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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.

Hirano N, Ohshima H, Sakashita H, Takahashi H - Nucleic Acids Res. (2007)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biological Science, Nihon University College of Bioresource Sciences, Fujisawa-shi, Kanagawa 252-8510, Japan.

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.

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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 (Vmax and Km) 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.
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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 (Vmax and Km) 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.

Mentions: Only limited information on the sequence preference of Endo IV has been available (4,12), and the small number of Endo IV-related proteins in the genome sequence databases has restricted the amount of insight provided by such proteins into the mechanism of sequence recognition by Endo IV. Recently, we have found that a 5′-dC1dN2dN3dN4dN5dC6-3′ (dC1–dC6) tract is crucial for the efficient cleavage by wild-type Endo IV (Figure 5A) (H. Ohshima, N. Hirano and H. Takahashi, submitted for publication). A third dC residue located at the dN2dN3dN4dN5 region of a dC1–dC6 tract improves the affinity of Endo IV to substrates, and a 5′-dCdTdT-3′ sequence at the dN2dN3dN4dN5 region restricts and enhances the Endo IV cleavage at the dC1 site (Figures 4A and 5a) (H. Ohshima, N. Hirano and H. Takahashi, submitted for publication).Figure 5.


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.

Hirano N, Ohshima H, Sakashita H, Takahashi H - Nucleic Acids Res. (2007)

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 (Vmax and Km) 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.
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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 (Vmax and Km) 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.
Mentions: Only limited information on the sequence preference of Endo IV has been available (4,12), and the small number of Endo IV-related proteins in the genome sequence databases has restricted the amount of insight provided by such proteins into the mechanism of sequence recognition by Endo IV. Recently, we have found that a 5′-dC1dN2dN3dN4dN5dC6-3′ (dC1–dC6) tract is crucial for the efficient cleavage by wild-type Endo IV (Figure 5A) (H. Ohshima, N. Hirano and H. Takahashi, submitted for publication). A third dC residue located at the dN2dN3dN4dN5 region of a dC1–dC6 tract improves the affinity of Endo IV to substrates, and a 5′-dCdTdT-3′ sequence at the dN2dN3dN4dN5 region restricts and enhances the Endo IV cleavage at the dC1 site (Figures 4A and 5a) (H. Ohshima, N. Hirano and H. Takahashi, submitted for publication).Figure 5.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biological Science, Nihon University College of Bioresource Sciences, Fujisawa-shi, Kanagawa 252-8510, Japan.

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.

Show MeSH
Related in: MedlinePlus