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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 denB(W88R) and denB(S176N) alleles on E. coli growth. Cultures of KH5402-1 cells harboring either pBR322 or pBRdenBam (A) or pBRW88Ram or pBRS176Nam (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.
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Figure 1: Effect of denB(W88R) and denB(S176N) alleles on E. coli growth. Cultures of KH5402-1 cells harboring either pBR322 or pBRdenBam (A) or pBRW88Ram or pBRS176Nam (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.

Mentions: To know whether the denB(W88R) and denB(S176N) alleles allow the growth of E. coli cells, we transformed KH5402-1 cells with a plasmid-containing denB mutant alleles in which an amber mutation had been introduced at the codon for Tyr38 (TAC). Cells cultured at 30°C would thus be expected to produce the full-length Endo IV mutant enzymes, whereas those cultured at 42°C would not. We previously showed that culture at 30°C of KH5402-1 cells transformed with pBRdenBam reduced colony-forming ability compared with that apparent at 42°C, and most (99%) of the original colonies that formed at 30°C did not give rise to colonies in the presence of the selection drug (ampicillin) at 42°C (4). These results indicated that most colonies that formed at 30°C consisted of cells lacking pBRdenBam and that most cells that expressed wild-type Endo IV were not viable. Here we examined the effects of denB expression on the growth of KH5402-1 cells transformed with pBRdenBam. KH5402-1 cells transformed with pBRdenBam revealed essentially no growth at 30°C, but normal growth at 42°C, and KH5402-1 cells transformed with pBR322 grow normally both at 30 and 42°C (Figure 1A). In contrast, the growth at 42 or 30°C of KH5402-1 cells transformed with pBR322 containing amber mutant forms of denB(W88R) or denB(S176N) alleles did not differ substantially from that of those transformed with pBR322 alone (Figure 1B). These results suggested that E. coli cells expressing the intact Endo IV mutant enzymes, Endo IV(W88R) and Endo IV(S176N), were viable and that, in contrast to wild-type Endo IV, these mutant enzymes do not show detrimental effect on the host cells.Figure 1.


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 denB(W88R) and denB(S176N) alleles on E. coli growth. Cultures of KH5402-1 cells harboring either pBR322 or pBRdenBam (A) or pBRW88Ram or pBRS176Nam (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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Effect of denB(W88R) and denB(S176N) alleles on E. coli growth. Cultures of KH5402-1 cells harboring either pBR322 or pBRdenBam (A) or pBRW88Ram or pBRS176Nam (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.
Mentions: To know whether the denB(W88R) and denB(S176N) alleles allow the growth of E. coli cells, we transformed KH5402-1 cells with a plasmid-containing denB mutant alleles in which an amber mutation had been introduced at the codon for Tyr38 (TAC). Cells cultured at 30°C would thus be expected to produce the full-length Endo IV mutant enzymes, whereas those cultured at 42°C would not. We previously showed that culture at 30°C of KH5402-1 cells transformed with pBRdenBam reduced colony-forming ability compared with that apparent at 42°C, and most (99%) of the original colonies that formed at 30°C did not give rise to colonies in the presence of the selection drug (ampicillin) at 42°C (4). These results indicated that most colonies that formed at 30°C consisted of cells lacking pBRdenBam and that most cells that expressed wild-type Endo IV were not viable. Here we examined the effects of denB expression on the growth of KH5402-1 cells transformed with pBRdenBam. KH5402-1 cells transformed with pBRdenBam revealed essentially no growth at 30°C, but normal growth at 42°C, and KH5402-1 cells transformed with pBR322 grow normally both at 30 and 42°C (Figure 1A). In contrast, the growth at 42 or 30°C of KH5402-1 cells transformed with pBR322 containing amber mutant forms of denB(W88R) or denB(S176N) alleles did not differ substantially from that of those transformed with pBR322 alone (Figure 1B). These results suggested that E. coli cells expressing the intact Endo IV mutant enzymes, Endo IV(W88R) and Endo IV(S176N), were viable and that, in contrast to wild-type Endo IV, these mutant enzymes do not show detrimental effect on the host cells.Figure 1.

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