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Molecular Biology

Rad3 Helicase in Homologous Recombination and Single Stranded DNA Degradation

Alpha Anders ('11); Mentor: Maria Christina Negritto

Abstract: Transcription Factor IIH (TFIIH) is a key protein complex that plays a role in basal transcription and nucleotide excision repair (NER). In humans mutations in the TFIIH’s helicase subunit, XPD, results in the UV light sensitive diseases Xeroderma Pigmentosum (XP), Cockayne syndrome and Trichothiodystrophy (TTD). Saccharomyces cervisiae's highly conserved homologue to XPD is Rad3. Rad3 can be mutated to mimic the mutations that cause these human diseases (Negritto 2009). The homologous mutations tested are rad3-G595R, rad3-A596P, rad3-R660C. The latter two generate TTD when mutated in XPD. Rad3 has also been shown to be involved in Short Sequence Recombination (SSR). The mutant rad3-G595R shows heightened levels of SSR and decreased single stranded DNA degradation at DSBs (Bailis et al. 1995). This study will investigate the levels of SSR and evaluate the extent of single stranded DNA degradation in the three mutants through an insertion deletion assay and pulse field gel electrophoresis.
Funding provided by The Fletcher Jones Foundation

The Role of Helicase Rad3 and the TFIIH Complex in Double Strand Break Repair in S. Cerevisiae

Jessica Deas ('11); Mentor: Tina Negritto

Abstract: Rad3 (XPD in humans) is a 5’-3’ DNA helicase, one of ten subunits of the multifunctional eukaryotic transcription factor IIH (TFIIH). TFIIH is essential for transcription initiation and in the nucleotide excision repair (NER) pathway of DNA damage. We hypothesized that TFIIH also plays a role in the double strand break (DSB) repair pathway and performed chromatin immunoprecipitation (ChIP) to determine whether TFIIH physically localizes to the site of DSBs. Homing endonucleases under inducible transcriptional control were used to create a DSB at the His3 locus in S. cerevisiae; ChIP results indicate that there is an increase in the amount of TFIIH present at the gene after a break occurs. We also investigated whether mutations in Rad3 affect its repair activity; in the Rad3-G595R mutant, TFIIH does not appear to be recruited to the site of a DSB, but further experiments are required to confirm this observation.
Funding provided by Howard Hughes Medical Institute

Biochemical Characterization of Two Novel Homing Endonucleases

David DiTullio ('11); Matthew Sazinsky; Mentor: Lenny Seligman

Abstract removed upon request.

FoxO, Senescence and Stress in Hydra

Patrick Halliday ('11) Mentor: Daniel Martinez

Abstract: A family of proteins known as Forkhead Box transcription factors (FoxO proteins) are thought to play a potential role in regulating senescence (aging) in the freshwater Cnidarian, Hydra. In evolutionarily similar organisms, these proteins have already been shown to regulate genes involved in cell growth, proliferation, differentiation and longevity. All previous studies concerning the behavior of FoxO in Hydra have been carried out using either H. magnipaillata or H. vulgaris. Both these species exhibit negligible senescence (lack of aging). However, H. oligactis, a species closely related to H. vulgaris, shows signs of aging following sexual reproduction (inducible senescence). In H. vulgaris, FoxO was shown to be localized to the nucleus during periods of stress, where it is able to regulate the expression of genes involved in stress response. Using similar techniques, we are now focusing upon characterizing the behavior of FoxO in the sexually inducible animal, H. oligactis.
Funding provided by Howard Hughes Medical Institute

Reverse Aging? Meiotic Resetting of the Aging Clock in Saccharomyces cerevisiae

Janice Joo ('11); Jasmine Kim ('11); Mentor: Laura Hoopes

Abstract: Saccharomyces cerevisiae can be induced into meiosis by nutrient starvation and then restored to mitotic growth when returned to rich media. Our lab has found that when aged 8 generation cells are induced into meiosis and then returned to mitotic growth, their life spans resemble those of young cells. This suggests that the aging clocks of the 8g cells are meiotically reset. Life span analyses indicate that the reset happens between 1 and 4 hours in meiosis. We hypothesize that the reset mechanism happens during DNA replication, and southern blots using BrdU incorporation are currently being performed to determine the time of DNA replication. In addition, we are performing microarrays on RNA from cells reset after 2 and 3 hours to determine changes in gene regulation. Statistical analysis is needed to determine trends in up and downregulation of certain genes.
Funding provided by Howard Hughes Medical Institute (JJ), The Fletcher Jones Foundation (JK)

Possible Induction of Apoptosis by Common Food Preservatives BHA and BHT

Tyler Petersen ('11); Mentors: Tina Negritto, Cynthia Selassie

Abstract: This study shows preliminary results supporting previous work by the Negritto lab showing BHT to cause damage to Saccharomyces cerevisiae in a way which induces apoptosis. Nuclear stains show BHT to cause malformed nuclei, and a 1.5 fold increase in fluorescence using a TUNEL technique compared to the DMSO control, pointing towards apoptosis. BHA on the other hand acts in a still unknown method, showing somewhat degraded nuclei, but a decreased fold fluorescence in the TUNEL assay, pointing towards another mechanism of action. Using a fused RAD52, a double strand break protein, and green fluorescent protein we are able to see foci where the RAD52-GFP localizes in the cell.
Funding provided by The Fletcher Jones Foundation

Recruitment of TFIIH to the site of Double Stranded Breaks at the SAM1 loci in S. Cerevisiae

Paige Wolstencroft ('12); Mentor: Tina Negritto

Abstract: Transcription factor IIH (TFIIH) is a ten subunit complex involved in both transcription initiation and lesion repair that contains two DNA helicases: XPD and XPB. Its involvement in the repair of double stranded breaks (DSBs), through the nucleotide excision repair pathway (NER) is unknown, but is important given the fatal diseases (XP, CS and TTD) linked to genetic mutations within the NER pathway. Rad3, the S. Cerevisiae homolog for XPD, is used to study the recruitment of TFIIH to the site of DSBs. When a DSB is induced at the Sam loci in S. Cerevisiae there is a greater than 2-fold increase in Rad3 between TP - 60 (before induction) and TP 30 (90 minutes after induction). Rad3 levels remain elevated through TP 120 and then decline as the DSB is repaired. This indicates that TFIIH is involved in the repair of DSBs at the Sam loci in S. Cerevisiae.
Funding provided by The Fletcher Jones Foundation

Selection of Yeast Aging Genes

Jocelyn Young ('11); Mentor: Laura Mays Hoopes

Abstract: Identification of genes involved in the aging of Saccharomyces cerevisiae may be accomplished through the use of barcode strains, a collection of deletion mutants in which each gene is replaced by a unique barcode of DNA flanked by two PCR handles utilized to amplify the barcode. By comparing the DNA barcodes from pre- and post-aging strains on a microarray, deletion strains that are being depleted and those being enriched over the aging process may be determined. To gain more insight, lifespan dissections were completed of strains deleted for SUR4, ACE2, MAC1, ELM1, SRB2, GUP1, GAS1, HTL1, ALG3, and REG1, genes found to be considerably affected by aging. Strains deleted for ELM1 and genes involved in telomere maintenance experienced decreased lifespans while the mac1 and alg3 deletion strains experienced increased lifespans. Thus, ELM1, MAC1, ALG3, as well as genes involved in telomere maintenance may play significant roles in yeast aging.
Funding provided by Rose Hills Foundation

Research at Pomona