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Nicholas DiBenedettoDetermining the Dynamics of Genomic Instability in T7 Schizosaccharomyces pombe MutantsNicholas DiBenedetto and Daniel Broek, Ph.D.

AbstractGenetic instability has been shown in many different types of human cancers, consisting of alterations to nucleic acid sequence or rearrangement of chromosomal material. Ribosomal DNA (rDNA) in eukaryotic genomes are repeating regions that are responsible for ribosome structure and function. Due to their highly transcribed nature, rDNA sites are very fragile and prone to breakage, and subsequent fusion or recombination of rDNA has been suggested in cancer and other genetic disorders such as Robertsonion translocations, hematologic malignancies, and neurodegenerative disorders (1,2,3). In an effort to prevent double stranded breakages, rDNA is replicated unidirectionally (in the same direction as transcription), so as to prevent collision of replication and transcription machinery. Unidirectional replication is achieved via replication fork barriers (RFBs) which are found in each rDNA repeat. In our lab, strains of Schizosaccharomyces pombe (S. pombe) named the T series were found to have expansion of rDNA material. Pulsed-Field Gel Electorphoresis (PFGE) was done to show the expansion of rDNA on chromosome III. The current hypothesis is that T7 (a strain of T series) harbors mutations that affect the stability of rDNA. The two theories that explain the mechanism by which expansion of rDNA occurs are as follows; (1) double stranded breaks and recombination occur on two different chromosome IIIs (diploid) and are ligated together via non-homologous end joining (NHEJ), or (2) double stranded breaks at rDNA repeat sites causes unequal crossover between sister chromatids. In support of these hypotheses, certain genes that were found to have point mutations in the T series, such as CENP-B homolog apb1 and gcn5, genetically interact with proteins sap1 and reb1 which are important to the stability of RFBs in S. pombe (4,5).ScaI Restriction Enzyme

ScaI restriction enzyme digests DNA on chromosomes I, II, and III in S. pombe into very small fragments, leaving just the rDNA from chromosome III. This allows us to visualize the rDNA via PFGE and asses whether or not expansion of rDNA material is occurring. Point Mutations in T Series Affecting Genetic InstabilityGeneProteinFunctionAbp1CenP-B centromere binding proteinGenetic interaction with reb1 and pnk1 (kinase involved in DNA repair)Gcn5SAGA complex histone deacetylaseGenetic interaction with sap1, reb1 (RFB stability proteins), rad51, and pnk1Cdc22Ribonucleotide reductaseMutants exhibit depleted dNTP pool resulting in RFB collapse and DS breaks

Whole genome sequencing revealed 11 point mutations (3 show here) that may affect the stability of RFBs in rDNA.T7 Banding Patterns PFGE of T7 ScaI RE Digests

Models of rDNA Expansion and ContractionReplication Fork Barriers

S. pombe contain 100s of rDNA repeats at the ends of chr III. Replication occurs bidirectionally at autonomous replication sites (ars), however RFBs ensure that replication only proceeds in a unidirectional manner. RFBs ensure that replication and transcription occur in the same direction so that they do not collide and cause DSB. Sap1 and reb1 proteins interact with the RFB and help to stabilize it. If the mutant genes identified in the T series interact with sap1 and reb1, then it could cause RFB collapse and subsequent DSBs.References1) Stimpson, K. M., Sullivan, L. L., Kuo, M. E., & Sullivan, B. A. (2014). Nucleolar Organization, Ribosomal DNA Array Stability, and Acrocentric Chromosome Integrity Are Linked to Telomere Function. PLoS ONE, 92) Kobayashi, S., Taki, T., Nagoshi, H., Chinen, Y., Yokokawa, Y., Kanegane, H. ... Taniwaki, M. (2014). Identification of novel fusion genes with 28S ribosomal DNA in hematologic malignancies. International Journal of Oncology, 44, 1193-1198.3) Qiao, Y., Mondal, K., Trapani, V., Wen, J., Carpenter, G., Wildin, R., Price, E. M., Gibbons, R. J., Eichmeyer, J., Jiang, R., DuPont, B., Martell, S., Lewis, S. M. E., Robinson, W. P., O'Driscoll, M., Wolf, F. I., Zwick, M. E. and Rajcan-Separovic, E. (2014), Variant ATRX Syndrome with Dysfunction of ATRX and MAGT1 Genes. Hum. Mutat., 35: 5862. 4) Zaratiegui, M., Vaughn, M. W., Irvine, D. V., Goto, D., Watt, S., Bhler, J., Martienssen, R. A. (2011). CENP-B preserves genome integrity at replication forks paused by Retrotransposon LTR. Nature, 469(7328), 112115. doi:10.1038/nature096085) Ryan, C. J., Roguev, A., Patrick, K., Xu, J., Jahari, H., Tong, Z., Krogan, N. J. (2012). Hierarchical modularity and the evolution of genetic interactomes across species. Molecular Cell, 46(5), 691704. 8

Conclusions and Future Directions22001600112510209458257857506806105854503652852252200160011251020945825785750680610585450365285225T7 cultures were grown as shown to the right to allow possible expansion/contraction of rDNA repeats. DNA plugs were made from two individual colonies and from the whole plate (labeled A, B, and Plate) and assessed via PFGE as shown above. The different banding patterns of rDNA in T7 clearly indicate changes of rDNA sizes. Different banding patterns should be noted especially within the same revertant strain, such as T7R3 and T7R5 (gel on the right). In lanes where there are no clear bands present, it is possible that there are great amounts of heterogeneity, which would result in a smear rather than a band. These results support the hypothesis that T7 harbors a mutation that affects rDNA stability.Point mutations have been identified within the T series, some of which have been shown to interact with proteins sap1 and reb1.RFBs play a very important role in the stability of rDNA repeats during replication and transcription. Collapse of the RFB leads to DSB and recombination of chromosome III in various different ways.If genetic interactions occur between the mutant genes discovered in the T7 and sap1 and reb1, then RFB collapse is much more probable, thus leading to abnormal rearrangement of rDNA.Continuation of PFGE will be done to further analyze rDNA content in T7. Hydroxyurea (HU) assays deplete cells of dNTPs, which causes a stall of replication machinery and subsequent DSB. Cells with faulty RFBs would likely be sensitive to HU and therefore assays will be done in the future.Grow T7 12 days

Plate

Select 5 colonies

Grow 24 hours

Plate

Select 2 colonies and pool of remaining colonies from one plate

Prepare plugs and assess via PFGE

T7R1 Single AT7R2 Single AT7R2 PlateT7R3 Single AT7R3 Single BT7R4 Single BT7R5 Single BT7R5 PlateUpper Band780800750610660700810790Lower Band390395380370370385410425

The table above quantifies the different sizes of the bands in T7. Note that lanes on the gel that did not show banding were omitted from the table.