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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 1 μM DNA frayed duplex. Measurements were carried out at 37°C in 30 mM MES buffer (pH 7.0) containing 100 mM Na+, 0.5 mM Na2EDTA, RecG (0 nM) and ATP (0 mM) (black), RecG (50 nM) and ATP (0.1 mM) (red), RecG (100 nM) and ATP (0.2 mM ) (green), RecG (150 nM) and ATP (0.3 mM, blue), RecG (200 nM) and ATP (0.4 mM, cyan), and RecG (250 nM) and ATP (0.5 mM) (pink), at (a) 0 wt% PEG 200 and (b) 40 wt% PEG 200.
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fig3: CD spectra of 1 μM DNA frayed duplex. Measurements were carried out at 37°C in 30 mM MES buffer (pH 7.0) containing 100 mM Na+, 0.5 mM Na2EDTA, RecG (0 nM) and ATP (0 mM) (black), RecG (50 nM) and ATP (0.1 mM) (red), RecG (100 nM) and ATP (0.2 mM ) (green), RecG (150 nM) and ATP (0.3 mM, blue), RecG (200 nM) and ATP (0.4 mM, cyan), and RecG (250 nM) and ATP (0.5 mM) (pink), at (a) 0 wt% PEG 200 and (b) 40 wt% PEG 200.

Mentions: The CD spectrum of the DNA in the presence of 100 mM Na+ without PEG 200 was characterized by a positive peak at 275 nm and negative peaks at 211 nm and 247 nm, a spectrum typical of the B-form conformation [50] (Figure 3). We then added RecG and ATP to the DNA. At 50 nM RecG and 0.1 mM ATP, we observed a decrease in intensity of the positive peak at 275 nm and a red shift such that negative peaks were located at 215 nm and 250 nm (Figure 3(a)). There was no signal from RecG due to the low concentration. Spectra were recorded after successive additions of RecG and ATP, and after each addition we observed a decrease in intensity of the positive peak. At 250 nM RecG and 0.5 mM ATP, there was complete loss in original B-form conformation of the DNA, indicating unwinding of the duplex. Previous report described the role of RecG in the processing of stalled replication forks, and acted by reversing the fork past the damage to create a four-way junction that allows template switching and lesion bypass [24]. It has also been reported that RecG unwinds both the leading and lagging strand duplex arms of a three-way junction and the unwinding of these arms was found to be coordinated [24]. In our study, as frayed duplex contains only single-stranded long overhangs at both the terminus, therefore, we propose that RecG should bind to the frayed duplex (as it mimics the replication fork) and should convert the duplex into single strands. Therefore, to confirm the same we have recorded the CD spectra of single stands constituting the frayed duplex under dilute and molecular crowding conditions (Figure S7). The CD spectrum of each single strand of frayed duplex in the presence of 100 mM Na+, 100 mM Na+ and 1 mM Mg2+, and 5 mM Mg2+ with and without PEG 200 was characterized by a positive peak at 274 nm and negative peaks at 244 nm. These CD signatures indicate that each single strand of the frayed duplex folds into typical of the B-form conformation due to the formation of intramolecular structure. To better understand the structures of frayed duplex after unwinding by RecG, secondary structures of single strands were predicted using M-fold [51, 52]. The monomers of frayed duplex showed 3 short complementary stems with a varying number of bases in loops along with short dangling ends at opposite terminus in each strand (Figure S8). When CD spectra were recorded after successive additions of RecG and ATP, and after each addition, we observed a decrease in intensity of the positive peak (Figure 3 and Figure S9). At 250 nM RecG and 0.5 mM ATP, there was complete loss in original B-form conformation of the DNA, indicating unwinding of the duplex into single strands solely due to the enzymatic activity of RecG in the presence of ATP. Earlier studies indicated that the substrate specificity of RecG was critically dependent on the concentrations of ATP and MgCl2, and under certain conditions, RecG preferentially unwound three strand junctions of DNA [1, 11]. Here, we observed functional activity of RecG in the presence of Na+ and absence of Mg2+.


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 1 μM DNA frayed duplex. Measurements were carried out at 37°C in 30 mM MES buffer (pH 7.0) containing 100 mM Na+, 0.5 mM Na2EDTA, RecG (0 nM) and ATP (0 mM) (black), RecG (50 nM) and ATP (0.1 mM) (red), RecG (100 nM) and ATP (0.2 mM ) (green), RecG (150 nM) and ATP (0.3 mM, blue), RecG (200 nM) and ATP (0.4 mM, cyan), and RecG (250 nM) and ATP (0.5 mM) (pink), at (a) 0 wt% PEG 200 and (b) 40 wt% PEG 200.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: CD spectra of 1 μM DNA frayed duplex. Measurements were carried out at 37°C in 30 mM MES buffer (pH 7.0) containing 100 mM Na+, 0.5 mM Na2EDTA, RecG (0 nM) and ATP (0 mM) (black), RecG (50 nM) and ATP (0.1 mM) (red), RecG (100 nM) and ATP (0.2 mM ) (green), RecG (150 nM) and ATP (0.3 mM, blue), RecG (200 nM) and ATP (0.4 mM, cyan), and RecG (250 nM) and ATP (0.5 mM) (pink), at (a) 0 wt% PEG 200 and (b) 40 wt% PEG 200.
Mentions: The CD spectrum of the DNA in the presence of 100 mM Na+ without PEG 200 was characterized by a positive peak at 275 nm and negative peaks at 211 nm and 247 nm, a spectrum typical of the B-form conformation [50] (Figure 3). We then added RecG and ATP to the DNA. At 50 nM RecG and 0.1 mM ATP, we observed a decrease in intensity of the positive peak at 275 nm and a red shift such that negative peaks were located at 215 nm and 250 nm (Figure 3(a)). There was no signal from RecG due to the low concentration. Spectra were recorded after successive additions of RecG and ATP, and after each addition we observed a decrease in intensity of the positive peak. At 250 nM RecG and 0.5 mM ATP, there was complete loss in original B-form conformation of the DNA, indicating unwinding of the duplex. Previous report described the role of RecG in the processing of stalled replication forks, and acted by reversing the fork past the damage to create a four-way junction that allows template switching and lesion bypass [24]. It has also been reported that RecG unwinds both the leading and lagging strand duplex arms of a three-way junction and the unwinding of these arms was found to be coordinated [24]. In our study, as frayed duplex contains only single-stranded long overhangs at both the terminus, therefore, we propose that RecG should bind to the frayed duplex (as it mimics the replication fork) and should convert the duplex into single strands. Therefore, to confirm the same we have recorded the CD spectra of single stands constituting the frayed duplex under dilute and molecular crowding conditions (Figure S7). The CD spectrum of each single strand of frayed duplex in the presence of 100 mM Na+, 100 mM Na+ and 1 mM Mg2+, and 5 mM Mg2+ with and without PEG 200 was characterized by a positive peak at 274 nm and negative peaks at 244 nm. These CD signatures indicate that each single strand of the frayed duplex folds into typical of the B-form conformation due to the formation of intramolecular structure. To better understand the structures of frayed duplex after unwinding by RecG, secondary structures of single strands were predicted using M-fold [51, 52]. The monomers of frayed duplex showed 3 short complementary stems with a varying number of bases in loops along with short dangling ends at opposite terminus in each strand (Figure S8). When CD spectra were recorded after successive additions of RecG and ATP, and after each addition, we observed a decrease in intensity of the positive peak (Figure 3 and Figure S9). At 250 nM RecG and 0.5 mM ATP, there was complete loss in original B-form conformation of the DNA, indicating unwinding of the duplex into single strands solely due to the enzymatic activity of RecG in the presence of ATP. Earlier studies indicated that the substrate specificity of RecG was critically dependent on the concentrations of ATP and MgCl2, and under certain conditions, RecG preferentially unwound three strand junctions of DNA [1, 11]. Here, we observed functional activity of RecG in the presence of Na+ and absence of Mg2+.

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