Genome-destabilizing effects associated with top1 loss or accumulation of top1 cleavage complexes in yeast.

Topoisomerase 1 (Top1), a Type IB topoisomerase, functions to relieve transcription- and replication-associated torsional stress in DNA. We investigated the effects of Top1 on genome stability in Saccharomyces cerevisiae using two different assays. First, a sectoring assay that detects loss of heterozygosity (LOH) on a specific chromosome was used to measure reciprocal crossover (RCO) rates. Features of individual RCO events were then molecularly characterized using chromosome-specific microarrays. In the second assay, cells were sub-cultured for 250 generations and LOH was examined genome-wide using microarrays. Though loss of Top1 did not destabilize single-copy genomic regions, RCO events were more complex than in a wild-type strain. In contrast to the stability of single-copy regions, sub-culturing experiments revealed that top1 mutants had greatly elevated levels of instability within the tandemly-repeated ribosomal RNA genes (in agreement with previous results). An intermediate in the enzymatic reaction catalyzed by Top1 is the covalent attachment of Top1 to the cleaved DNA. The resulting Top1 cleavage complex (Top1cc) is usually transient but can be stabilized by the drug camptothecin (CPT) or by the top1-T722A allele. We found that increased levels of the Top1cc resulted in a five- to ten-fold increase in RCOs and greatly increased instability within the rDNA and CUP1 tandem arrays. A detailed analysis of the events in strains with elevated levels of Top1cc suggests that recombinogenic DNA lesions are introduced during or after DNA synthesis. These results have important implications for understanding the effects of CPT as a chemotherapeutic agent.

Characterization of null and hypomorphic alleles of the Drosophila l(2)dtl/cdt2 gene: Larval lethality and male fertility.

The Drosophila lethal(2)denticleless (l(2)dtl) gene was originally reported as essential for embryogenesis and formation of the rows of tiny hairs on the larval ventral cuticle known as denticle belts. It is now well-established that l(2)dtl (also called cdt2) encodes a subunit of a Cullin 4-based E3 ubiquitin ligase complex that targets a number of key cell cycle regulatory proteins, including p21, Cdt1, E2F1 and Set8, to prevent replication defects and maintain cell cycle control. To investigate the role of l(2)dtl/cdt2 during development, we characterized existing l(2)dtl/cdt2 mutants and generated new deletion alleles, using P-element excision mutagenesis. Surprisingly, homozygous l(2)dtl/cdt2 mutant embryos developed beyond embryogenesis, had intact denticle belts, and lacked an observable embryonic replication defect. These mutants died during larval stages, affirming that loss of l(2)dtl/cdt2 function is lethal. Our data show that L(2)dtl/Cdt2 is maternally deposited, remains nuclear throughout the cell cycle, and has a previously unreported, elevated expression in the developing gonads. We also find that E2f1 regulates l(2)dtl/cdt2 expression during embryogenesis, possibly via several highly conserved putative E2f1 binding sites near the l(2)dtl/cdt2 promoter. Finally, hypomorphic allele combinations of the l(2)dtl/cdt2 gene result in a novel phenotype: viable, low-fertility males. We conclude that "denticleless" is a misnomer, but that l(2)dtl/cdt2 is an essential gene for Drosophila development.