The protein gp74 from the bacteriophage HK97 functions as a HNH endonuclease

Abstract

The last gene in the genome of the bacteriophage HK97 encodes the protein gp74. We present data in this article that demonstrates, for the first time, that gp74 possesses HNH endonuclease activity. HNH endonucleases are small DNA binding and digestion proteins characterized by two His residues and an Asn residue. We demonstrate that gp74 cleaves lambda phage DNA at multiple sites and that gp74 requires divalent metals for its endonuclease activity. We also present intrinsic tryptophan fluorescence data that show direct binding of Ni(2+) to gp74. The activity of gp74 in the presence of Ni(2+) is significantly decreased below neutral pH, suggesting the presence of one or more His residues in metal binding and/or DNA digestion. Surprisingly, this pH-dependence of activity is not seen with Zn(2+) , suggesting a different mode of binding of Zn(2+) and Ni(2+) . This difference in activity may result from binding of a second Zn(2+) ion by a putative zinc finger in gp74 in addition to binding of a Zn(2+) ion by the HNH motif. These studies define the biochemical function of gp74 as an HNH endonuclease and provide a platform for determining the role of gp74 in life cycle of the bacteriophage HK97.

Figure 1

Gp74-mediated digestion of lambda phage DNA in the presence of Ni²⁺ ions. Fractions containing gp74 from the Superdex 75 gel filtration column were assayed for endonuclease activity in the presence of Ni²⁺. The top panel shows the elution profile of gp74 from the Superdex 75 column. Fractions between 13.5 and 17.0 mL, indicated by dashed lines at the bottom of the trace, were tested. The bottom panel shows the agarose gel (1%) electrophoresis analysis of the digestion reactions from each fraction. Digestion reactions were allowed to proceed for 6 h. The concentrations of Ni²⁺ ions and lambda (λ) DNA in the reactions were 0.5 mM and 25 μg/mL, respectively. In each reaction, 26.3 μL of the gp74 fraction was used in a total reaction volume of 200 μL. A volume of 26.3 μL was chosen, so that gp74 concentration would be 24 μg/mL in the reaction corresponding to the elution fraction with the highest gp74 concentration. For the control reaction containing λ DNA and gp74 only, 26.3 μL from the fraction with the greatest amount of gp74 was used.

Figure 2

Concentration-dependence of gp74-mediated digestion of lambda phage DNA with Ni²⁺ ions. Agarose gel (1%) electrophoresis analysis of the time course of the lambda phage DNA digestion reactions is shown. The upper panel shows control reactions in which the DNA (50 μg/mL) was incubated in 20 mM HEPES, pH 7.0 alone, or in the presence of either gp74 (48 μg/mL) or 1 mM Ni²⁺ ions. There is no digestion of lambda phage under these conditions. The lower panel depicts digestion experiments in which lambda phage DNA was incubated with both gp74 and Ni²⁺ ions at concentrations of 0.5–5 mM. The smear of DNA in these gels indicates that in the presence of Ni²⁺, gp74 mediates digests λ DNA at multiple sites.

Figure 3

Gp74 also digests lambda phage DNA in the presence of Zn²⁺. Agarose gel (1%) electrophoresis analysis of the time course of the lambda phage DNA digestion by gp74 in the presence of 0.5–5 mM Zn²⁺ ions. As with the Ni²⁺ digestion experiments, the concentration of λ DNA was 50 μg/mL and that of gp74 was 48 μg/mL. Reactions were performed in 20 mM HEPES, pH 7.0. As seen in the presence of Ni²⁺, gp74 mediates digestion of λ DNA at multiple sites (smear) in the presence of Zn²⁺, albeit with reduced efficiency.

Figure 4

The activity of gp74 in the presence of Ni²⁺, but not Zn²⁺, is mediated by pH. Agarose gel (1%) electrophoresis analysis of lambda phage DNA digestion by gp74 in the presence of (A) Ni²⁺ ions or (B) Zn²⁺ ions. At pH 5, reactions contained 25 μg/mL of λ DNA, 48 μg/mL of gp74, and 0.5 mM of either Ni²⁺ or Zn²⁺ ions. At pH 6–8, reactions contained 50 μg/mL of lambda phage DNA, 48 μg/mL of gp74, and 0.5 mM of either Ni²⁺ or Zn²⁺ ions.

Figure 5

Circular dichroism spectra of gp74. CD spectra were recorded of 2 μM gp74 in 20 mM Na⁺ phosphate at pH values of 5 (red), 6 (purple), 7 (blue), or 8 (green). CD spectra are an average of 5 scans with absorbance measurements every 0.2 nm. All CD spectra were blank corrected.

Figure 6

Intrinsic tryptophan fluorescence data indicates direct binding of Ni²⁺ to gp74. (A) Emission spectra of 1 μM gp74 in the absence (solid line) and presence of 1M Ni²⁺ ions (dotted line). (B) Binding of Ni²⁺ ions to gp74 as monitored by intrinsic tryptophan fluorescence. The data (solid circles) were fit assuming a 1:1 complex (solid line),⁴² as described in Fluorescence metal-binding studies.