Faculty Bibliography

L1 elements are polyA retrotransposons which inhabit the human genome. Recent work has defined an endonuclease (L1 EN) encoded by the L1 element required for retrotransposition. We report the sequence specificity of this nicking endonuclease and the physical basis of its DNA recognition. L1 endonuclease is specific for the unusual DNA structural features found at the TpA junction of 5'(dTn-dAn) x 5'(dTn-dAn) tracts. Within the context of this sequence, substitutions which generate a pyrimidine-purine junction are tolerated, whereas purine-pyrimidine junctions greatly reduce or eliminate nicking activity. The A-tract conformation of the DNA substrate 5' of the nicked site is required for L1 EN nicking. Chemical or physical unwinding of the DNA helix enhances L1 endonuclease activity, while disruption of the adenine mobility associated with TpA junctions reduces it. Akin to the protein-DNA interactions of DNase I, L1 endonuclease DNA recognition is likely mediated by minor groove interactions. Unlike several of its homologues, however, L1 EN exhibits no AP endonuclease activity. Finally, we speculate on the implications of the specificity of the L1 endonuclease for the parasitic relationship between retroelements and the human genome.

Two artificial transposons have been constructed that carry a gene encoding Green Fluorescent Protein and can be used for generating libraries of GFP fusions in a gene of interest. One such element, AT2GFP, can be used to generate GFP insertions in frame with the amino acid sequence of the protein of interest, with a stop codon at the end of the GFP coding sequence; AT2GFP also contains a selectable marker that confers trimethoprim resistance in bacteria. The second element, GS, can be used to generate tribrid GFP fusions because there is no stop codon in the GFP transposon, and the resulting fusion proteins contain the entire amino acid sequence encoded by the gene. The GS element consists of a gfp open reading frame and a supF amber suppressor tRNA gene; the supF portion of the GS transposon can be utilized as a selectable marker in bacteria. Its sequence contains a fortuitous open reading frame, and thus it can be translated continuously with the gfp amino acid sequence. As a target for GFP insertions, we used a plasmid carrying the native Ty1 retrotransposon of the yeast Sacharomyces cerevisiae. The resulting multiple GFP fusions to Ty1 capsid protein Gag and Ty1 integrase were useful in determining the cellular localization of these proteins. Libraries of GFP fusions generated by transposition in vitro represent a novel and potentially powerful method to study the cell distribution and cellular localization signals of proteins.

The Tf2 retrotransposon, found in the fission yeast Schizosaccharomyces pombe, is nearly identical to its sister element, Tf1, in its reverse transcriptase-RNase H and integrase domains but is very divergent in the gag domain, the protease, the 5' untranslated region, and the U3 domain of the long terminal repeats. It has now been demonstrated that a neo-marked copy of Tf2 overexpressed from a heterologous promoter can mobilize into the S. pombe genome and produce true transposition events. However, the Tf2-neo mobilization frequency is 10- to 20-fold lower than that of Tf1-neo, and 70% of the Tf2-neo events are homologous recombination events generated independently of a functional Tf2 integrase. Thus, the Tf2 element is primarily dependent on homologous recombination with preexisting copies of Tf2 for its propagation. Finally, production of Tf2-neo proteins and cDNA was also analyzed; surprisingly, Tf2 was found to produce its reverse transcriptase as a single species in which it is fused to protease, unlike all other retroviruses and retrotransposons.

The Drosophila retroelement gypsy has a number of unusual features including an unusual LTR terminal sequence and an apparent target sequence preference. The ovo locus is a known hotspot for gypsy insertion. We examined the target sequence preference of gypsy within ovo by isolating 26 new insertions and sequencing the gypsy/ovo junctions. Insertions were found at multiple sites within the ovo locus. The insertions clustered within an approximately 150 bp region in the non-translated region of the ovo beta transcript, with most insertions falling within the first intron. There were seven sites of insertion within this region and these mostly conform to the consensus sequence YRYRYR (where Y = pyrimidine and R = purine). However, this target sequence is at best necessary but not sufficient to specify a hotspot, as there were several other sequences conforming to this consensus in the ovo locus that were not hit. The results indicate that gypsy may have a higher degree of target specificity than most infectious LTR retroelements.

The eukaryotic small nucleolar RNAs (snoRNAs) are involved in processing of pre-rRNA and modification of rRNA nucleotides. Some snoRNAs are derived from mono- or polycistronic transcription units, whereas others are encoded in introns of protein genes. The present study addresses the role of the RNA lariat-debranching enzyme (Dbr1p) in the synthesis and function of intronic snoRNAs in the yeast Saccharomyces cerevisiae. Intronic snoRNA production was determined to depend on Dbr1p. Accumulation of mature intronic snoRNAs is reduced in a dbr1 mutant; instead, intronic snoRNAs are "trapped" within host intron lariats. Interestingly, the extent of intronic snoRNA accumulation in the form of lariats in dbr1 cells varied among different intronic snoRNAs. Intronic snoRNAs encoded within shorter introns, such as U24 and snR38, accumulate more unprocessed lariat precursors than those encoded within longer introns, e.g., U18 and snR39. This correlation was corroborated by experiments conducted with model intron:U24 snoRNA constructs. These results support a splicing-dependent exonucleolytic pathway for the biosynthesis of intronic snoRNAs. Curiously, U24 in a lariat may be functional in directing methylation of ribosomal RNA.

Transcriptional silencing in Saccharomyces cerevisiae occurs at the silent mating-type loci HML and HMR, at telomeres, and at the ribosomal DNA (rDNA) locus RDN1. Silencing in the rDNA occurs by a novel mechanism that depends on a single Silent Information Regulator (SIR) gene, SIR2. SIR4, essential for other silenced loci, paradoxically inhibits rDNA silencing. In this study, we elucidate a regulatory mechanism for rDNA silencing based on the finding that rDNA silencing strength directly correlates with cellular Sir2 protein levels. The endogenous level of Sir2p was shown to be limiting for rDNA silencing. Furthermore, small changes in Sir2p levels altered rDNA silencing strength. In rDNA silencing phenotypes, sir2 mutations were shown to be epistatic to sir4 mutations, indicating that SIR4 inhibition of rDNA silencing is mediated through SIR2. Furthermore, rDNA silencing is insensitive to SIR3 overexpression, but is severely reduced by overexpression of full-length Sir4p or a fragment of Sir4p that interacts with Sir2p. This negative effect of SIR4 overexpression was overridden by co-overexpression of SIR2, suggesting that SIR4 directly inhibits the rDNA silencing function of SIR2. Finally, genetic manipulations of SIR4 previously shown to promote extended life span also resulted in enhanced rDNA silencing. We propose a simple model in which telomeres act as regulators of rDNA silencing by competing for limiting amounts of Sir2 protein.