The Structure of Kinetoplast DNA

Abstract

1 Degradation of highly purified kinetoplast DNA (kDNA) networks with restriction endonucleases yields ‘extra’ bands in agarose gels that are absent from digests of mini-circles. Each of the five endonucleases tested, i.e. AluI, HapII, EcoRI, Hsu and HindII + III, yields a unique set of ‘extra’ bands. The ‘extra’ bands consist of linear DNA; they are not mini-circle oligomers and their added molecular weights, calculated from mobility in gels, are around 2 × 10⁷. Double digests with two restriction endonucleases yield a new set of ‘extra’ bands, showing that the ‘extra’ bands obtained with different enzymes are all derived from the same complex component of kDNA. In digests of ³²P-labelled kDNA an average of 2.3% of the radioactivity is recovered in the ‘extra’ bands. 2 Treatment of kDNA networks with the single-strand-specific S₁ nuclease of Aspergillus oryzue preferentially releases a linear DNA with a molecular weight of 26 × 10⁶, calculated from mobility in gels. We present evidence that the ‘extra’ bands obtained with restriction endonucleases are derived from this component. 3 DNA-DNA renaturation analysis of fragmented kDNA shows the presence of a minor complex component with a complexity of about 3 × 10⁷, making up less than 10% of the total kDNA. 4 From these results we conclude that 3–5% of the kDNA consists of a homogeneous class of maxi-circles catenated in the mini-circle network. The molecular weight of these maxi-circles is about 26 × 10⁶ and they contain a unique, non-repetitive, non-mini-circle nucleotide sequence. This component is a prime candidate for the true mitochondrial DNA of trypanosomes.