Studies on the mechanism of A/T to C/G transversion inmutT mutant of Escherichia coli and identification of a new F factor genesut1 that can suppress mutT mutation.

Abstract

Mutations in the mutT gene of E. coli result in elevated A/T to C/G transversions that can arise through the formation of A/G and/or T/C replication intermediates. Although it has been shown that A/G is the preferred intermediate, mutT gene function is required during replication and MutT is a dGTPase, the precise function of MutT in fidelity is not clear. In order to examine if MuT can proofread A/G (template/primer) mispairs, an in vitro 3{dollar}\sp\prime{dollar} {dollar}\to{dollar} 5{dollar}\sp\prime{dollar} A/G specific exonuclease assay was established. Protein extracts from mutT1 and mutT{dollar}\sp{lcub}+{rcub}{dollar} cells were used to extend a primed ssDNA substrate DNA containing G at the primer terminus, paired with template A. The results indicate that the mutT1 cell extracts may be deficient in processing of A/G mispairs compared to mutT{dollar}\sp+{dollar} extracts. A second study shows that the MicA dependent A/G to C/G post replication mismatch correction contributes to the observed mutation rate in mutT cells. A mutT1 micA double mutant was constructed. The mutation rate of the double mutant was no more than mutT1 single mutant. The data indicates that MicA can convert G/A mismatches to C/G in a mutT cell. Furthermore, a new gene sut1 which can decrease the mutation rate of mutT cells in vivo has been isolated. The gene has no homology to the mutT gene and probably is not sum44 (a suppressor of mutT1 that maps near mutT). Genetic and physical mapping analyses failed to locate the sut1 gene on the E. coli chromosome. The gene was shown to be derived from the F factor of E. coli and mapped to the f7 EcoRI fragment between repF1B and the pif operon. The mutT suppression activity resides in a 520 bp NcoI-EcoRV fragment. Sequence analysis reveals several small open reading frames (ORF) of less than 70 amino acids. A small RNA 95 nt long was identified from clones containing the NcoI-EcoRV fragment. However, further deletion into the RNA coding sequence had no effect on the ability to complement mutT mutation. There is also an 80 bp sequence upstream of the NcoI-EcoRV fragment that is 80% homologous to the colE1 replication primer RNAII. The significance is unclear.