Degradation of ancient DNA

Since the appearance of the first reports describing the isolation and cloning of DNA from a 140-year-old preserved quagga [1] and from a 2 400 year-old Egyptian mummy [2], ancient DNA has been extracted from a number of organisms preserved in a variety of ways. Sources include preserved museum specimens, frozen mammoths, a 17 million year-old Magnolia leaf, and a 130 million yearold weevil entombed in amber. In each case, the extracted DNA was present as low molecular weight fragments, typically 100–200 basepairs (bp) long [3,4]. Ancient DNA is also extensively modified by hydrolysis and oxidation, leading to deletions, oxidized pyrimidines and intermolecular and intramolecular cross-linking of molecules. But regardless of the conditions of preservation of the specimen or its age, the size of DNA fragments recovered is remarkably similar. There is no observable correlation between the age of the sample and the length of the fragments. For example, Svante Pääbo examined DNA from a sample of 4 year-old dried pork, a 100 year-old marsupial wolf, a 13 000 year-old ground sloth and several mummies, and found an average fragment size of 100–200 bp [4]. If the postmortem degradation of DNA proceeds at an approximately constant rate, an older sample would be predicted to contain shorter fragments than a more recent one. This is not the case, so there cannot be continuous degradation. None of the papers describing the extraction of ancient DNA addressed the reason, or suggested an explanation, for the relatively constant fragment size. Here, we address the question of why ancient DNA undergoes degradation to small fragments regardless of age, and suggest an explanation for the phenomenon. The nuclear DNA of all eukaryotes is found as chromatin, the basic unit of which, the nucleosome, consists of DNA and associated histone proteins. The histones protect the DNA from degradation by nucleases, and treatment of chromatin with nucleases produces DNA fragments of about 160 bp in length, the length depending on the species. The fragment size reflects the length of DNA wrapped around a single nucleosome core [5]. Prokaryotes, mitochondria and chloroplasts do not have histones, but their DNA is associated with histone-like proteins and forms nucleosome-like structures [6,7]; the Escherichia coli HU protein protects 120–160 bp fragments of DNA from digestion by nucleases [6]. On the death of a cell, its DNA is degraded, resulting in a distinct pattern [8]. Regardless of the cause of death, the chromatin is fragmented by endonuclease cleavage of the vulnerable region between nucleosomes (linker DNA), leading to the formation of oligonucleosomesized fragments (reviewed in [9,10]). (This fragment size has been demonstrated for cell death by apoptosis — programmed cell death — but the condition of DNA after cell death by necrosis is not clear.) Little is know about the fate of mitochondrial, chloroplast, or prokaryotic DNA upon death. It has been shown, however, that following necrosis, condensed material and calcification of the mitochondria are apparent (reviewed in [11,12]). The size of DNA fragments extracted from ancient sources is approximately that protected by the nucleosome core (100–200 bp). This may be a coincidence. We speculate, however, that upon death nucleases degrade the DNA at the linker regions between nucleosomes. Later, when the histones are degraded, the nucleases themselves would be similarly affected, so the DNA would not be further fragmented by enzymatic degradation. Dehydration of the tissue, as well as changes in pH, temperature and salt concentration, might contribute to protecting the DNA from further fragmentation. The DNA can then apparently remain stable for many thousands, or even millions, of years.