Repair of chromosome breaks by Nonhomologous end joining (NHEJ) determines the effectiveness of many cancer therapies, and is essential for assembly of antigen receptor genes (immunoglobulins and T cell receptors) required for adaptive immunity. This proposal addresses if polymerases specifically to NHEJ primarily add RNA in the course of this DNA repair pathway - a violation of the central dogma of molecular biology.
Aim 1 experiments address the extent RNA is incorporated during general double strand break repair, as well as whether RNA incorporation is regulated by cell cycle stage. Cas9 nuclease will be used to target breaks to a site in chromosome 6. The fraction of RNA in products of repair of this break will be determined by sequencing products before and after specific destruction of products with embedded RNA. Cells in different cell cycle phases will be purified, and the fraction of RNA in repair products in these different populations compared to determine if cell cycle phase influences RNA incorporation.
Aim 2 experiments will investigate why RNA is incorporated during NHEJ. They will further explore preliminary data arguing RNA incorporation by NHEJ polymerases is required to resolve a subset of broken ends by the NHEJ ligase. The nucleotide added by NHEJ polymerases will be systematically varied, and effects on ligation assessed. This will address if some nucleotide analogs ? many of which are already used in the clinic for chemotherapy and as antivirals -stimulate ligation, while other nucleotide analogs have the opposite effect (poisoning of ligation). We will also determine if the poisoning of NHEJ's ligation step by these analogs could be used to manipulate the response to radiation therapy.
Aim 3 addresses the consequences of the RNA that is now embedded in the repaired chromosome. RNA in genomic DNA is rapidly replaced with DNA by ribonucleotide excision repair (RER):
Aim 3 experiments will address what mechanisms help prevent RER from re-breaking chromosomes with embedded RNA. This proposal thus seeks to determine if we must reconsider how much of NHEJ functions, and addresses novel biological and potentially clinically significant consequences of this new mechanism.
The proposal investigates if polymerases add RNA, not DNA, in the course of repair of chromosome breaks; how often it happens, why this may initially make repair more accurate and efficient, but then make the repaired chromosome more fragile and contribute to aberration. Insights from this work may suggest a way to take drugs already widely used in the clinic as antivirals, and repurpose them to improve radiation therapy.