Abstract 2125: A new method for assessing fidelity of DSB repair shows involvement of different DNA repair mechanisms at specific sites in human genome
Authors: Evdokimova Viktoria N. , Gandhi Manoj , Bakkenist Christopher J. , Nikiforov Yuri E. 
Published: August 2012

Abstract
Introduction: Two major mechanisms of double-strand break (DSB) repair are non-homologous end joining (NHEJ) and homologous recombination (HR). Whereas NHEJ is error-prone and causes small deletions and other mutations at the site of repair, HR is highly fidelic. Here, we introduce a new method to identify mutations at the sites of DSB repair and apply it to study the fidelity of repair at different sites in human genome. Design: To enable the induction of DSBs at endogenous sequences in the somatic genome of TPC-1 human thyroid cells, the cells were transduced with a HA-ER-I-PpoI retrovirus (gift of Dr. Michael Kastan). DSBs were induced by treating cells with 4-hydroxytamoxifen (4-OHT) which causes I-PpoI to enter the nucleus. I-PpoI induction for 6h produced multiple γH2AX foci in about 75% of cells. We investigated the incidence of mutations following I-PpoI induction at two DNA sites. One has an I-PpoI site located within a coding gene, DAB1, on 1p32-p31and a second has an I-PpoI site within an intergenic region on 2q21 that we designate as 2qIG. qPCR analysis revealed that 9-16% of target sequences were cleaved at 6h post I-PpoI induction, which is consistent with previous observations that showed that I-PpoI cleaves about 10% of the target sites in human cells. Mutations at the I-PpoI cleavage sites were detected after reducing the presence of wild-type amplicons by digesting DNA and subsequent PCR products with I-PpoI prior to the resolution of PCR products by capillary electrophoresis or prior to cloning and sequencing. Results: Mutations at the sites of cleavage by I-PpoI were determined after induction of I-PpoI for 6h, 24h, and 48h. After removal of the majority (&gt;97%) of wild-type sequences by digesting with I-PpoI, resolution of the PCR products by capillary electrophoresis showed the presence of various smaller size amplicons at the intergenic 2qIG site at each time interval and in all three replicates of the experiment, but no detectable abnormal size amplicons at the DAB1 gene site. Sequence analysis of cloned PCR products from one experiment revealed that 157 out of 252 sequences of the intergenic 2qIG site were mutant, whereas all 167 clones containing the DAB1 site were wild-type (P&lt;0.0001). Most of the mutant 2qIG sequences had 2-27nt deletions and the ligation sequences frequently had microhomology that involved one or two nucleotides, which is highly characteristic of NHEJ. Conclusions: We have established a new method to assess mutations arising during the repair of DSBs and demonstrate that mutations occur at sites of DSB repair in intergenic DNA but not at sites of DSB repair in coding DNA. This suggests that repair of DSBs in coding and non-coding DNA may involve different mechanisms.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2125. doi:1538-7445.AM2012-2125