A presentation on how a particular type of DNA damage is repaired earned Molecular, Cellular, and Developmental Biology doctoral student Zhuobin "Ben" Liang the award for the "best platform presentation" by a graduate student at the 16th Annual Midwest DNA Repair Symposium in May. Liang works with Lyle Simmons, MCDB, and Thomas E. Wilson of the UM Medical School Departments of Pathology and Human Genetics.
Liang uses yeast as a model system to investigate how the structures of a double-strand break (DSB) in the DNA affect repair. DSB repair is often made by the rejoining of the broken ends. However, particular forms of the break can complicate rejoining and lead to mutations. Liang developed a system to cause breaks with diverse structures at a specific locus in the yeast genome. He then used various techniques to study how different break structures changed the repair mechanism.
The title of his presentation, which he was invited to give at the meeting, is "Kinetic study of chromosomal double-strand breaks with diverse break structures using high-resolution techniques." Co-authors are Sivakumar Nallasivam and Thomas E. Wilson.
This year’s meeting was held at Wayne State University on May 17-18. Researchers from throughout the Midwest gathered to discuss DNA repair after environmental damage and mutagenesis.
Non-homologous end joining (NHEJ) is the dominant double-strand break (DSB) repair pathway in cells with limited or no 5’ resection. DSBs often harbor diverse break structures that can complicate rejoining and lead to mutations. To better understand how overhang polarity affects repair, we engineered an efficient system to induce site-specific 5’-overhanging DSBs (5’ DSBs) in the S. cerevisiae genome using zinc finger nucleases (ZFNs). Improved activity of our ZFN system allows us to study for the first time the repair kinetics of 5’ DSBs by chromatin immunoprecipitation and next-generation sequencing. Surprisingly, NHEJ factors, including Yku80, Pol4 and Dnl4, had significantly higher recruitment to ZFN-induced 5’ DSBs as compared to HO-induced 3’ DSBs in the same locus. Consistently, NHEJ efficiency was also higher at ZFN-induced 5’ DSBs. Exonucleases involved in 5’ resection, such as Exo1, have been demonstrated in vitro to have varying activity on substrates with different overhang polarities. We thus hypothesize that 3’ and 5’ DSBs have different kinetics of end-processing affecting the stability and/or activity of NHEJ. We are in the process of analyzing the kinetics of end-processing using our newly developed ligation-mediated qPCR at single-nucleotide resolution. In addition, we demonstrate that yeast Tyrosyl-DNA phosphodiesterase 1 (Tdp1) was recruited at a low level exclusively to 5’ DSBs and that its recruitment was antagonized by Ku. Conversely, overexpression of Tdp1 weakly compromised NHEJ. These findings suggest that Tdp1 competes with NHEJ at 5’ DSBs. Moreover, sequencing of chromosomal 5’-DSB joints has not to date revealed evidence for Tdp1-mediated suppression of insertional mutagenesis as observed in plasmid studies. In summary, our study provides new insights of how overhang polarity at genomic DSBs influences end-processing and repair outcomes.