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Structure and function of the regulatory C-terminal HRDC domain from Deinococcus radiodurans RecQ.

Killoran MP, Keck JL - Nucleic Acids Res. (2008)

Bottom Line: The structure reveals unusual electrostatic surface features that distinguish it from other HRDC domains.Mutation of individual residues in these regions affects the DNA binding affinity of DrRecQ and its ability to unwind a partial duplex DNA substrate.Taken together, the results suggest the unusual electrostatic surface features of the DrRecQ HRDC domain may be important for inter-domain interactions that regulate structure-specific DNA binding and help direct DrRecQ to specific recombination/repair sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706-1532, USA.

ABSTRACT
RecQ helicases are critical for maintaining genome integrity in organisms ranging from bacteria to humans by participating in a complex network of DNA metabolic pathways. Their diverse cellular functions require specialization and coordination of multiple protein domains that integrate catalytic functions with DNA-protein and protein-protein interactions. The RecQ helicase from Deinococcus radiodurans (DrRecQ) is unusual among RecQ family members in that it has evolved to utilize three 'Helicase and RNaseD C-terminal' (HRDC) domains to regulate its activity. In this report, we describe the high-resolution structure of the C-terminal-most HRDC domain of DrRecQ. The structure reveals unusual electrostatic surface features that distinguish it from other HRDC domains. Mutation of individual residues in these regions affects the DNA binding affinity of DrRecQ and its ability to unwind a partial duplex DNA substrate. Taken together, the results suggest the unusual electrostatic surface features of the DrRecQ HRDC domain may be important for inter-domain interactions that regulate structure-specific DNA binding and help direct DrRecQ to specific recombination/repair sites.

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DrRecQ variants have reduced affinity for ssDNA and dup-3′ DNA. (A) The positions of alanine-substitution mutations are indicated on ribbons (left) and surface (right) representations of the HRDC #3 structure. Residues in close proximity to a phosphate ion in the crystal structure are shown in blue, with those comprising the acidic patch in red. (B–E) Fraction of DNA bound with either ssDNA (B and D) or dup-3′ (C and E) by DrRecQ variants with mutations in the phosphate binding site (B and C) or D816 in the acidic patch (D and E). DrRecQ (closed diamonds), K805A (open squares), R805A (closed circles) and D816A (open circles). A trend line for DrRecQ is shown in each plot for reference.
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Figure 2: DrRecQ variants have reduced affinity for ssDNA and dup-3′ DNA. (A) The positions of alanine-substitution mutations are indicated on ribbons (left) and surface (right) representations of the HRDC #3 structure. Residues in close proximity to a phosphate ion in the crystal structure are shown in blue, with those comprising the acidic patch in red. (B–E) Fraction of DNA bound with either ssDNA (B and D) or dup-3′ (C and E) by DrRecQ variants with mutations in the phosphate binding site (B and C) or D816 in the acidic patch (D and E). DrRecQ (closed diamonds), K805A (open squares), R805A (closed circles) and D816A (open circles). A trend line for DrRecQ is shown in each plot for reference.

Mentions: To test whether residues involved in phosphate binding or formation of the acidic patch affect the function of DrRecQ, we created single alanine-substitution mutations altering these regions in the full-length protein (Figure 2A). Purified DrRecQ variants were tested in vitro for their ability to bind DNA by observing DrRecQ-dependent changes in the fluorescence anisotropy of a fluoroscein-labeled ssDNA or dup-3′ DNA. Gel-shift analysis was used to test binding to synthetic HJ DNA because we had previously observed multiple binding events by DrRecQ variants using fluorescence anisotropy that made interpretation of the results difficult (18). Our hypothesis was that basic residues that bound phosphate in the HRDC #3 crystal structure could play roles in DNA binding and that their mutation to alanine could result in decreased DNA binding affinity. DrRecQ bound ssDNA with a Kd,app of 1.3 ± 0.5 nM (Figure 2B, Table 2). Substitution of alanine at either K805 or R806 resulted in modest 2-fold binding defects (Kd,app of 2.5 ± 0.6 nM and 2.7 ± 0.2 nM for each mutant, respectively) although only the defect in R806A ssDNA binding appeared to be statistically significant, with a P-value of 0.0078 (Figure 2B, Table 2). Likewise, the Kd,app for binding to the dup-3′ substrate was weakened 2-fold or less from 143.6 ± 23.4 nM for DrRecQ to 279.0 ± 17.7 nM for K805A and 177.9 ± 98.3 nM for R806A (Figure 2C, Table 2). Only the K805A mutant showed a statistically significant difference from WT on the dup-3′ substrate (P = 0.0013). We also observed modestly reduced HJ binding affinity for the K805A variant relative to DrRecQ, suggesting mutation of this residue compromises stable association with HJ DNA (Figure 3). These results indicate that phosphate binding residues in HRDC #3 could play a minor role in DNA binding, although the relatively modest binding defects of their variants suggest the remainder of DrRecQ provides the major DNA binding surfaces on the enzyme.Figure 2.


Structure and function of the regulatory C-terminal HRDC domain from Deinococcus radiodurans RecQ.

Killoran MP, Keck JL - Nucleic Acids Res. (2008)

DrRecQ variants have reduced affinity for ssDNA and dup-3′ DNA. (A) The positions of alanine-substitution mutations are indicated on ribbons (left) and surface (right) representations of the HRDC #3 structure. Residues in close proximity to a phosphate ion in the crystal structure are shown in blue, with those comprising the acidic patch in red. (B–E) Fraction of DNA bound with either ssDNA (B and D) or dup-3′ (C and E) by DrRecQ variants with mutations in the phosphate binding site (B and C) or D816 in the acidic patch (D and E). DrRecQ (closed diamonds), K805A (open squares), R805A (closed circles) and D816A (open circles). A trend line for DrRecQ is shown in each plot for reference.
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Figure 2: DrRecQ variants have reduced affinity for ssDNA and dup-3′ DNA. (A) The positions of alanine-substitution mutations are indicated on ribbons (left) and surface (right) representations of the HRDC #3 structure. Residues in close proximity to a phosphate ion in the crystal structure are shown in blue, with those comprising the acidic patch in red. (B–E) Fraction of DNA bound with either ssDNA (B and D) or dup-3′ (C and E) by DrRecQ variants with mutations in the phosphate binding site (B and C) or D816 in the acidic patch (D and E). DrRecQ (closed diamonds), K805A (open squares), R805A (closed circles) and D816A (open circles). A trend line for DrRecQ is shown in each plot for reference.
Mentions: To test whether residues involved in phosphate binding or formation of the acidic patch affect the function of DrRecQ, we created single alanine-substitution mutations altering these regions in the full-length protein (Figure 2A). Purified DrRecQ variants were tested in vitro for their ability to bind DNA by observing DrRecQ-dependent changes in the fluorescence anisotropy of a fluoroscein-labeled ssDNA or dup-3′ DNA. Gel-shift analysis was used to test binding to synthetic HJ DNA because we had previously observed multiple binding events by DrRecQ variants using fluorescence anisotropy that made interpretation of the results difficult (18). Our hypothesis was that basic residues that bound phosphate in the HRDC #3 crystal structure could play roles in DNA binding and that their mutation to alanine could result in decreased DNA binding affinity. DrRecQ bound ssDNA with a Kd,app of 1.3 ± 0.5 nM (Figure 2B, Table 2). Substitution of alanine at either K805 or R806 resulted in modest 2-fold binding defects (Kd,app of 2.5 ± 0.6 nM and 2.7 ± 0.2 nM for each mutant, respectively) although only the defect in R806A ssDNA binding appeared to be statistically significant, with a P-value of 0.0078 (Figure 2B, Table 2). Likewise, the Kd,app for binding to the dup-3′ substrate was weakened 2-fold or less from 143.6 ± 23.4 nM for DrRecQ to 279.0 ± 17.7 nM for K805A and 177.9 ± 98.3 nM for R806A (Figure 2C, Table 2). Only the K805A mutant showed a statistically significant difference from WT on the dup-3′ substrate (P = 0.0013). We also observed modestly reduced HJ binding affinity for the K805A variant relative to DrRecQ, suggesting mutation of this residue compromises stable association with HJ DNA (Figure 3). These results indicate that phosphate binding residues in HRDC #3 could play a minor role in DNA binding, although the relatively modest binding defects of their variants suggest the remainder of DrRecQ provides the major DNA binding surfaces on the enzyme.Figure 2.

Bottom Line: The structure reveals unusual electrostatic surface features that distinguish it from other HRDC domains.Mutation of individual residues in these regions affects the DNA binding affinity of DrRecQ and its ability to unwind a partial duplex DNA substrate.Taken together, the results suggest the unusual electrostatic surface features of the DrRecQ HRDC domain may be important for inter-domain interactions that regulate structure-specific DNA binding and help direct DrRecQ to specific recombination/repair sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706-1532, USA.

ABSTRACT
RecQ helicases are critical for maintaining genome integrity in organisms ranging from bacteria to humans by participating in a complex network of DNA metabolic pathways. Their diverse cellular functions require specialization and coordination of multiple protein domains that integrate catalytic functions with DNA-protein and protein-protein interactions. The RecQ helicase from Deinococcus radiodurans (DrRecQ) is unusual among RecQ family members in that it has evolved to utilize three 'Helicase and RNaseD C-terminal' (HRDC) domains to regulate its activity. In this report, we describe the high-resolution structure of the C-terminal-most HRDC domain of DrRecQ. The structure reveals unusual electrostatic surface features that distinguish it from other HRDC domains. Mutation of individual residues in these regions affects the DNA binding affinity of DrRecQ and its ability to unwind a partial duplex DNA substrate. Taken together, the results suggest the unusual electrostatic surface features of the DrRecQ HRDC domain may be important for inter-domain interactions that regulate structure-specific DNA binding and help direct DrRecQ to specific recombination/repair sites.

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