Investigating the Role of Hob1 in Repairing Double Stranded DNA Breaks in the Fission Yeast, Schizosaccharomyces pombe Sarah Ozturk, Andy Mai, Daitoku Sakamuro, Ph.D., and Amy Abdulovic ‐Cui Ph.D . Department of Biological Sciences and the Department of Biochemistry & Molecular Biology, Georgia Regents University, Augusta, GA.
DNA & Genomic Integrity • DNA • Mutations, lesions • Cancer, disease • Restoring DNA http://www.euroimmun.us/img/dsDNS_12.jpg http://www.nwfsc.noaa.gov/publications/scipubs/techmemos/tm17/figures/p arkfig2.gif
Double Stranded Breaks • Causes – X-rays – Gamma radiation • 1 single double stranded break is lethal to cell http://blog.ctl.net/wp- content/uploads/2013/04/photo.jpg
Damage and Repair • DNA strand breaks • Checkpoint pathways activated • Repair Pathways – Homologous Recombination – Non-Homologous End Joining https://achemicallife.files.wordpress.com/2012/12/ dna_breaks.png
Homologous Recombination • Sister chromatids as template – More accurate, higher precision – Non mutagenic • Only occurs after DNA synthesis https://teaching.ncl.ac.uk/bms/wiki/ima ges/d/d2/Sister_Chromatids.JPG
MRN protein arrives to break site, checkpoint Picture edited from: Brandsma and van van Gent Genome Integrity 2012, 3:9 http://www.genomeintegrity.com/content/3/1/9
Rad51 interacts with single stranded overhangs Picture edited from: Brandsma and van van Gent Genome Integrity 2012, 3:9 http://www.genomeintegrity.com/content/3/1/9
Strand invasion of sister chromatid Picture edited from: Brandsma and van van Gent Genome Integrity 2012, 3:9 http://www.genomeintegrity.com/content/3/1/9
Bidirectional replication producing two identical double stranded DNA Picture edited from: Brandsma and van van Gent Genome Integrity 2012, 3:9 http://www.genomeintegrity.com/content/3/1/9
Non-Homologous End Joining • Process of joining two DNA ends together • No sister chromatid involved • Nuclease activity, mutagenic – Lose DNA information • Insert picture with DNA mutation http://www.nwfsc.noaa.gov/publications/scipubs/techme mos/tm17/figures/parkfig2.gif
Ku proteins interact with ends of break Picture edited from: Brandsma and van van Gent Genome Integrity 2012, 3:9 http://www.genomeintegrity.com/content/3/1/9
Ku recruits other proteins to site Nuclease activity trims flaps to allow for ligation Nuclease Picture edited from: Brandsma and van van Gent Genome Integrity 2012, 3:9 http://www.genomeintegrity.com/content/3/1/9
Ligation occurs and forms 1 copy of mutagenic double stranded DNA Nuclease Picture edited from: Brandsma and van van Gent Genome Integrity 2012, 3:9 http://www.genomeintegrity.com/content/3/1/9
Bin1 interacts with Ku Bin1 proteins No Bin1 Bin1 Nuclease NHEJ No NHEJ (Mutagenic, (Apoptosis or cancer) HR) Picture edited from: Brandsma and van van Gent Genome Integrity 2012, 3:9 http://www.genomeintegrity.com/content/3/1/9
Hob1 • Homolog of mammalian Bin1 • Role in inhibiting Non-Homologous End Joining (NHEJ)
Hypothesis • Hypothesis: Hob1 plays a fundamental role in S. pombe by inhibiting non-homologous end joining.
Schizosaccharomyces pombe • Fission yeast • Eukaryotic model system • Manipulation • 2-4 hr generation time • Conserved repair pathways http://www.ucl.ac.uk/gee/images/carousel-2014/Sz-pombe- strains
Methods • Genetically in yeast, in vivo 1.) Extract E.coli plasmid 2) Linearize plasmid 3) Transform plasmid into S. pombe 4) Allow for selective growth 5) Assess the success of the transformation
Plasmid: pAL19 • Transcriptional start site • Ampicillin • ARS
2) Linearize 1.) Extract plasmid with plasmid restriction from E.coli enzyme
Heat and Chemical Shock Leu- v NHEJ
Density of Counted NHEJ Rate Total Cells Colonies (Density/C (cells / mL) olonies) 2.6x10 -6 Wild Type 2.45x10^7 59 <4.1x10 -8 hob1 Δ 2.2x10^7 0 100 fold DECREASE in NHEJ Promoting DSB repair
#1 Possible Reasons for Data • There was not a control used to test the intake of DNA into the yeast. – In future experiments, a control plasmid will be used to verify that transformation occurred.
Experiment Control plasmid with Design Using Linearized Ura Plasmid Control Yeast able to grow on plates without uracil Ura- v Pal19 isolated from E. coli Leu- v Yeast able to grow on DNA rejoined to form Transformed linearized plates without leucine circular plasmid and plasmid and control control is present
#2 Possible Reasons for Data • Yeast Hob1 could behave differently than mammalian Bin1 – Multicellular – Unicellular • Homologous recombination – Prevalent in yeast – Investigate role of Hob1
Summary • Hob1 role in NHEJ • Hypothesized that it would behave similar to Bin1 • Experiment – Extracted and cut plasmid – Transformed into fission yeast – Selectively grew to assess NHEJ repair • 100 fold reduction in NHEJ rate – Hob1 induces DSB repair
Thanks to Dr. Sakamuro and Dr. Abdulovic-Cui lab group!
References • Bennardo, Nicole, Anita Cheng, Nick Huang, and Jeremy M. Stark. "Alternative-NHEJ Is a Mechanistically Distinct Pathway of Mammalian Chromosome Break Repair." PLOS Genetics. N.p., 27 June 2008. Web. 23 Sept. 2014 • Brandsma, I., & Van Gent, D. (2012, January 1). Pathway choice in DNA double strand break repair: Observations of a balancing act. Retrieved from http://www.genomeintegrity.com/content/pdf/2041-9414-3-9.pdf • Forsburg, S. L. "THE YEASTS SACCHAROMYCES CEREVISIAE AND SCHIZOSACCHAROMYCES POMBE: MODELS FOR CELL BIOLOGY RESEARCH." Gravitational and Space Biology (2005): n. pag. Web. • Ramalingam, A., Farmer, G., Stamato, T., & Prendergast, G. (2007, August 1). Bin1 Interacts with and Restrains the DNA End-Binding Protein Complex Ku. Retrieved from http://www.tandfonline.com/doi/abs/10.4161/cc.6.15.4514 • Routhier, E., Donover1,2, P., & Prendergast, G. (2002, October 22). Hob1 , the fission yeast homolog of Bin1, is dispensable for endocytosis or actin organization, but required for the response to starvation or genotoxic stress. Retrieved from http://www.nature.com/onc/journal/v22/n5/full/1206162a.html
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