What question did this study set out to answer?

To identify the specific genetic mutations in five classical temperature-sensitive Escherichia coli dnaB mutants and characterize their corresponding biochemical defects at permissive and nonpermissive temperatures.

February 1, 1995Journal of BacteriologyOpen Access

Biochemical characterization of Escherichia coli temperature-sensitive dnaB mutants dnaB8, dnaB252, dnaB70, dnaB43, and dnaB454

View Full Paper

Ask AI

Bookmark

Authors

DSDaman SalujaUniversity of Delhi GGG N GodsonColumbia University Irving Medical Center

Discussion

Member takes

Implication

In vitro study reveals how point mutations impair enzymatic functions in Escherichia coli DnaB mutants at elevated temperatures, suggesting distinct domain-specific regulatory roles.

Key Points

To identify the specific genetic mutations in five classical temperature-sensitive Escherichia coli dnaB mutants and characterize their corresponding biochemical defects at permissive and nonpermissive temperatures.
Isolated and sequenced dnaB genes from mutants PC8 (dnaB8), RS162 (dnaB252), CR34/454 (dnaB454), HfrH165/70 (dnaB70), and CR34/43 (dnaB43), then overexpressed recombinant proteins in BL21 (DE3) cells.
Assayed purified mutant proteins for primer RNA synthesis, ATPase activity, and helicase activity at 30°C and 42°C.
Measured single-stranded DNA (ssDNA) binding capability across temperatures via gel shift assays using a 5-(N-(p-azidobenzoyl)-3-aminoallyl)-dUMP photoaffinity analog.
Mutants dnaB8, dnaB454, and dnaB43 shared an identical Ala130Val mutation in the hinge region, exhibiting negligible primer RNA synthesis, ATPase, and helicase activities at 42°C.
The dnaB70 mutant harbored a Met242Ile change near the ATP-binding site and displayed approximately 25% of wild-type ATPase activity at 30°C that remained stable at 42°C, while DnaB252 (Gly299Asp) matched wild-type activity across all assays.
All mutant proteins retained ssDNA binding at both 30°C and 42°C, proving that thermal inactivation of enzymatic activity does not stem from defective substrate binding.

Cite This Study

Saluja et al. (1995) studied this question.

synapsesocial.com/papers/6a8c137352e1a91077e9901c https://doi.org/10.1128/jb.177.4.1104-1111.1995

View Full Paper

Ask AI

Bookmark

February 1, 1995Journal of BacteriologyOpen Access

Biochemical characterization of Escherichia coli temperature-sensitive dnaB mutants dnaB8, dnaB252, dnaB70, dnaB43, and dnaB454

View Full Paper

Ask AI

Bookmark

Authors

DSDaman SalujaUniversity of Delhi GGG N GodsonColumbia University Irving Medical Center

Discussion

Member takes

Implication

In vitro study reveals how point mutations impair enzymatic functions in Escherichia coli DnaB mutants at elevated temperatures, suggesting distinct domain-specific regulatory roles.

Key Points

To identify the specific genetic mutations in five classical temperature-sensitive Escherichia coli dnaB mutants and characterize their corresponding biochemical defects at permissive and nonpermissive temperatures.
Isolated and sequenced dnaB genes from mutants PC8 (dnaB8), RS162 (dnaB252), CR34/454 (dnaB454), HfrH165/70 (dnaB70), and CR34/43 (dnaB43), then overexpressed recombinant proteins in BL21 (DE3) cells.
Assayed purified mutant proteins for primer RNA synthesis, ATPase activity, and helicase activity at 30°C and 42°C.
Measured single-stranded DNA (ssDNA) binding capability across temperatures via gel shift assays using a 5-(N-(p-azidobenzoyl)-3-aminoallyl)-dUMP photoaffinity analog.
Mutants dnaB8, dnaB454, and dnaB43 shared an identical Ala130Val mutation in the hinge region, exhibiting negligible primer RNA synthesis, ATPase, and helicase activities at 42°C.
The dnaB70 mutant harbored a Met242Ile change near the ATP-binding site and displayed approximately 25% of wild-type ATPase activity at 30°C that remained stable at 42°C, while DnaB252 (Gly299Asp) matched wild-type activity across all assays.
All mutant proteins retained ssDNA binding at both 30°C and 42°C, proving that thermal inactivation of enzymatic activity does not stem from defective substrate binding.

Cite This Study

Saluja et al. (1995) studied this question.

synapsesocial.com/papers/6a8c137352e1a91077e9901c https://doi.org/10.1128/jb.177.4.1104-1111.1995

Also Consider

Synapse has enriched 5 closely related papers on similar clinical questions. Consider them for comparative context:

View Full Paper

Ask AI

Bookmark