the extended sequence of templates with five consecutive Ts (A, C, E, G, I, K) or As (B, D, F, H, J, L) is indicated to the right of each gel panel. All lanes with reactions lacking polymerase are indicated as ‘pr’ and reactions with no additional nucleotide are indicated by dash (–).
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gks233-F3: Specificity of nucleotide incorporation by mutant pol Vs. Reactions catalyzed by F10L (A–D), Y11A (E–H) or Y11F (I–L) in the presence 100 µM of each individual dNTP (A, B, E, F, I, J) or rNTP (C, D, G, H, K, L) were carried out for 5 min. All reactions contained 1 mM ATP. The identity of the nucleotide incorporated is shown below each lane and the extended sequence of templates with five consecutive Ts (A, C, E, G, I, K) or As (B, D, F, H, J, L) is indicated to the right of each gel panel. All lanes with reactions lacking polymerase are indicated as ‘pr’ and reactions with no additional nucleotide are indicated by dash (–).

Mentions: Misincorporation assays (Figure 3 and Supplementary Figure S2) were unambiguous for F10L, i.e. they show that this enzyme is quite accurate, although it is still able to incorporate ATP opposite template T, C and G (Figure 3A–D and Supplementary Figure S2A–D). Unexpectedly, misincorporation of ATP opposite C and G (Supplementary Figure S2A–D) is actually more efficient than opposite the complementary T (Figure 3A–D). Misincorporation patterns for wild-type pol V and Y11F were very similar with slight, if any, increase in fidelity of dNTP incorporation for the Y11F mutant (c.f. Figures 1 and 3I–L; Supplementary Figure S2I–S2L). Nevertheless, both enzymes are highly error-prone and capable of incorporation of multiple wrong dNTPs outcompeting excessive amounts of ATP (as judged by the unique pattern of product distribution in various lanes).Figure 3.


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)

Specificity of nucleotide incorporation by mutant pol Vs. Reactions catalyzed by F10L (A–D), Y11A (E–H) or Y11F (I–L) in the presence 100 µM of each individual dNTP (A, B, E, F, I, J) or rNTP (C, D, G, H, K, L) were carried out for 5 min. All reactions contained 1 mM ATP. The identity of the nucleotide incorporated is shown below each lane and the extended sequence of templates with five consecutive Ts (A, C, E, G, I, K) or As (B, D, F, H, J, L) is indicated to the right of each gel panel. All lanes with reactions lacking polymerase are indicated as ‘pr’ and reactions with no additional nucleotide are indicated by dash (–).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks233-F3: Specificity of nucleotide incorporation by mutant pol Vs. Reactions catalyzed by F10L (A–D), Y11A (E–H) or Y11F (I–L) in the presence 100 µM of each individual dNTP (A, B, E, F, I, J) or rNTP (C, D, G, H, K, L) were carried out for 5 min. All reactions contained 1 mM ATP. The identity of the nucleotide incorporated is shown below each lane and the extended sequence of templates with five consecutive Ts (A, C, E, G, I, K) or As (B, D, F, H, J, L) is indicated to the right of each gel panel. All lanes with reactions lacking polymerase are indicated as ‘pr’ and reactions with no additional nucleotide are indicated by dash (–).
Mentions: Misincorporation assays (Figure 3 and Supplementary Figure S2) were unambiguous for F10L, i.e. they show that this enzyme is quite accurate, although it is still able to incorporate ATP opposite template T, C and G (Figure 3A–D and Supplementary Figure S2A–D). Unexpectedly, misincorporation of ATP opposite C and G (Supplementary Figure S2A–D) is actually more efficient than opposite the complementary T (Figure 3A–D). Misincorporation patterns for wild-type pol V and Y11F were very similar with slight, if any, increase in fidelity of dNTP incorporation for the Y11F mutant (c.f. Figures 1 and 3I–L; Supplementary Figure S2I–S2L). Nevertheless, both enzymes are highly error-prone and capable of incorporation of multiple wrong dNTPs outcompeting excessive amounts of ATP (as judged by the unique pattern of product distribution in various lanes).Figure 3.

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