Page 29 - Hormel Report 2021

mechanisms of genome maintenance, determine genome-wide roles of this remodeler in protecting chromatin domains and identify additional regula- tors/pathways cooperating with HELLS in protecting cells from detrimental consequences of genotoxic stress. Our studies will provide important insights into the origin of chromosomal rearrangements found in cancer and ICF syndrome. In addition, our studies will characterize new synthetic lethal and synthetic rescue interactions in genome mainte- nance pathways that can be exploited as targets in pursuing new therapeutic strategies.
New tools in cancer risk identification and prevention. My laboratory is developing new assays and methods for integrative, high resolution, genome-wide studies of DNA damage and repair, focusing on clinically relevant DNA alkylation dam- age. DNA alkylation damage is cytotoxic, mutagenic and pre-carcinogenic, and represents one of the most abundant types of DNA lesions induced by environmental carcinogens, chemotherapeutics, by-products of cellular metabolism or inflammation. We have developed quantitative, cell based assay (alk-BER) to measure formation of DNA alkylation damage and its repair in genomic DNA (Fig.1).
Figure 1: Schematic outline of alk-BER assay. The assay involves exposing the cells to MMS (step 1), isolation of total genomic DNA (step 2), conversion of MMS-induced methylated bases to SSBs with damage specific enzymes AAG and APE1 (step 3), separation of DNA fragments containing SSBs by alkaline agarose gel electrophoresis (step 4), gel staining, imaging, and quantitation of MDAs (step 5).
| 29
In order to better understand contribution of DNA alkylation to cancer development and treatment,
we are developing novel NGS sequencing methods – Alkyl DNA Adducts (ADA-SMRT) – that enable
high resolution, genome-wide mapping of DNA alkylation formation and repair (Fig.2). The goal
of these studies is to uncover genomic hot spots
of alkyl DNA adducts and their contribution to somatic mutations or chromosomal rearrangements found in cancer and other diseases.
Publications:
• Li Y., Mao P., Basenko EY., Lewis Z., Smerdon M., Czaja W. (2021) Versatile Cell-Based Assay for Measuring Base Excision Repair of DNA Alkylation Damage. Scientific Reports (accepted)
• Czaja, W., Miller, K.Y., Miller, B.L., Skinner, M.K. (2014) Structural and functional conservation of fungal MatA and human SRY sex determining transcription factors. Nature Communications. 17;5:5434.
• Czaja, W., Bensasson, D., Bergman, C.M., Garfinkel, D.J. (2020) Evolution of Ty1 copy number control in yeast by horizontal transfer and recombination. PLoS Genet. 16(2): e1008632.
Figure 2: Outline of ADA-SMRT. Cells are treated with alkylating agents (1), genomic DNA is isolated and SMRT sequencing library is prepared (2 and 3), SMRT sequencing is performed, and locations of damaged DNA bases are mapped (4 and 5) in genome-wide scale.