The Escherichia coli polB locus is identical to dinA, the structural gene for DNA polymerase II. Characterization of Pol II purified from a polB mutant.

Abstract:

Escherichia coli DNA polymerase II (Pol II) is a member of the group B, "alpha-like" family of DNA polymerases. Pol II is encoded by the damage-inducible dinA gene and exhibits SOS induction under the control of Lex A repressor. The polB gene was originally designated as the structural gene for Pol II based on the absence of detectable Pol II activity in cell lysates prepared from a strain containing the mutant polB100 allele. Because polB and dinA mapped at different chromosomal locations, it remained an open question whether polB, in addition to lexA, might be involved in regulating the expression of Pol II. We have cloned and sequenced the polB100 mutant allele, including adjacent surrounding sequences, and have expressed the mutant dinA gene from Pol B100 on a high copy number plasmid. Our sequence data reveal that polB and dinA represent the same gene and that the original transduction mapping of polB was inaccurate. We purified the mutant Pol B100 polymerase and show that it retains 5 to 10% of the wild-type level of polymerase activity. The Pol B100 mutation, Gly401 --> Asp401, is not located within any of the five conserved domains that define group B polymerases. Pol B100 retains a wild-type level of 3' --> 5' exonuclease activity. We suggest that the normal level of exonucleolytic proofreading associated with the mutant Pol B100 enzyme may explain the repeated failures, over the past two decades, to detect phenotypes in polB mutant strains.

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