The Genome Recombination/Regulation Section focuses on three topics: functions that control the stability of genomes, functions that control aspects of gene expression, and the development of a yeast based system to study HIV-1. Studies on genome instability. Our studies on genome stability grew out of an analysis of the programmed genome rearrangement that controls cell type in yeast. That process is initiated by a site specific double strand break (DSB). We used derivatives of this system to study DSB repair and recently established two new principles: mitotic recombination is associated with a relatively high error rate, caused in part by the involvement of error prone polymerases; and the some DSB repair leads to the formation of palindromes. We documented a thousand-fold elevated level of base substitutions among recombinants and showed that these are largely the result of the translesion polymerase encoded by the REV3 gene (polz). A second pathway leads to an elevated level of single base deletions/additions. The involvement of error prone polymerases in recombination may provide an explanation for the origin of somatic hypermutation of immunoglobulin genes. The origin of palindromic gene amplifications has been an unsolved mystery. Such amplifications are common in tumor cells. We demonstrated that the palindromes are formed in our system by a novel kind of nonhomologous end joining (NHEJ)which is independent of some of the recombination functions that are required for most NHEJ events. Our analysis of the palindromic DNAs required the development of new systems for their cloning and sequencing. We plan to extend those studies to the isolation and characterization of palindromic sequences from mammalian genomes. Studies on gene expression. Our studies on gene expression grew out of our analysis of retrotransposition events. We are developing methods to monitor the fidelity of transcription and the functions that contribute to the accuracy of that process. One such method involves monitoring the fidelity of retrotransposition. We demonstrated that the fidelity of retrotransposition is a thousand fold lower than simple genome duplication. This process reflects the fidelity of transcription and of reverse transcription. We have isolated mutations that reduce the fidelity of retrotransposition and are determining whether they directly affect the accuracy of transcription. We are also monitoring the fidelity of transcription in a variety of RNA polymerase mutant strains. These studies on the fidelity of transcription led us into the characterization of transcription factors involved in initiation and elongation. TFIIS is an elongation factor that has been implicated in the restart of stalled transcription complexes. It stimulates the removal of mispaired bases from the 3' end of the RNA in vitro. Hence it is a strong candidate for a function involved in the fidelity of transcription. Our studies established a new role for the elongation factor TFIIS in initiation. HIV-1 Reverse Transcriptase Several years ago, we developed hybrid Ty/HIV-1 elements (HART) that have HIV-1 RT substituted for the Ty1 RT. These elements replicate in yeast and are dependent on the polymerase and RNase activities of HIV-1 RT. Replication of the HART elements is sensitive to the non-nucleoside class of HIV-1 RT inhibitors (NNRTI) while HART elements containing HIV-1 RT domains from resistant viruses are themselves resistant. We developed a luciferase based high throughput assay to screen for compounds that inhibit the replication of HART elements. We are also using HART elements to detect RT domains that confer resistant to efavirenz among the viral sequences recovered from patient blood. This approach promises to provide an early detection system for the emergence of NNRTI resistant viruses in patients.
