Faculty Bibliography

FKBP12 is a conserved member of the prolyl-isomerase enzyme family and serves as the intracellular receptor for FK506 that mediates immunosuppression in mammals and antimicrobial actions in fungi. To investigate the cellular functions of FKBP12 in Saccharomyces cerevisiae, we employed a high-throughput assay to identify mutations that are synthetically lethal with a mutation in the FPR1 gene, which encodes FKBP12. This screen identified a mutation in the HOM6 gene, which encodes homoserine dehydrogenase, the enzyme catalyzing the last step in conversion of aspartic acid into homoserine, the common precursor in threonine and methionine synthesis. Lethality of fpr1 hom6 double mutants was suppressed by null mutations in HOM3 or HOM2, encoding aspartokinase and aspartate beta-semialdehyde dehydrogenase, respectively, supporting the hypothesis that fpr1 hom6 double mutants are inviable because of toxic accumulation of aspartate beta-semialdehyde, the substrate of homoserine dehydrogenase. Our findings also indicate that mutation or inhibition of FKBP12 dysregulates the homoserine synthetic pathway by perturbing aspartokinase feedback inhibition by threonine. Because this pathway is conserved in fungi but not in mammals, our findings suggest a facile route to synergistic antifungal drug development via concomitant inhibition of FKBP12 and Hom6.

LTR-containing retrotransposons reverse transcribe their RNA genomes, and the resulting cDNAs are integrated into the genome by the element-encoded integrase protein. The yeast LTR retrotransposon Ty1 preferentially integrates into a target window upstream of tDNAs (tRNA genes) in the yeast genome. We investigated the nature of these insertions and the target window on a genomic scale by analyzing several hundred de novo insertions upstream of tDNAs in two different multicopy gene families. The pattern of insertion upstream of tDNAs was nonrandom and periodic, with peaks separated by approximately 80 bp. Insertions were not distributed equally throughout the genome, as certain tDNAs within a given family received higher frequencies of upstream Ty1 insertions than others. We showed that the presence and relative position of additional tDNAs and LTRs surrounding the target tDNA dramatically influenced the frequency of insertion events upstream of that target.

LINE-1 (L1) elements are retrotransposons that comprise large fractions of mammalian genomes. Transcription through L1 open reading frames is inefficient owing to an elongation defect, inhibiting the robust expression of L1 RNA and proteins, the substrate and enzyme(s) for retrotransposition. This elongation defect probably controls L1 transposition frequency in mammalian cells. Here we report bypassing this transcriptional defect by synthesizing the open reading frames of L1 from synthetic oligonucleotides, altering 24% of the nucleic acid sequence without changing the amino acid sequence. Such resynthesis led to greatly enhanced steady-state L1 RNA and protein levels. Remarkably, when the synthetic open reading frames were substituted for the wild-type open reading frames in an established retrotransposition assay, transposition levels increased more than 200-fold. This indicates that there are probably no large, rigidly conserved cis-acting nucleic acid sequences required for retrotransposition within L1 coding regions. These synthetic retrotransposons are also the most highly active L1 elements known so far and have potential as practical tools for manipulating mammalian genomes.

LINE-1 (L1) elements are the most abundant autonomous retrotransposons in the human genome, accounting for about 17% of human DNA. The L1 retrotransposon encodes two proteins, open reading frame (ORF)1 and the ORF2 endonuclease/reverse transcriptase. L1 RNA and ORF2 protein are difficult to detect in mammalian cells, even in the context of overexpression systems. Here we show that inserting L1 sequences on a transcript significantly decreases RNA expression and therefore protein expression. This decreased RNA concentration does not result from major effects on the transcription initiation rate or RNA stability. Rather, the poor L1 expression is primarily due to inadequate transcriptional elongation. Because L1 is an abundant and broadly distributed mobile element, the inhibition of transcriptional elongation by L1 might profoundly affect expression of endogenous human genes. We propose a model in which L1 affects gene expression genome-wide by acting as a 'molecular rheostat' of target genes. Bioinformatic data are consistent with the hypothesis that L1 can serve as an evolutionary fine-tuner of the human transcriptome.

The silent information regulator protein (Sir2) and its homologs (collectively known as sirtuins) are NAD+-dependent deacetylase enzymes involved in chromosome stability, gene silencing and cell aging in eukaryotes and archaea. The discovery that sirtuin-dependent protein deacetylation is a NAD+-consuming reaction established a link with the energy generation systems of the cell. This link to metabolism was recently extended to the post-translational control of the activity of short-chain fatty acyl-coenzyme A (adenosine monophosphate-forming) synthetases in bacteria and yeast. The crystal structure of the Sir protein complexed with a peptide of a protein substrate provided insights into how sirtuins interact with their protein substrates.

Sir2 proteins form a family of NAD(+)-dependent protein deacetylases required for diverse biological processes, including transcriptional silencing, suppression of rDNA recombination, control of p53 activity, regulation of acetyl-CoA synthetase, and aging. Although structures of Sir2 enzymes in the presence and absence of peptide substrate or NAD(+) have been determined, the role of the enzyme in the mechanism of deacetylation and NAD(+) cleavage is still unclear. Here, we present additional structures of Sir2Af2 in several differently complexed states: in a productive complex with NAD(+), in a nonproductive NAD(+) complex with bound ADP-ribose, and in the unliganded state. We observe a new mode of NAD(+) binding that seems to depend on acetyl-lysine binding, in which the nicotinamide ring of NAD(+) is buried in the highly conserved "C" pocket of the enzyme. We propose a detailed structure-based mechanism for deacetylation and nicotinamide inhibition of Sir2 consistent with mutagenesis and enzymatic studies.

We describe a new synthetic lethality analysis by microarray (SLAM) technique that uses approximately 4,600 Saccharomyces cerevisiae haploid deletion mutants with molecular 'bar codes' (TAGs). We used SGS1 and SRS2, two 3'-->5' DNA helicase genes, as 'queries' to identify their redundant and unique biological functions. We introduced these 'query mutations' into a haploid deletion pool by integrative transformation to disrupt the query gene in every cell, generating a double mutant pool. Optimization of integrative transformation efficiency was essential to the success of SLAM. Synthetic interactions defined a DNA helicase genetic network and predicted a role for SRS2 in processing damaged replication forks but, unlike SGS1, not in rDNA replication, DNA topology or lagging strand synthesis. SGS1 and SRS2 have synthetic defects with MRC1 but not RAD9, suggesting that SGS1 and SRS2 function in a parallel pathway with MRC1 to transduce the DNA replication stress signal to the general DNA damage checkpoint pathway. Both helicase genes have rad51-reversible synthetic defects with 5'-->3' DNA helicase RRM3, suggesting that RRM3 helps prevent formation of toxic recombination intermediates. SLAM detects synthetic lethality efficiently and ranks candidate genetic interactions, making it an especially useful method.