We intend to define the factors which determine the susceptibility of particular nucleotide sites in DNA to induced mutation and to understand how protein factors act to modulate the process. There is increasing evidence to support the """"""""A"""""""" rule: the concept the DNA polymereases confronted with a non-instructional lesion will insert an adenine nucleotide. However, the exceptions reported with particular mutagens indicate that even lesions which block DNA synthesis may not be completely non-instructional. We plan a systematic study of the specificity of induced mutation to better understand the interaction of lesion, polymerase, accessory proteins and neighboring nucleotide sequence in the generation of induced mutation. We will refine our system for measurement of mutation in vitro, using an M13 vector containing a lac complementing insert adn with a uracil-containing (+) strand annealed to a normal (-) strand without the insert so that the lac complementing region forms part of a single stranded gap. The (+) strand is reacted wit mutagen so that lesions are present also in the gapped region. Viable phage are produced only after in vitro DNA synthesis to close the gap. We have already demonstrated mutagenesis in vitro on an aminofluorene-containing template using a modified T7 DNA polymerase. We observed both G-->T transversions and single base deletions. A hot spot for -1G deletions occurred at an GGGAAAACCC sequence. We will modify our methodology to provide a more efficient, positive selection system. We plan the following experiments: a) insertion of out of frame segments of defined sequence at the 5' end of the lac insert. These segments will be mutagenized both randomly and by site directed mutagenesis in order to better define the rules which determine the susceptibility of sequences to frame shift mutagenesis; b) determination of the ability of different polymerases and accessory factors to affect the mutagenic spectrum. We wish to determine the role of processivity and proofreading in mutagenesis on non- instructive templates and to investigate the role DNA binding proteins such as recA and single strand binding protein in bypass and mutation; and c) study of the termination pattern and mutation specificity on a template containing monofunctional psoralen adducts which require SOS functions for mutagenesis, since such adducts have been show in vivo to induce mutants not predicted by the """"""""A rule"""""""".
