The effects of DNA sequence on enzymatic function has been an area of extensive research. Recent studies reveal strong DNA sequence effects on the 3'-exonuclease activity of Rrp1. This sequence specificity has been precisely defined with oligonucleotide substrates that reveal inhibition of Rrp1 in pyrimidine repeats and at purine-pyrimidine junctions. In addition, a 200-300-fold slower catalytic rate is measured for Rrp1 progressing through repeated thymidine nucleotides compared to repeated adenosine nucleotides. A manuscript describing this work was recently submitted for publication. In recent enzymatic studies of Rrp1, a 3'-phosphodiesterase assay was developed with higher sensitivity and specificity than standard assay methods. This assay has now been successfully applied to identify new repair activities in extracts from yeast and Drosophila embryos. A novel repair enzyme with properties similar to Drosophila Rrp1 has been extensively purified from extracts of the yeast, Saccharomyces cerevisiae. Partial purification yields a protein fraction enriched in a Mg++-dependent 3'-phosphodieserase with tightly associated class II AP endonuclease. The active protein in the partially purified fraction has now been tentatively identified, and further characterization of this yeast protein is in progress. A survey of adult and embryo Drosophila extracts for the presence of DNA repair functions whose specificity overlap with Rrp1 has been carried out using these enzyme assays. Two potentially novel Drosophila enzymes are detected and are currently being further characterized.
