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Single-molecule analysis reveals two separate DNA-binding domains in the Escherichia coli UvrA dimer.

Wagner K, Moolenaar G, van Noort J, Goosen N - Nucleic Acids Res. (2009)

Bottom Line: Using single-molecule analysis we show that dimerization of UvrA in the presence of ATP is significantly higher than with ADP or nonhydrolyzable ATPgammaS, suggesting that the active UvrA dimer contains a mixture of ADP and ATP.Apparently ATP binding or hydrolysis is not needed to discriminate between DNA ends and internal sites.A significant number of complexes could be detected where one UvrA dimer bridges two DNA ends implying the presence of two separate DNA-binding domains, most likely present in each monomer.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Genetics, Leiden Institute of Chemistry, Leiden University, Leiden, the Netherlands.

ABSTRACT
The UvrA protein is the initial damage-recognizing factor in bacterial nucleotide excision repair. Each monomer of the UvrA dimer contains two ATPase sites. Using single-molecule analysis we show that dimerization of UvrA in the presence of ATP is significantly higher than with ADP or nonhydrolyzable ATPgammaS, suggesting that the active UvrA dimer contains a mixture of ADP and ATP. We also show that the UvrA dimer has a high preference of binding the end of a linear DNA fragment, independent on the presence or type of cofactor. Apparently ATP binding or hydrolysis is not needed to discriminate between DNA ends and internal sites. A significant number of complexes could be detected where one UvrA dimer bridges two DNA ends implying the presence of two separate DNA-binding domains, most likely present in each monomer. On DNA containing a site-specific lesion the damage-specific binding is much higher than DNA-end binding, but only in the absence of cofactor or with ATP. With ATPgammaS no discrimination between a DNA end and a DNA damage could be observed. We present a model where damage recognition of UvrA depends on the ability of both UvrA monomers to interact with the DNA flanking the lesion.

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Distribution of UvrA complexes on damaged (gray bars) and undamaged DNA (white bars), when incubated in absence of cofactor (A) and in presence of ATP (B), ATPγS (C) or ADP (D). The total number of complexes counted is shown in each plot. The occurrence probability is the observed probability of UvrA binding within a range of positions, and the complex position is shown as relative distance to the DNA center (see ‘Materials and methods’ section). Complexes with relative distance to the DNA center >0.4 are defined as bound to a DNA end.
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Figure 6: Distribution of UvrA complexes on damaged (gray bars) and undamaged DNA (white bars), when incubated in absence of cofactor (A) and in presence of ATP (B), ATPγS (C) or ADP (D). The total number of complexes counted is shown in each plot. The occurrence probability is the observed probability of UvrA binding within a range of positions, and the complex position is shown as relative distance to the DNA center (see ‘Materials and methods’ section). Complexes with relative distance to the DNA center >0.4 are defined as bound to a DNA end.

Mentions: Analysis of the position of UvrA on the DNA revealed that the majority of the UvrA–DNA complexes were located at the end of the DNA fragment (Figure 5A). The distribution of internal-bound UvrA versus end-bound protein was independent of the cofactor used (Figure 6 and Table 3), showing that the cofactor does not play a role in the discrimination between internal sites and DNA ends. Most likely the partly single-stranded nature of DNA ends enhances the affinity for UvrA. The specificity of UvrA for a DNA end versus an internal site was calculated to be a factor ∼400 [Equation (4), ‘Materials and methods’ section].Figure 5.


Single-molecule analysis reveals two separate DNA-binding domains in the Escherichia coli UvrA dimer.

Wagner K, Moolenaar G, van Noort J, Goosen N - Nucleic Acids Res. (2009)

Distribution of UvrA complexes on damaged (gray bars) and undamaged DNA (white bars), when incubated in absence of cofactor (A) and in presence of ATP (B), ATPγS (C) or ADP (D). The total number of complexes counted is shown in each plot. The occurrence probability is the observed probability of UvrA binding within a range of positions, and the complex position is shown as relative distance to the DNA center (see ‘Materials and methods’ section). Complexes with relative distance to the DNA center >0.4 are defined as bound to a DNA end.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2665241&req=5

Figure 6: Distribution of UvrA complexes on damaged (gray bars) and undamaged DNA (white bars), when incubated in absence of cofactor (A) and in presence of ATP (B), ATPγS (C) or ADP (D). The total number of complexes counted is shown in each plot. The occurrence probability is the observed probability of UvrA binding within a range of positions, and the complex position is shown as relative distance to the DNA center (see ‘Materials and methods’ section). Complexes with relative distance to the DNA center >0.4 are defined as bound to a DNA end.
Mentions: Analysis of the position of UvrA on the DNA revealed that the majority of the UvrA–DNA complexes were located at the end of the DNA fragment (Figure 5A). The distribution of internal-bound UvrA versus end-bound protein was independent of the cofactor used (Figure 6 and Table 3), showing that the cofactor does not play a role in the discrimination between internal sites and DNA ends. Most likely the partly single-stranded nature of DNA ends enhances the affinity for UvrA. The specificity of UvrA for a DNA end versus an internal site was calculated to be a factor ∼400 [Equation (4), ‘Materials and methods’ section].Figure 5.

Bottom Line: Using single-molecule analysis we show that dimerization of UvrA in the presence of ATP is significantly higher than with ADP or nonhydrolyzable ATPgammaS, suggesting that the active UvrA dimer contains a mixture of ADP and ATP.Apparently ATP binding or hydrolysis is not needed to discriminate between DNA ends and internal sites.A significant number of complexes could be detected where one UvrA dimer bridges two DNA ends implying the presence of two separate DNA-binding domains, most likely present in each monomer.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Genetics, Leiden Institute of Chemistry, Leiden University, Leiden, the Netherlands.

ABSTRACT
The UvrA protein is the initial damage-recognizing factor in bacterial nucleotide excision repair. Each monomer of the UvrA dimer contains two ATPase sites. Using single-molecule analysis we show that dimerization of UvrA in the presence of ATP is significantly higher than with ADP or nonhydrolyzable ATPgammaS, suggesting that the active UvrA dimer contains a mixture of ADP and ATP. We also show that the UvrA dimer has a high preference of binding the end of a linear DNA fragment, independent on the presence or type of cofactor. Apparently ATP binding or hydrolysis is not needed to discriminate between DNA ends and internal sites. A significant number of complexes could be detected where one UvrA dimer bridges two DNA ends implying the presence of two separate DNA-binding domains, most likely present in each monomer. On DNA containing a site-specific lesion the damage-specific binding is much higher than DNA-end binding, but only in the absence of cofactor or with ATP. With ATPgammaS no discrimination between a DNA end and a DNA damage could be observed. We present a model where damage recognition of UvrA depends on the ability of both UvrA monomers to interact with the DNA flanking the lesion.

Show MeSH
Related in: MedlinePlus