Biochemical analysis of the substrate specificity and sequence preference of endonuclease IV from bacteriophage T4, a dC-specific endonuclease implicated in restriction of dC-substituted T4 DNA synthesis

Abstract

Endonuclease IV encoded by denB of bacteriophage T4 is implicated in restriction of deoxycytidine (dC)-containing DNA in the host Escherichia coli. The enzyme was synthesized with the use of a wheat germ cell-free protein synthesis system, given a lethal effect of its expression in E.coli cells, and was purified to homogeneity. The purified enzyme showed high activity with single-stranded (ss) DNA and denatured dC-substituted T4 genomic double-stranded (ds) DNA but exhibited no activity with dsDNA, ssRNA or denatured T4 genomic dsDNA containing glucosylated deoxyhydroxymethylcytidine. Characterization of Endo IV activity revealed that the enzyme catalyzed specific endonucleolytic cleavage of the 5' phosphodiester bond of dC in ssDNA with an efficiency markedly dependent on the surrounding nucleotide sequence. The enzyme preferentially targeted 5'-dTdCdA-3' but tolerated various combinations of individual nucleotides flanking this trinucleotide sequence. These results suggest that Endo IV preferentially recognizes short nucleotide sequences containing 5'-dTdCdA-3', which likely accounts for the limited digestion of ssDNA by the enzyme and may be responsible in part for the indispensability of a deficiency in denB for stable synthesis of dC-substituted T4 genomic DNA.

Figure 1

SDS–PAGE analysis of the cell-free synthesis and purification of Endo IV. Endo IV was synthesized as a GST fusion protein by in vitro translation and purified as described in Materials and Methods. Samples at various stages of the purification procedure were subjected to SDS–PAGE on a 12% gel and stained with Coomassie brilliant blue. Lane 1, molecular size standards; lane 2, a translation mixture incubated in the absence of mRNA; lane 3, a translation mixture incubated in the presence of mRNA; lanes 4 and 5, the soluble and insoluble fractions, respectively, of the translation mixture incubated in the presence of mRNA; lanes 6 and 7, the flow-through fractions of a glutathione–Sepharose 4B MicroSpin column for the sample in lane 4 before and after, respectively, treatment of column-bound proteins with PreScission protease. The bands corresponding to the GST-Endo IV fusion protein in lane 3 and the purified Endo IV protein in lane 7 are indicated by asterisks.

Figure 2

Substrate specificity of Endo IV. The activity of Endo IV (1, 0.5, 0.2, 0.1 or 0.05 μg/ml) was assayed with phiX174 RFI circular dsDNA (A), phiX174 RFI linear dsDNA (B), phiX174 virion circular ssDNA (C), linear ssRNA used for the in vitro synthesis of the GST-Endo IV fusion protein (D), heat-denatured T4 genomic dsDNA containing gluc-dHMC (E), or heat-denatured dC-substituted T4 (T4dC) genomic dsDNA (F) as the substrate (5 μg/ml). The reaction products were separated by electrophoresis on a 1.0% agarose gel and stained with SYBR Gold. Lanes M and (–) contain a 1 kb DNA ladder and a reaction mixture incubated in the absence of the enzyme, respectively.

Figure 3

Cleavage of a phiX174-based oligonucleotide by Endo IV. A 45 base oligonucleotide based on the sequence of phiX174 ssDNA and labeled at the 5′ end with Cy5 was used as the substrate at a concentration of 10 μM for assay of the activity of Endo IV (6.6, 3.3, 1.3, 0.66 or 0.33 μg/ml). The reaction products were separated by electrophoresis on a 10% polyacrylamide gel containing 7 M urea and visualized with an image analyzer. Lane (–) represents a reaction mixture incubated in the absence of enzyme. Lane M represents a mixture of oligonucleotides labeled at the 5′ end with Cy5 and with sequences identical to those of residues 1–18, 1–27, 1–32, 1–34, 1–38, 1–41 and 1–45 of the substrate. Cleavage sites of the substrate are indicated by arrows with a size proportional to the relative extent of cleavage at the corresponding position. Each dC in the substrate sequence is shown in boldface.

Figure 4

Sequence preference of Endo IV. The activity of Endo IV was determined by measurement of the amount of acid-soluble nucleotides released from the substrate (10 μM). A series of 19 base 5′-(dA)₈dDdCdD(dA)₈-3′ oligonucleotides (A) and a series of 21 base 5′-(dA)₈dDdTdCdAdD(dA)₈-3′ oligonucleotides (B) were used as substrates, where dD represents dA, dT or dG. The relative activity was calculated by dividing the enzymatic activity for each oligonucleotide by that for 5′-(dA)₈dAdCdA(dA)₈-3′ or (dC)₂₅ in (A) or by that for 5′-(dA)₈dAdTdCdAdA(dA)₈-3′ or (dC)₂₅ in (B). Data are means from two independent experiments.