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Complex activities of the human Bloom's syndrome helicase are encoded in a core region comprising the RecA and Zn-binding domains.

Gyimesi M, Harami GM, Sarlós K, Hazai E, Bikádi Z, Kovács M - Nucleic Acids Res. (2012)

Bottom Line: We performed a quantitative mechanistic analysis of truncated BLM constructs that are shorter than the previously identified minimal functional module.Surprisingly, we found that a BLM construct comprising only the two conserved RecA domains and the Zn(2+)-binding domain (residues 642-1077) can efficiently perform all mentioned HR-related activities.The results demonstrate that the Zn(2+)-binding domain is necessary for functional interaction with DNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, ELTE-MTA Momentum Motor Enzymology Research Group, Eötvös University, Pázmány P. s. 1/c, H-1117 Budapest, Hungary.

ABSTRACT
Bloom's syndrome DNA helicase (BLM), a member of the RecQ family, is a key player in homologous recombination (HR)-based error-free DNA repair processes. During HR, BLM exerts various biochemical activities including single-stranded (ss) DNA translocation, separation and annealing of complementary DNA strands, disruption of complex DNA structures (e.g. displacement loops) and contributes to quality control of HR via clearance of Rad51 nucleoprotein filaments. We performed a quantitative mechanistic analysis of truncated BLM constructs that are shorter than the previously identified minimal functional module. Surprisingly, we found that a BLM construct comprising only the two conserved RecA domains and the Zn(2+)-binding domain (residues 642-1077) can efficiently perform all mentioned HR-related activities. The results demonstrate that the Zn(2+)-binding domain is necessary for functional interaction with DNA. We show that the extensions of this core, including the winged-helix domain and the strand separation hairpin identified therein in other RecQ-family helicases, are not required for mechanochemical activity per se and may instead play modulatory roles and mediate protein-protein interactions.

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BLM1077 disrupts D-loop structures. Unwinding reactions using Cy3-labelled (asterisks) D-loop DNA substrates with a 41-nt 3′-invading strand producing 21 bp of dsDNA region and a 20-nt 5′-ssDNA tail (3-INV, A), or a 41-nt 3′-invading strand with a 20-nt complementary strand annealed to the 5′-ssDNA-tail (DL, B) (schematic representations of structures are shown). The DL sample contained 24% of 3-INV substrate (lower band, indicated), which could not be separated by chromatography. Different concentrations (0–1 µM) of BLM1077 were incubated with the DNA substrates for 15 min at 37°C in the presence of 2 mM ATP. Samples were then deproteinised and run on 12% acrylamide gel. (C) Dependence of the extent of unwinding of the labelled strand (%) on BLM1077 concentration. 3-INV disruption (filled square) showed similar BLM1077 concentration dependence to that of DL substrate (open circle). The grey plot indicates the presence of 3-INV fraction in the DL sample.
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gks008-F5: BLM1077 disrupts D-loop structures. Unwinding reactions using Cy3-labelled (asterisks) D-loop DNA substrates with a 41-nt 3′-invading strand producing 21 bp of dsDNA region and a 20-nt 5′-ssDNA tail (3-INV, A), or a 41-nt 3′-invading strand with a 20-nt complementary strand annealed to the 5′-ssDNA-tail (DL, B) (schematic representations of structures are shown). The DL sample contained 24% of 3-INV substrate (lower band, indicated), which could not be separated by chromatography. Different concentrations (0–1 µM) of BLM1077 were incubated with the DNA substrates for 15 min at 37°C in the presence of 2 mM ATP. Samples were then deproteinised and run on 12% acrylamide gel. (C) Dependence of the extent of unwinding of the labelled strand (%) on BLM1077 concentration. 3-INV disruption (filled square) showed similar BLM1077 concentration dependence to that of DL substrate (open circle). The grey plot indicates the presence of 3-INV fraction in the DL sample.

Mentions: Synthesis-dependent strand annealing (SDSA) is one of the major routes of HR-based DNA repair in which BLM plays essential roles (Supplementary Figure S1) (1,53). In this pathway, disruption of D-loops is followed by re-annealing of the extended invading strand to the other 5′-resected end of the original DSB. To test whether these activities are retained in BLM1077, we performed strand annealing (Figure 4C–F) and D-loop disruption assays (Figure 5).Figure 5.


Complex activities of the human Bloom's syndrome helicase are encoded in a core region comprising the RecA and Zn-binding domains.

Gyimesi M, Harami GM, Sarlós K, Hazai E, Bikádi Z, Kovács M - Nucleic Acids Res. (2012)

BLM1077 disrupts D-loop structures. Unwinding reactions using Cy3-labelled (asterisks) D-loop DNA substrates with a 41-nt 3′-invading strand producing 21 bp of dsDNA region and a 20-nt 5′-ssDNA tail (3-INV, A), or a 41-nt 3′-invading strand with a 20-nt complementary strand annealed to the 5′-ssDNA-tail (DL, B) (schematic representations of structures are shown). The DL sample contained 24% of 3-INV substrate (lower band, indicated), which could not be separated by chromatography. Different concentrations (0–1 µM) of BLM1077 were incubated with the DNA substrates for 15 min at 37°C in the presence of 2 mM ATP. Samples were then deproteinised and run on 12% acrylamide gel. (C) Dependence of the extent of unwinding of the labelled strand (%) on BLM1077 concentration. 3-INV disruption (filled square) showed similar BLM1077 concentration dependence to that of DL substrate (open circle). The grey plot indicates the presence of 3-INV fraction in the DL sample.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351180&req=5

gks008-F5: BLM1077 disrupts D-loop structures. Unwinding reactions using Cy3-labelled (asterisks) D-loop DNA substrates with a 41-nt 3′-invading strand producing 21 bp of dsDNA region and a 20-nt 5′-ssDNA tail (3-INV, A), or a 41-nt 3′-invading strand with a 20-nt complementary strand annealed to the 5′-ssDNA-tail (DL, B) (schematic representations of structures are shown). The DL sample contained 24% of 3-INV substrate (lower band, indicated), which could not be separated by chromatography. Different concentrations (0–1 µM) of BLM1077 were incubated with the DNA substrates for 15 min at 37°C in the presence of 2 mM ATP. Samples were then deproteinised and run on 12% acrylamide gel. (C) Dependence of the extent of unwinding of the labelled strand (%) on BLM1077 concentration. 3-INV disruption (filled square) showed similar BLM1077 concentration dependence to that of DL substrate (open circle). The grey plot indicates the presence of 3-INV fraction in the DL sample.
Mentions: Synthesis-dependent strand annealing (SDSA) is one of the major routes of HR-based DNA repair in which BLM plays essential roles (Supplementary Figure S1) (1,53). In this pathway, disruption of D-loops is followed by re-annealing of the extended invading strand to the other 5′-resected end of the original DSB. To test whether these activities are retained in BLM1077, we performed strand annealing (Figure 4C–F) and D-loop disruption assays (Figure 5).Figure 5.

Bottom Line: We performed a quantitative mechanistic analysis of truncated BLM constructs that are shorter than the previously identified minimal functional module.Surprisingly, we found that a BLM construct comprising only the two conserved RecA domains and the Zn(2+)-binding domain (residues 642-1077) can efficiently perform all mentioned HR-related activities.The results demonstrate that the Zn(2+)-binding domain is necessary for functional interaction with DNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, ELTE-MTA Momentum Motor Enzymology Research Group, Eötvös University, Pázmány P. s. 1/c, H-1117 Budapest, Hungary.

ABSTRACT
Bloom's syndrome DNA helicase (BLM), a member of the RecQ family, is a key player in homologous recombination (HR)-based error-free DNA repair processes. During HR, BLM exerts various biochemical activities including single-stranded (ss) DNA translocation, separation and annealing of complementary DNA strands, disruption of complex DNA structures (e.g. displacement loops) and contributes to quality control of HR via clearance of Rad51 nucleoprotein filaments. We performed a quantitative mechanistic analysis of truncated BLM constructs that are shorter than the previously identified minimal functional module. Surprisingly, we found that a BLM construct comprising only the two conserved RecA domains and the Zn(2+)-binding domain (residues 642-1077) can efficiently perform all mentioned HR-related activities. The results demonstrate that the Zn(2+)-binding domain is necessary for functional interaction with DNA. We show that the extensions of this core, including the winged-helix domain and the strand separation hairpin identified therein in other RecQ-family helicases, are not required for mechanochemical activity per se and may instead play modulatory roles and mediate protein-protein interactions.

Show MeSH
Related in: MedlinePlus