Isolation And Characterization Of Human Genes Affecting

Environmental Health Sciences

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Abstract

The yeast S. cerevisiae is an invaluable in vivo test tube for examining human gene functions and disease including, including cell signaling, DNA metabolism and mitochondrial function. We have focused on 3 human genes with broad health significance: the Friedreich's ataxia gene frataxin, the FLAP endonuclease FEN1 (also see Project 65073), and the tumor suppressor p53. The mitochondrial protein frataxin helps maintain appropriate iron levels in the mitochondria. A deficiency in humans causes Friedreich?s ataxia. Absence of frataxin in yeast (yfh1-) results in loss of mitochondrial DNA, apparently due to radicals generated by excess iron, and a ?petite? phenotype. The 10-fold excess iron found in mitochondria of yfh1- mutants correlates with increased iron levels in tissues of FRDA patients. Our observations that yfh1- cells grew slower than spontaneous petites and were sensitive to both the alkylating agent MMS and the replication inhibitor hydroxyurea led us to hypothesize the occurrence of nuclear as well as mitochondrial DNA damage. We tested this assessing induction of a nuclear damage inducible gene. Only the yfh1- colonies exhibited induction, indicating substantial nuclear DNA damage. Also there was increased spontaneous chromosomal mutation and recombination. The excess iron found in the mitochondria of yfh1- cells implies that the nuclear DNA damage is mediated by reactive oxygen species (ROS). The following results strongly support this view: damage inducible response the presence of the radical scavenger N-acetylcysteine, increased mutation in a peroxidase double mutant gpx1- yfh1-, or when yfh1- is combined with deletions of the base excision repair genesNTG1, NTG2, APN1. There was a 2-4 fold increase in H2O2 in supernatants of yfh1- cells using a fluorescence-based assay. This suggested that the nuclear DNA damage in yfh1- cells might arise fromH2O2 generated by excess iron in the mitochondria. H2O2could traverse the mitochondrial membrane barrier and damage nuclear DNA via Fenton Chemistry. This is the first demonstration of altered mitochondrial function leading to changes in genome stability. We also developed a rheostatable system to regulate the frataxin levels, since the human disease FRDA occurs as a result of a deficiency and not lack of the protein. Our lab has developed a system to regulate the expression of genes to varying levels under a Gal promoter by varying galactose in the medium This system will enable us to mimic the human disease situation of low levels of the protein making yeast a better model for the disease. FEN1. To address in vivo mechanisms of flap cleavage we developed a screen to identify and characterize randomly generated (siteless directed mutagenesis) Fen1 mutations that are toxic when expressed in yeast. We identified 17, and characterized 12 toxic mutants that resulted in various levels of growth inhibition and reduced survival. Surprisingly, about half of the mutations were found within the loop domain of the protein, suggesting that this small, flexible region plays an important role in Fen1 activity. This region is considered to directly interact with the single- stranded 5'-flap and position it into the active site. Toxic mutants caused G2 arrest and cell death and were unable to repair MMS lesions. Through a collaboration with the Hubscher lab in Zurich, we found that all the mutant proteins retained flap binding. Unlike the catalytic site mutants, which lacked cleavage of any 5? flaps, the loop mutants exhibited partial ability to cut 5? flaps when an adjacent single nucleotide 3? flap was present. Through a combination of genetic approaches and biochemical analyses with various flap substrates, we established that the flexible loop is essential for flap cleavage by hFEN1. P53. Given the broad spectrum of p53 functions as a transcription factor and the many different p53 alleles with single amino acid changes that are aberrantly expressed in cancer cells, a detailed knowledge of the functional status of p53 mutants could have clinical value, especially for therapies tailored to specific tumors. Although several methods have been attempted to classify p53 mutants, based on physical/chemical, or immunological/structural parameters, it is not presently possible to predict a priori the behavior of a mutant protein. p53 transactivation activity can be assessed easily in yeast using an ADE2 red/white colony color reporter assay (corresponding to nonfunctional/functional p53). Transcription is initiated by p53 binding at response elements (20-22 bp) placed upstream from the ADE2 gene. The assay evaluates the effect of amino acid changes at p53 codons 53-364, where 99% of all tumor mutations are reported. Surprisingly, in a small screen of bladder cancer material about half the p53 alleles resulted in a partial-function phenotype. To examine the general incidence of partial-function mutations, we mutagenized p53 and identified alleles with reduced transactivation activities towards any of the response elements p21, RGC and MDM2. 57 mutants with different single a.a. changes were identified. The mutations were uniformly distributed across the p53 region analyzed that covers the transactivation domain II, proline rich region, DNA binding and tetramerization domains. Interestingly, 65% of these appear in the tumor p53 database and correspond to 2% of all p53 tumor missense mutations. Therefore, nearly 20% of tumor p53 mutants are likely to have partial function. Among 5 mutants tested in A human p53-null Saos-2 cell line all showed partial function in transactivation and colony suppression assays. Thus the yeast based assessment of the p53 transcription factor has revealed novel features of tumor associated p53 mutations. Little is known about the mechanisms that determine p53 transactivation specificity in vivo. We developed a system in yeast that addresses transactivation capacity of WT and mutant p53 proteins from 30 different p53 Response Elements (REs) under conditions where all other factors, such as chromatin, are kept constant. To take into account the levels of p53 expression in the transactivation assay, we developed rheostatable regulation of p53 expression (see above). The p53 transactivation capacity toward each of thirty 20-22 bp REs were ranked using a simple phenotypic assay and other biochemical assays. The REs were from p53-target genes involved in cell cycle control, apoptosis, DNA repair, and p53 regulation, thus representing a broad functional matrix of p53 targets. Surprisingly, there was as much as a 1000-fold difference in transactivation. WT p53 had weak activity towards half the apoptotic REs. Thus, the intrinsic DNA binding affinity, as well as p53 protein levels, are important contributors to p53-induced differential transactivation. We have also analyzed 20 tumor-associated p53 alleles for transactivation with this broad array of p53 REs. Interestingly several mutations showed residual function with at least half of the REs examined. Using the rheostatable system, we devised a simple screen to identify p53 mutants exhibiting increased transactivation (supertrans) compared to WT. While some were more active than WT p53 with all REs, others exhibited altered sequence specificity and level of transactivation. These mutants will help us understand p53 functions in human cells as well as help to assess how p53 mutations may give rise to cancer. We are addressing the impact of ectopic expression of the supertrans p53 mutants in human cell lines, including transformed and non-transformed cells with different p53 status. Cell cycle progression, apoptosis, DNA repair, and activation of p53 targets are being investigated. Genome-wide gene expression studies using microarray technologies will be used to probe and better understand the global change