The Ser176 of T4 endonuclease IV is crucial for the restricted and polarized dC-specific cleavage of single-stranded DNA implicated in restriction of dC-containing DNA in host Escherichia coli

Abstract

Endonuclease (Endo) IV encoded by denB of bacteriophage T4 is an enzyme that cleaves single-stranded (ss) DNA in a dC-specific manner. Also the growth of dC-substituted T4 phage and host Escherichia coli cells is inhibited by denB expression presumably because of the inhibitory effect on replication of dC-containing DNA. Recently, we have demonstrated that an efficient cleavage by Endo IV occurs exclusively at the 5'-proximal dC (dC1) within a hexameric or an extended sequence consisting of dC residues at the 5'-proximal and the 3'-proximal positions (dCs tract), in which a third dC residue within the tract affects the polarized cleavage and cleavage rate. Here we isolate and characterize two denB mutants, denB(W88R) and denB(S176N). Both mutant alleles have lost the detrimental effect on the host cell. Endo IV(W88R) shows no enzymatic activity (<0.4% of that of wild-type Endo IV). On the other hand, Endo IV(S176N) retains cleavage activity (17.5% of that of wild-type Endo IV), but has lost the polarized and restricted cleavage of a dCs tract, indicating that the Ser176 residue of Endo IV is implicated in the polarized cleavage of a dCs tract which brings about a detrimental effect on the replication of dC-containing DNA.

Figure 1.

Effect of denB(W88R) and denB(S176N) alleles on E. coli growth. Cultures of KH5402-1 cells harboring either pBR322 or pBRdenB^am (A) or pBRW88R^am or pBRS176N^am (B) were grown overnight in LB-Thy-Amp liquid broth at 42°C, and the cells were then used to inoculate fresh LB-Thy-Amp liquid broth and incubated at 42°C or 30°C. Samples were removed at the indicated times for determination of OD₆₀₀. Data are means of values from two independent experiments.

Figure 2.

Effect of dC tract length on enzymatic activities of wild-type and mutant (S176N or W88R) forms of Endo IV. Enzymatic activity was determined by measurement of the amount of acid-soluble nucleotides released from the substrate (10 μM). All substrates with the exception of [(dC)₄₅]_45/45 contained 25 nt. The specific activity of the wild-type (WT) enzyme with the [(dC)₂₅]_25/25 substrate was ∼8.0 U/mg. Relative activity was calculated by dividing the enzymatic activity of each Endo IV enzyme observed with each substrate by that apparent with the wild-type enzyme and [(dC)₂₅]_25/25 as substrate. Data are means of values from two independent experiments.

Figure 3.

Analysis and comparison of the sequence preferences of wild-type and S176N mutant forms of Endo IV. The V_max (A) and K_m (B) of the wild-type (WT) and S176N mutant forms of Endo IV were determined with series of 25-base oligonucleotides [dCdYdYdYdYdC]_6/25 (where dY represents dT or dC) and shown by black columns (WT) and white columns (S176N) in the figure. Data are means of values from two independent experiments.

Figure 4.

Comparison of cleavage patterns of oligonucleotide containing the 5′-dCdTdCdTdTdC-3′ corresponding to a T4 DNA sequence by wild-type and S176N mutant forms of Endo IV. A 45-base oligonucleotide (Cy5–T4A) based on the sequence of T4 DNA and labeled at its 5′ end with Cy5 was used as the substrate (10 μM) for assay of the activity of wild-type (0.5, 1.0, 2.0 or 4.0 μg/ml) (A) or S176N mutant (1.0, 2.0, 4.0 or 8.0 μg/ml) (B) forms of Endo IV. The reaction products were separated by electrophoresis on a 10% polyacrylamide gel containing 7 M urea and were 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 their 5′ ends with Cy5 and with sequences identical to those of residues 1 to 19, 1 to 21 or 1 to 24 of the substrate. Cleavage sites of the substrate are indicated by arrows, and dC sites in dG₁₉–dC₂₀–dT₂₁, dT₂₁–dC₂₂–dT₂₃ and dT₂₄–dC₂₅–dA₂₆ are indicated by a, b and c, which correspond to the dC₁, dC₃ and dC₆ sites of the 5′-dC₁dT₂dC₃dT₄dT₅dC₆-3′ tract, respectively.

Figure 5.

Schematic representation of the dCs tract model for restricted dC-specific cleavage by Endo IV and the role of Ser¹⁷⁶. (A) A dCs tract is shown as a horizontal line with two closed boxes (dC₆ and dC₁) from the 3′ to 5′ direction. A downward arrow indicates the point where an endonucleolytic cleavage by Endo IV occurs and a rightward arrow indicates the element enhancing and restricting the enzymatic activity (V_max and K_m) of Endo IV. Endo IV binds to both dC₁ and dC₆ residues, and especially the Ser¹⁷⁶ residue of the enzyme contributes to recognition of a dC residue located at the dN₂dN₃dN₄dN₅ region of a dCs tract and leads the enzyme to exhibit cytotoxicity and recognize the 5′-dCdTdT-3′ trinucleotide element enhancing the cleavage activity at the dC₁ site. This interaction is required for the restricted and polarized cleavage at the dC₁ site by Endo IV. (B) Replacement of Ser¹⁷⁶ with Asn disrupts the interaction between Endo IV and a dC residue located at the dN₂dN₃dN₄dN₅ region, resulting in the losses of restricted and polarized cleavage at the dC₁ site and cytotoxicity.