Repairing Double Stranded DNA Breaks in the Fission Yeast, - - PowerPoint PPT Presentation

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Repairing Double Stranded DNA Breaks in the Fission Yeast, - - PowerPoint PPT Presentation

Oct 10, 2022 45 likes •326 views

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

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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.

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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

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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

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  • DNA strand breaks
  • Checkpoint pathways

activated

  • Repair Pathways

– Homologous Recombination – Non-Homologous End Joining

Damage and Repair

https://achemicallife.files.wordpress.com/2012/12/ dna_breaks.png

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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

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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

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Rad51 interacts with single stranded

  • verhangs

Picture edited from: Brandsma and van van Gent Genome Integrity 2012, 3:9 http://www.genomeintegrity.com/content/3/1/9

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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

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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

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  • Process of joining two DNA ends together
  • No sister chromatid involved
  • Nuclease activity, mutagenic

– Lose DNA information

  • Insert picture with DNA mutation

Non-Homologous End Joining

http://www.nwfsc.noaa.gov/publications/scipubs/techme mos/tm17/figures/parkfig2.gif

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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

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Nuclease

Ku recruits other proteins to site Nuclease activity trims flaps to allow for ligation

Picture edited from: Brandsma and van van Gent Genome Integrity 2012, 3:9 http://www.genomeintegrity.com/content/3/1/9

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Nuclease

Ligation occurs and forms 1 copy

  • f mutagenic double stranded

DNA

Picture edited from: Brandsma and van van Gent Genome Integrity 2012, 3:9 http://www.genomeintegrity.com/content/3/1/9

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Nuclease

Bin1 Bin1 interacts with Ku proteins

Picture edited from: Brandsma and van van Gent Genome Integrity 2012, 3:9 http://www.genomeintegrity.com/content/3/1/9

Bin1 No Bin1 NHEJ (Mutagenic, cancer) No NHEJ (Apoptosis or HR)

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Hob1

  • Homolog of mammalian Bin1
  • Role in inhibiting Non-Homologous End Joining

(NHEJ)

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Hypothesis

  • Hypothesis: Hob1 plays a fundamental role in
  • S. pombe by inhibiting non-homologous end

joining.

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  • Fission yeast
  • Eukaryotic model system
  • Manipulation
  • 2-4 hr generation time
  • Conserved repair pathways

Schizosaccharomyces pombe

http://www.ucl.ac.uk/gee/images/carousel-2014/Sz-pombe- strains

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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

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Plasmid: pAL19

  • Transcriptional start site
  • Ampicillin
  • ARS
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1.) Extract plasmid from E.coli 2) Linearize plasmid with restriction enzyme

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Leu- v Heat and Chemical Shock NHEJ

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Density of Total Cells (cells / mL) Counted Colonies NHEJ Rate (Density/C

  • lonies)

Wild Type 2.45x10^7 59 2.6x10-6 hob1Δ 2.2x10^7 <4.1x10-8 100 fold DECREASE in NHEJ

Promoting DSB repair

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#1 Possible Reasons for Data

  • There was not a control used to test the intake
  • f DNA into the yeast.

– In future experiments, a control plasmid will be used to verify that transformation occurred.

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Leu- v Pal19 isolated from E. coli Control plasmid with Ura Linearized Plasmid Transformed linearized plasmid and control DNA rejoined to form circular plasmid and control is present Yeast able to grow on plates without leucine Ura- v Yeast able to grow on plates without uracil

Experiment Design Using Control

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#2 Possible Reasons for Data

  • Yeast Hob1 could behave differently than

mammalian Bin1

– Multicellular – Unicellular

  • Homologous recombination

– Prevalent in yeast – Investigate role of Hob1

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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

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Thanks to Dr. Sakamuro and Dr. Abdulovic-Cui lab group!

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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
  • f 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