Double strand breaks (DSBs) as indicators of genomic instability in PATRR-mediated translocations

Abstract

Genomic instability contributes to a variety of potentially damaging conditions, including DNA-based rearrangements. Breakage in the form of double strand breaks (DSBs) increases the likelihood of DNA damage, mutations and translocations. Certain human DNA regions are known to be involved in recurrent translocations, such as the palindrome-mediated rearrangements that have been identified at the breakpoints of several recurrent constitutional translocations: t(11;22)(q23;q11), t(17;22)(q11;q11) and t(8;22) (q24;q11). These breakpoints occur at the center of palindromic AT-rich repeats (PATRRs), which suggests that the structure of the DNA may play a contributory role, potentially through the formation of secondary cruciform structures. The current study analyzed the DSB propensity of these PATRR regions in both lymphoblastoid (mitotic) and spermatogenic cells (meiotic). Initial results found an increased association of sister chromatid exchanges (SCEs) at PATRR regions in experiments that used SCEs to assay DSBs, combining SCE staining with fluorescence in situ hybridization (FISH). Additional experiments used chromatin immunoprecipitation (ChIP) with antibodies for either markers of DSBs or proteins involved in DSB repair along with quantitative polymerase chain reaction to quantify the frequency of DSBs occurring at PATRR regions. The results indicate an increased rate of DSBs at PATRR regions. Additional ChIP experiments with the cruciform binding 2D3 antibody indicate an increased rate of cruciform structures at PATRR regions in both mitotic and meiotic samples. Overall, these experiments demonstrate an elevated rate of DSBs at PATRR regions, an indication that the structure of PATRR containing DNA may lead to increased breakage in multiple cellular environments.

Figure 1

Detection of SCEs at PATRR sites in lymphocytes using UV-Hoechst exposure coupled with FISH probes. (A) Model depicting an SCE event at the PATRR regions on chromosomes 11 (solid) and 22 (stippled) on both normal and t(11;22) chromosomes with FISH probes for PATRR 11 (green) and PATRR22 (red). (B) Representative metaphase spreads derived from normal peripheral blood lymphocytes visualized by UV-Hoescht plus Giemsa and UV-Hoescht plus FISH probes for PATRR 8 (green) and PATRR 22 (red). The green arrow marks an SCE event at PATRR8. (C) Representative metaphase spreads derived from lymphocytes of a t(11;22) balanced translocation carrier. Images are visualized either by UV-Hoescht plus Giemsa or by UV-Hoescht plus FISH probes for PATRR11 (green) and PATRR22 (red). The black arrows identify an SCE event at PATRR11 on the der11 chromosome. In (B) and (C), black and white images of DAPI counterstained chromosomes enable visualization of SCEs with greater contrast.

Figure 2

Median fold increase box plots for hESC-derived spermatogenic cells over the background 6q26. Data are from multiple ChIP assays using DSB-associated antibodies for γH2AX, Rad51 and NBS1 and testing for their association at PATRR regions on chromosomes 17, 8, 11 and 22.

Figure 3

Median fold increase box plots for normal lymphoblastoid and hESC-derived spermatogenic cells over the 6q26 background. Data are from multiple ChIP assays using the cruciform binding antibody 2D3.