Production of UV-induced Frameshift Mutations in Vitro by DNA Polymerases Deficient in 3′ → 5′ Exonuclease Activity

Abstract

In order to study the conversion of UV lesions into frameshift and base substitution mutations, M13mp2 phage DNA was altered by the addition of extra pyrimidines, or by construction of a nonsense codon preceded by a run of pyrimidines within the β-galactosidase complementing region. The normal sequence 5′ GTC GTT TTA CAA 3′ was changed to GTC GTT T TTA CAA (MIDT) of GTC GTT C TTA CAA (MIDC) to study frameshifts an to GTC GTT CTT TAA (OCHRE) to study reversion of the ochre (TAA) codon. Escherichia coli pol I Kf and T7 DNA polymerase mutant enzymes devoid of 3′ → 5′ exonuclease activity produce UV-induced revertants at higher frequency than did their exonuclease proficient counterparts. Removal of cyclobutane dimers with photolyase before in vitro synthesis did not greatly affect mutant frequency although which treatment led to significantly increased DNA synthesis by the wild-Type T7 DNA polymerase on UV-irradiated substrate. Reversions of the in frame ochre sequence GTT CTT TAA produced by the Δ28 T7 DNA polymerase were mainly by base substitution in the TAA codon. About half of the E. coli Kf exo - enzyme ochre revertants had a TTA deletion. Five mutant T7 DNA polymerases with varying exonuclease activity gave revertant frequencies that correlated better with published values of enzyme velocity than with exonuclease activity or with measured bypass synthesis. Our data indicate that loss of proofreading activity increase the frequency of UV-induced frameshifts, but lack of such activity is not sufficient for their production. We suggest that frameshifts occur more frequently when nucleotide addition opposite the lesion is slow. The same lesion can give rise to a different spectrum of mutations depending on the polymerase.