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. This year, we focused on the analysis of DNA palindromes. These head to head sequences are highly unstable sequences. Some tumor cells undergo gene amplification by unknown mechanisms that generate palindromes. The instability of these sequences makes it nearly impossible to clone palindromes in bacteria. Similarly, the secondary structures that can be adopted by palindromic DNAs make them very difficult to sequence. Hence, it is has not been possible to analyze these palindromes in detail or study their origins. It remains possible that some of regions of the human genome that have proven impossible to clone are palindromic regions. This year, we made progress in three important areas related to the study of palindromes. First, identified yeast strains that tolerate palindromes. Second, we developed a recombination substrate that generates palindromes and identified a class of recombinants that is almost exclusively palindromes. This breakthrough means we can screen for recombination functions that are required for the production palindromes. Third, we developed a method that allows us to sequence palindromic DNAs. Combined these tools allow us to address the unknown mechanism by which palindromic gene amplifications are formed. 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. 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. In addition to detecting very low levels of well known efavirenz resistant variants, we have been able to detect novel variants with moderate levels of efivirenz resistance which may have a role in the evolution of resistant HIV.
