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. 2002 Jun 1;30(11):2280-9.
doi: 10.1093/nar/30.11.2280.

Bacillus subtilis bacteriophage SPP1 hexameric DNA helicase, G40P, interacts with forked DNA

Silvia Ayora  1 , Frank Weise, Pablo Mesa, Andrzej Stasiak, Juan C Alonso
Affiliations

Bacillus subtilis bacteriophage SPP1 hexameric DNA helicase, G40P, interacts with forked DNA

Silvia Ayora et al. Nucleic Acids Res. .

Abstract

SPP1-encoded replicative DNA helicase gene 40 product (G40P) is an essential product for phage replication. Hexameric G40P, in the presence of AMP-PNP, preferentially binds unstructured single-stranded (ss)DNA in a sequence-independent manner. The efficiency of ssDNA binding, nucleotide hydrolysis and the unwinding activity of G40P are affected in a different manner by different nucleotide cofactors. Nuclease protection studies suggest that G40P protects the 5' tail of a forked molecule, and the duplex region at the junction against exonuclease attack. G40P does not protect the 3' tail of a forked molecule from exonuclease attack. By using electron microscopy we confirm that the ssDNA transverses the centre of the hexameric ring. Our results show that hexameric G40P DNA helicase encircles the 5' tail, interacts with the duplex DNA at the ss-double-stranded DNA junction and excludes the 3' tail of the forked DNA.

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Figures

Figure 1
Figure 1
ATPase activity of G40P. (A, B and C) The effect of polynucleotide effector concentration on G40P ATPase activity is shown. Reactions were as described in the Materials and Methods. ATPase activity of G40P is presented as turnover in percent versus concentration of effector nucleotides. (A) ssDNA as effector. The results for poly(dA) are shown enlarged in the small window; (B and C) dsDNA and RNA as effector, respectively. (D) ATPase activity in the presence (circles) and absence (squares) of ssDNA as a function of ATP concentration. (E) Effect of different cations on the ATPase activity of G40P. Filled squares or bars denote results in the presence of ssDNA, and empty squares or bars indicate results in absence of ssDNA.
Figure 2
Figure 2
Effect of ribonucleotides on the G40P activities. (A) NTPase activity of G40P in the presence of ssDNA as a function of NTP concentration. (B) Kapp of G40P for a 50-nt ssDNA in the presence of 2 mM NTP. (C) DNA unwinding activity of G40P in the presence of the 2 mM NTP. The symbols + and – denote the presence or absence of G40P. H denotes heated susbtrate. The data represent the average of three independent experiments. Experimental details are described in the Materials and Methods.
Figure 3
Figure 3
G40P–DNA complex formation. (A) Kapp of G40P for 51- or 50-nt [γ-32P]ssDNA (2 nM) in the absence or the presence of ATP or AMP-PNP measured by EMSA. The Kapp of G40P for 21-, 18- and 16-nt [γ-32P]ssDNA segments, in the presence of AMP-PNP, were also measured. (B) EMSA of G40P with a 21-nt [γ-32P]ssDNA (G40P concentrations doubling from 0.25 to 32 nM) and with a 51-nt [γ-32P]ssDNA segment (G40P concentrations doubling from 3 to 384 nM) were assayed. The two types of complexes obtained are denoted by I and II, and FD denotes the ssDNA in the absence of G40P. (C) Kapp of G40P for [γ-32P]-single-arm or [γ-32P]-forked substrates in the presence of AMP-PNP, measured by EMSA, are shown. All Kapp data represent the average of three independent experiments. The poly(dA)16 and poly(dA)25 regions are denoted as an oblique line. (D) EMSA of G40P with a 5′ single-arm substrate (G40P concentrations doubling from 6 to 50 nM) and with 3′ single-arm substrate (G40P concentrations doubling from 25 to 200 nM). The complexes obtained are denoted by I and FD denotes the protein-free DNA.
Figure 4
Figure 4
G40P protection of the ssDNA arms of a forked molecule. G40P (12.5 and 25 nM) was incubated with 2 nM of double-arm substrate labelled at the 5′ end (A) or at the 3′ end (B) of the duplex in the presence or in the absence of 1 mM AMP-PNP. After 15 min incubation at 37°C, ExoVII (0.2 U) was added and the incubation was continued for 15 min at 37°C. The digestions were stopped and samples separated by 10% dPAGE and analysed by autoradiography. G40P encircling the 5′ tail of the 39/16 forked substrate is depicted not at scale. The symbols + and – denote the presence or absence of the indicated product. The asterisk denotes the labelled end. The arrow indicates the direction of exonucleolytic digestion. [γ-32P]poly(dA) is used as a 1-nt molecular weight ladder.
Figure 5
Figure 5
G40P protection of the duplex region of the 39/16 forked molecule. G40P (12.5 and 25 nM) was incubated with 2 nM of forked molecule labelled at the 3′ (A) or at the 5′ end (B) of the ssDNA tail, in the presence or in the absence of 1 mM AMP-PNP. After 15 min incubation a 37°C, ExoVI (1 U) (A) or ExoIII (1 U) (B) was added and the incubation was continued for 15 min at 37°C. The digestions were stopped and samples separated by 15% dPAGE and analysed by autoradiography. G40P encircling the 5′ tail of the 39/16 forked substrate is depicted not at scale. The symbols + and – denote the presence or absence of the indicated product. The asterisk denotes the labelled end. The arrow indicates the direction of exonucleolytic digestion. [γ-32P]poly(dA) is used as a 1-nt molecular weight ladder.
Figure 6
Figure 6
Electron micrographs of G40P bound to viral M13mp18 DNA in the presence of AMP-PNP. ssM13mp18 DNA (0.2 nM) was incubated, in buffer C, with G40P (50 nM) during 15 min at 37°C, and the complexes were visualised by EM. The regions of ssDNA entering the G40P inner channel are denoted by arrows (a–l). Three controls of ssM13mp18 in the absence of G40P are shown in (m–o).
Figure 7
Figure 7
Proposed model of unwinding of DNA by G40P. G40P binds ssDNA in a 5′ → 3′ polarity through the inner channel, contacting the 5′-ssDNA arm, whereas there is no interaction with the 3′ arm which is fully excluded. G40P interacts with dsDNA at the junction. Protein conformational changes and ATP hydrolysis would lead to base pair separation. Then, the hexamer would translocate and bind to the newly separated stretch of the strand.
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References

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