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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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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.
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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.

Mentions: The denB product of T4dC phages would not be expected to possess enzymatic activity, because Endo IV catalyzes endonucleolytic cleavage of dC-containing ssDNA. To confirm this expectation, we examined the enzymatic activities of Endo IV(W88R), Endo IV(S176N) and wild-type Endo IV with 45-base 5′-(dC)45-3′ ([(dC)45]45/45), 25-base 5′-(dC)25-3′ ([(dC)25]25/25) and various oligonucleotides as substrates listed in Table 1. The activity of wild-type Endo IV decreased as the length of dC tract decreased from 15 to 5, and its enzymatic activity with [dC]1/25 did not differ from that with [dCdCdCdCdC]5/25 (Figure 2). As expected, Endo IV(W88R) showed essentially no Endo IV activity, which was <0.4% of that of wild-type Endo IV with [(dC)25]25/25. In contrast, Endo IV(S176N) showed a substantial level of Endo IV activity, which was 17.5% of that of wild-type Endo IV with [(dC)25]25/25 and equal to that of wild-type Endo IV with [dCdCdCdCdC]5/25, even though the denB(S176N) allele allowed the synthesis of T4dC genomic DNA and the growth of host cells. In addition, the activity of wild-type Endo IV increased markedly (by a factor of 20) as the length of dC tract increased from five to six, whereas that of Endo IV(S176N) did not (by a factor of 2). These results indicated that the S176N mutation might affect the sequence preference of Endo IV.Figure 2.


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)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175332&req=5

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.
Mentions: The denB product of T4dC phages would not be expected to possess enzymatic activity, because Endo IV catalyzes endonucleolytic cleavage of dC-containing ssDNA. To confirm this expectation, we examined the enzymatic activities of Endo IV(W88R), Endo IV(S176N) and wild-type Endo IV with 45-base 5′-(dC)45-3′ ([(dC)45]45/45), 25-base 5′-(dC)25-3′ ([(dC)25]25/25) and various oligonucleotides as substrates listed in Table 1. The activity of wild-type Endo IV decreased as the length of dC tract decreased from 15 to 5, and its enzymatic activity with [dC]1/25 did not differ from that with [dCdCdCdCdC]5/25 (Figure 2). As expected, Endo IV(W88R) showed essentially no Endo IV activity, which was <0.4% of that of wild-type Endo IV with [(dC)25]25/25. In contrast, Endo IV(S176N) showed a substantial level of Endo IV activity, which was 17.5% of that of wild-type Endo IV with [(dC)25]25/25 and equal to that of wild-type Endo IV with [dCdCdCdCdC]5/25, even though the denB(S176N) allele allowed the synthesis of T4dC genomic DNA and the growth of host cells. In addition, the activity of wild-type Endo IV increased markedly (by a factor of 20) as the length of dC tract increased from five to six, whereas that of Endo IV(S176N) did not (by a factor of 2). These results indicated that the S176N mutation might affect the sequence preference of Endo IV.Figure 2.

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