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"Nick translation" in Escherichia coli rep strains deficient in DNA polymerase I activities
Abstract:
Using phiX1974 replicative form (RF) DNA as an in vivo probe, we have investigated the coordinated action of the 5' leads to 3' exonuclease and polymerase activities of DNA polymerase I in order to understand better its physiological role. We constructed double mutants containing the rep mutation (the replication of phiX174 RF does not occur in rep mutants) together with a mutation affecting DNA polymerase I, either polA12 or polA546ex. Using these mutants, which are believed to be thermosensitive in the polymerase function or the 5' leads to 3' exonuclease function respectively, we studied the kinetics of nick translation at the permissive and non-permissive temperatures in vivo. The substrate was the phiX174 replicative form DNA nicked by the phiX174 gene A protein. E. coli rep polA546ex gave the lowest rate of nick translation, although the ability to perform nick translation, at least as measured by our assay, was still present. E. coli rep polA12 showed a similar low rate at the non-permissive temperature but a rate close to the wild-type level at the permissive temperature. Formation of the parental replicative form molecule in either strain was affected little, even at the restrictive temperature. Our results suggest that DNA polymerase I may not play a major role in ongoing DNA replication.
Insights
Investigating DNA polymerase I
Area of Science:
- Molecular Biology
- Enzymology
- Bacteriology
Background:
- DNA polymerase I possesses both 5' to 3' exonuclease and polymerase activities.
- The physiological role of these coordinated activities in DNA replication remains incompletely understood.
Purpose of the Study:
- To investigate the in vivo role of DNA polymerase I's coordinated exonuclease and polymerase activities.
- To elucidate the physiological function of DNA polymerase I during DNA replication.
Main Methods:
- Construction of double mutants: E. coli rep mutants combined with thermosensitive DNA polymerase I mutations (polA12 and polA546ex).
- In vivo kinetic studies of nick translation using phiX174 replicative form DNA as a probe.
- Analysis of nick translation rates at permissive and non-permissive temperatures.
Main Results:
- E. coli rep polA546ex exhibited the slowest nick translation rate, indicating a role for the 5' to 3' exonuclease function.
- E. coli rep polA12 showed reduced nick translation at non-permissive temperatures, implicating polymerase function.
- Parental replicative form DNA formation was minimally affected by mutations, even at restrictive temperatures.
Conclusions:
- DNA polymerase I's exonuclease and polymerase activities are involved in nick translation.
- The study suggests DNA polymerase I may not be essential for ongoing DNA replication in vivo.
- The findings challenge the established view of DNA polymerase I's primary role in replication.