DNA Replication / Gene Amplification / Replication Proteins
Kapler’s broad research interests are concerned with the replication and transmission of eukaryotic chromosomes. The failure to completely replicate the genome during S phase or partially re-replicate chromosomes leads to genome instability- a hallmark of cancer cells. The central questions investigated in the laboratory are concerned with how replication initiation sites are established in chromosomes and how they are regulated during conventional (G1/S/G2/M) and alternative cell cycles, including endoreplication (gap-S-gap-S…) and locus-specific gene amplification. The laboratory uses the early branching model eukaryote, Tetrahymena thermophila, as its model system to study these questions. As a member of the Ciliophora lineage, Tetrahymena contains two nuclei within each cell, the diploid germline micronucleus and polyploid somatic macronucleus. The complex developmental program that generates a new macronucleus in exconjugants has provided fertile ground for studying the differential regulation of replication origin usage. A strength of the model system is Tetrahymena’s ability to support replication of naturally occurring and artificially engineered minichromosomes. These features have provides excellent opportunities to use forward and reverse genetic approaches to identify and characterize cis-acting determinants and trans-acting regulatory factors. While Tetrahymena employs conserve eukaryotic DNA replication machinery, including the origin recognition complex (ORC) and MCM2-7 replicative helicase, we have discovered novel regulatory factors, including an RNA molecule that selectively targets ORC to origins of replication in the rDNA minichromsome, which is amplified 9000-fold during development. The current focus of the lab is to use high throughput (nascent strand) DNA sequencing to generate a comprehensive map of replication initiation sites under different physiological conditions. We recently reported that ORC and MCM protein levels are reduced 50-fold under conditions of replication stress, and that upon removal of the stressor, cells replicate their entire macronuclear genome prior to replenishment of ORC and MCM subunits. Known origins are inactive during this highly unconventional S phase, yet new initiation events generate bidirectional replication forks and faithfully replicate chromosomes. Comparative analysis of origin usage under high and low ORC conditions should reveal ORC-dependent and ORC-independent replication initiation sites. Functional assays will be used to determine the underlying mechanism for ORC-independent replication initiation. The laboratory takes advantage of powerful molecular and genetic tools available in the Tetrahymena model system, including classic genetic analysis, an array of approaches for making gene knockouts and replacements, the ability to synchronize the cell cycle and development, single molecule DNA fiber imaging, molecular approaches to visualize DNA replication intermediates, high throughput RNA-Seq and nascent strand DNA sequencing. Finally, the functional analysis of genetically modified Tetrahymena is used to test models and has established new paradigms in the DNA replication field.
- Feng, L., Wang, G., Hamilton, E. P., Xiong, J., Yan, G., Chen, K., ... Liu, Y. (2017). A germline-limited piggyBac transposase gene is required for precise excision in Tetrahymena genome rearrangement. Nucleic Acids Research. 45(16), gkx652-–9502.
- Sandoval, P. Y., Lee, P., Meng, X., & Kapler, G. M. (2015). Checkpoint Activation of an Unconventional DNA Replication Program in Tetrahymena. PLOS Genetics. 11(7), e1005405–e1005405.
- Lee, P., Meng, X., & Kapler, G. M. (2015). Developmental Regulation of the Tetrahymena thermophila Origin Recognition Complex. PLOS Genetics. 11(1), e1004875–e1004875.
- Gao, S., Xiong, J., Zhang, C., Berquist, B. R., Yang, R., Zhao, M., ... Liu, Y. (2013). Impaired replication elongation in Tetrahymena mutants deficient in histone H3 Lys 27 monomethylation. Genes & development. 27(15), 1662–1679.
- Donti, T. R., Datta, S., Sandoval, P. Y., & Kapler, G. M. (2009). Differential targeting of Tetrahymena ORC to ribosomal DNA and non‐rDNA replication origins. The EMBO journal. 28(3), 223–233.
- Mohammad, M. M., Donti, T. R., Yakisich, J. S., Smith, A. G., & Kapler, G. M. (2007). Tetrahymena ORC contains a ribosomal RNA fragment that participates in rDNA origin recognition. The EMBO journal. 26(24), 5048–5060.
- Yakisich, J. S., Sandoval, P. Y., Morrison, T. L., & Kapler, G. M. (2006). TIF1 Activates the Intra-S-Phase Checkpoint Response in the Diploid Micronucleus and Amitotic Polyploid Macronucleus of Tetrahymena. MOLECULAR BIOLOGY OF THE CELL. 17(12), 5185–5197.
- Yakisich, J. S., & Kapler, G. M. (2006). Deletion of the Tetrahymena thermophila rDNA replication fork barrier region disrupts macronuclear rDNA excision and creates a fragile site in the micronuclear genome. Nucleic Acids Research. 34(2), 620–634.
- Morrison, T. L., Yakisich, J. S., Cassidy-Hanley, D., & Kapler, G. M. (2005). TIF1 Represses rDNA Replication Initiation, but Promotes Normal S Phase Progression and Chromosome Transmission in Tetrahymena. MOLECULAR BIOLOGY OF THE CELL. 16(6), 2624–2635.
- Yakisich, J. S., & Kapler, G. M. (2004). The effect of phosphoinositide 3-kinase inhibitors on programmed nuclear degradation in Tetrahymena and fate of surviving nuclei. Cell Death & Differentiation. 11(10), 1146–1149.
To Academic Professional Track Faculty