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Structural and Functional Characterization of RecG Helicase under Dilute and Molecular Crowding Conditions.

Saxena S, Nagatoishi S, Miyoshi D, Sugimoto N - J Nucleic Acids (2012)

Bottom Line: In an ATP-dependent reaction, the Escherichia coli RecG helicase unwinds DNA junctions in vitro.These distinct conformational behaviors were observed to be independent of Na(+) and Mg(2+).Our findings raise the possibility that the α-helix and β-strand forms of RecG are a preactive and an active structure with the helicase activity, respectively.

View Article: PubMed Central - PubMed

Affiliation: Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, 7-1-20 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan.

ABSTRACT
In an ATP-dependent reaction, the Escherichia coli RecG helicase unwinds DNA junctions in vitro. We present evidence of a unique protein conformational change in the RecG helicase from an α-helix to a β-strand upon an ATP binding under dilute conditions using circular dichroism (CD) spectroscopy. In contrast, under molecular crowding conditions, the α-helical conformation was stable even upon an ATP binding. These distinct conformational behaviors were observed to be independent of Na(+) and Mg(2+). Interestingly, CD measurements demonstrated that the spectra of a frayed duplex decreased with increasing of the RecG concentration both under dilute and molecular crowding conditions in the presence of ATP, suggesting that RecG unwound the frayed duplex. Our findings raise the possibility that the α-helix and β-strand forms of RecG are a preactive and an active structure with the helicase activity, respectively.

No MeSH data available.


Related in: MedlinePlus

CD spectra of 5 μM RecG. Measurements were carried out in 30 mM MES buffer (pH 7.0) containing 100 mM Na+ and 0.5 mM Na2EDTA (a) at 0 wt% PEG 200 without ATP at 4°C (black) and 37°C (green), or with 1 mM ATP at 4°C (red) and 37°C (blue), and (b) at 40 wt% PEG 200 without ATP at 4°C (black) and 37°C (green), or with 1 mM ATP at 4°C (red) and 37°C (blue), respectively.
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fig1: CD spectra of 5 μM RecG. Measurements were carried out in 30 mM MES buffer (pH 7.0) containing 100 mM Na+ and 0.5 mM Na2EDTA (a) at 0 wt% PEG 200 without ATP at 4°C (black) and 37°C (green), or with 1 mM ATP at 4°C (red) and 37°C (blue), and (b) at 40 wt% PEG 200 without ATP at 4°C (black) and 37°C (green), or with 1 mM ATP at 4°C (red) and 37°C (blue), respectively.

Mentions: Firstly, we used CD spectroscopy to study the structural states of RecG with and without ATP in the presence of different cations (Na+, Mg2+, or both Na+ and Mg2+) at 0 wt% and 40 wt% PEG 200, a neutral cosolute to study systematically the effects of molecular crowding. RecG was expressed in E. coli and purified by using ion exchange chromatography and affinity chromatography followed by dialysis. Figure 1(a) shows CD spectra at 4°C or 37°C of 5 μM RecG in the presence of 100 mM Na+ with or without ATP at 0 wt% PEG 200. The CD spectra without ATP displayed a positive peak at 198 nm and negative peaks at 208 nm and 220 nm, which are characteristic of an α-helix [23]. In the presence of 1 mM ATP, the CD spectra of RecG had a positive peak at 195 nm and a negative peak at 225 nm. These CD signatures are in agreement with formation of a β-strand structure [23].


Structural and Functional Characterization of RecG Helicase under Dilute and Molecular Crowding Conditions.

Saxena S, Nagatoishi S, Miyoshi D, Sugimoto N - J Nucleic Acids (2012)

CD spectra of 5 μM RecG. Measurements were carried out in 30 mM MES buffer (pH 7.0) containing 100 mM Na+ and 0.5 mM Na2EDTA (a) at 0 wt% PEG 200 without ATP at 4°C (black) and 37°C (green), or with 1 mM ATP at 4°C (red) and 37°C (blue), and (b) at 40 wt% PEG 200 without ATP at 4°C (black) and 37°C (green), or with 1 mM ATP at 4°C (red) and 37°C (blue), respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3420092&req=5

fig1: CD spectra of 5 μM RecG. Measurements were carried out in 30 mM MES buffer (pH 7.0) containing 100 mM Na+ and 0.5 mM Na2EDTA (a) at 0 wt% PEG 200 without ATP at 4°C (black) and 37°C (green), or with 1 mM ATP at 4°C (red) and 37°C (blue), and (b) at 40 wt% PEG 200 without ATP at 4°C (black) and 37°C (green), or with 1 mM ATP at 4°C (red) and 37°C (blue), respectively.
Mentions: Firstly, we used CD spectroscopy to study the structural states of RecG with and without ATP in the presence of different cations (Na+, Mg2+, or both Na+ and Mg2+) at 0 wt% and 40 wt% PEG 200, a neutral cosolute to study systematically the effects of molecular crowding. RecG was expressed in E. coli and purified by using ion exchange chromatography and affinity chromatography followed by dialysis. Figure 1(a) shows CD spectra at 4°C or 37°C of 5 μM RecG in the presence of 100 mM Na+ with or without ATP at 0 wt% PEG 200. The CD spectra without ATP displayed a positive peak at 198 nm and negative peaks at 208 nm and 220 nm, which are characteristic of an α-helix [23]. In the presence of 1 mM ATP, the CD spectra of RecG had a positive peak at 195 nm and a negative peak at 225 nm. These CD signatures are in agreement with formation of a β-strand structure [23].

Bottom Line: In an ATP-dependent reaction, the Escherichia coli RecG helicase unwinds DNA junctions in vitro.These distinct conformational behaviors were observed to be independent of Na(+) and Mg(2+).Our findings raise the possibility that the α-helix and β-strand forms of RecG are a preactive and an active structure with the helicase activity, respectively.

View Article: PubMed Central - PubMed

Affiliation: Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, 7-1-20 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan.

ABSTRACT
In an ATP-dependent reaction, the Escherichia coli RecG helicase unwinds DNA junctions in vitro. We present evidence of a unique protein conformational change in the RecG helicase from an α-helix to a β-strand upon an ATP binding under dilute conditions using circular dichroism (CD) spectroscopy. In contrast, under molecular crowding conditions, the α-helical conformation was stable even upon an ATP binding. These distinct conformational behaviors were observed to be independent of Na(+) and Mg(2+). Interestingly, CD measurements demonstrated that the spectra of a frayed duplex decreased with increasing of the RecG concentration both under dilute and molecular crowding conditions in the presence of ATP, suggesting that RecG unwound the frayed duplex. Our findings raise the possibility that the α-helix and β-strand forms of RecG are a preactive and an active structure with the helicase activity, respectively.

No MeSH data available.


Related in: MedlinePlus