AT-rich sequences flanking the 5′-end breakpoint of the 4977-bp deletion of human mitochondrial DNA are located between two bent-inducing DNA sequences that assume distorted structure in organello

Abstract

The 4977-bp deletion is the most common deletion among more than 90 large-scale deletions of human mitochondrial DNA (mtDNA) that are associated with aging and mitochondrial myopathies. The reason why the frequency of occurrence of this common deletion is so high in aged and myopathic human tissues is not clear. Since several studies proved that unusual DNA structures play very important roles in a number of recombination events, we hypothesized that some kind of unusual DNA structure may flank the breakpoints of the 4977-bp mtDNA deletion. We used two-dimensional (2-D) gel electrophoresis to assess the mobility abnormalities of the PCR-amplified DNA fragments encompassing the sequences of nucleotide position (np) 7901 to 9058 of human mtDNA. The results showed that the sequences of np 7901–8732 and np 8251–9058 exhibited retarded and increased mobilities, respectively, and the sequence of np 8285–8676 showed normal mobility in the 2-D gel. This indicates that the 5′-end breakpoint of the 4977-bp deletion is located within the junction site of two flanking bent-inducting DNA sequences. We confirmed this notion by using osmium tetroxide (OsO 4) to probe mtDNA in organello. The results showed that the two AT-rich sequences flanking the 5′-end breakpoint of the 4977-bp deletion are susceptible to OsO 4 modification. These findings suggest that the DNA sequences of the 5′-end breakpoint of the common mtDNA deletion are rendered to assume a more distorted structure than B-DNA by these two flanking bent-inducing DNA sequences in organello and thereby render this region to be more vulnerable to attack by reactive oxygen species and free radicals.