The goal of this project is to identify the critical functions of topoisomerase I (topo I) in S. cerevisiae through a molecular-genetic analysis of mutations that are inviable in combination with top 1 mutations. Topoisomerase I is presumed to be important to relieve torsional stress encountered during DNA replication and transcription. However, yeast mutants with a null mutation in the TOP 1 gene are viable. One explanation for the viability of top 1 mutants is that there are proteins providing overlapping functions. To identify genes encoding overlapping functions, we have isolated mutants that are inviable unless topo I is being expressed (tpr mutants for topo I-requiring). The TPR genes define at least five complementation groups. We have also found that top 1 single mutants have a previously unrecognized defect in ribosomal DNA replication. I. Molecular-genetic and biochemical analysis of TPR (topo I-requiring) genes. A. TPR1 (a gene encoding a protein homologous to TOP 1 over two short regions) We will examine DNA and RNA synthesis in top1 tpr1 (ts) mutants made in vitro to define what that function is. The Tpr1 protein will be immunolocalized to see if it is in the nucleolus like Top 1. To determine whether Tpr1 encodes a topoisomerase, assays will be performed on the purified protein. B. TPR2 (RNA polymerase II subunit RPB4) The fact that TOP1 can functionally replace an RNA polymerase II subunit suggests that a similar biochemical activity may be provided by that subunit. Transcription induced supercoiling of template DNA and mRNA start site efficiency in tpr2 mutants, and topoisomerase and DNA helicase assays on the Tpr2 protein will be performed to provide evidence for similar functions. C. TPR3, 4 and 5 (function currently unknown) The DNA sequences of TPR3, 4 and 5 will determine homologies between these and other genes. top1 tpr3, 4 and 5 (ts) double mutants will be examined for physiological defects (DNA, RNA synthesis, and cell-cycle arrest), and the examined for defects in nucleolar structure. II. Analysis of the rDNA replication defect in top1 and examination of tpr mutants We will examine the effect of cis-acting mutations in an rDNA cistron on topo I function during rDNA replication by constructing a mini-cistron with which point mutations and deletions can be readily introduced into the array. The mini-cistron will be used in combination with Fangman 2D replication gels and electron microscopy to examine rDNA replication intermediates in top1 and tpr mutants.
