Translesion DNA synthesis: polymerase response to altered nucleotides

Abstract

A system for the determination of the specificity of incorporation opposite lesions during DNA synthesis past damaged bases has been used as a model of mutation. The system, based on the dideoxynucleotide sequence method, uses lesions in the template strand as chain terminators. As a first approximation, such lesions constitute non-instructive sites in the DNA. DNA synthesis catalysed by bacteriophage T4 DNA polymerase terminates one nucleotide 3' to the lesion on the template strand. With other polymerases, synthesis may terminate opposite the lesion. The details of termination site depend on the enzyme, the metal ion (Mg2+ or Mn2+), the lesion and the particular nucleotide sequence. The sequence effect for termination on normal templates has two components, one ascribable to secondary structure, the other intrinsic to the sequence itself. DNA molecules terminated before lesions may be used as substrates in 'second stage' reactions in which elongation can be detected on the addition of particular dNTPs (deoxynucleoside triphosphates) to a reaction mixture. The specificity of elongation depends on the polymerase, on the activity of the 3'--greater than 5' editing nuclease and on the particular lesion. DNA polymerases have a preference for the addition of purines, particularly adenine, opposite non-instructional sites. This preference suggests an explanation for the specificity of base substitution mutations: treatments that produce non-instructional sites from purines will lead to transversions, treatments that affect pyrimidines lead to transitions. Even though a base is added opposite a lesion, further elongation may be rate limiting. Whether or not elongation occurs is dependent on the sequence 5' to the lesion on the template strand. The interactions of the factors affecting bypass: polymerase, lesion and sequence, may well result in an idiosyncratic behaviour for each mutable site.