Theory

In metabolically active cells, genomic damage is effectively repaired through complex enzymatic pathways; in dead or dormant cells such as bacterial endospores, damage will accumulate over time (Nicholson et al. 2000). Most fossil remains of one hundred to a

Martin B. Hebsgaard

Ancient DNA and Evolution Group, Department of Biology, University of Copenhagen,

Universitetsparken 15, DK-2100 Copenhagen, Denmark

[email protected]

R. Margesin (ed.) Permafrost Soils, Soil Biology 16,

DOI: 10.1007/978-3-540-69371-0, © Springer-Verlag Berlin Heidelberg 2009

few thousand years old do not contain amplifiable endogenous DNA. This indicates that DNA degradation must occur at a rapid tempo (Hofreiter et al. 2001a). It also indicates that the DNA molecule is relatively unstable compared to other cellular components (Lindahl 1993). The initial degradation process begins with cells being dissolved by cellular enzymes; subsequently, rupture of the cell releases nutrients, which support the growth of environmental microorganisms that contribute further to the degradation process (Nicholson et al. 2000). Rapid desiccation, freezing, and high salt concentrations can in special cases significantly reduce this enzymatic and microbial degradation. In cases like these, slower continuous processes such as hydrolysis, oxidation, and cross-linking will modify the DNA and finally render it irretrievable (Hofreiter et al. 2001b; Willerslev et al. 2004b; Pääbo et al. 2004; Willerslev and Cooper 2005) (Fig. 4.1).

[| cytosine

II thymine O

I adenine

N H H guanine

[| cytosine

N H H guanine

Fig. 4.1 a The DNA molecule is highly prone to spontaneous degradation processes such as hydrolysis and oxidation. Hydrolytic damage is responsible for breaks of the sugar backbone (1), for base loss (especially the purines, adenine and guanine = depurination) (2), and for the deami-nation of bases (cytosine, adenine, and guanine) (3). Oxidative damage perturbs the integrity of the DNA molecule by attacking the shared double bond of carbons C5 and C6 of pyrimidines (cytosine and thymine) (4) or the C4 (5), C5 (6) and C8 (7) carbons of purines. The sugar backbone can also be attacked (8). Hydrolytic and oxidative damage causes nicks, and blocking- or miscoding lesions. b A largely unrecognized DNA modification is crosslinking which includes intermolecular crosslinks such those of DNA and proteins (1) and interstrand crosslinks, i.e. between two DNA strands (2). Crosslinks prevent amplification, but might also stabilize the DNA molecule over time, so reducing fragmentation

Fig. 4.1 a The DNA molecule is highly prone to spontaneous degradation processes such as hydrolysis and oxidation. Hydrolytic damage is responsible for breaks of the sugar backbone (1), for base loss (especially the purines, adenine and guanine = depurination) (2), and for the deami-nation of bases (cytosine, adenine, and guanine) (3). Oxidative damage perturbs the integrity of the DNA molecule by attacking the shared double bond of carbons C5 and C6 of pyrimidines (cytosine and thymine) (4) or the C4 (5), C5 (6) and C8 (7) carbons of purines. The sugar backbone can also be attacked (8). Hydrolytic and oxidative damage causes nicks, and blocking- or miscoding lesions. b A largely unrecognized DNA modification is crosslinking which includes intermolecular crosslinks such those of DNA and proteins (1) and interstrand crosslinks, i.e. between two DNA strands (2). Crosslinks prevent amplification, but might also stabilize the DNA molecule over time, so reducing fragmentation

The key question is whether we can predict the long-term survival of DNA, and what environmental conditions and genomic protection mechanisms allow the DNA to survive longest on the Earth's biosphere. Several attempts have been made to predict long-term DNA survival, such as amino acid racemization (Poinar et al. 1996), thermal age (Smith et al. 2001), and extrapolations from DNA in solution (Pââbo and Wilson 1988; Willerslev et al. 2004a). These models are in general too simple; for example, they assume that hydrolytic depurination is the only significant type of DNA damage, even though other modifications such as crosslinking have been shown to be more important for the retrieval of DNA under certain conditions (Rivkina et al. 2000; Willerslev et al. 2004a; Hansen et al. 2006) (Fig. 4.1).

Further, some bacterial cells have continuous metabolic activity, allowing genomic repair over time which extends the long-term survival of metabolically active cells compared to cells under dormancy (Johnson et al. 2007). Thus, predicting DNA survival remains complicated, among other things because the rates of DNA degradation under various environmental conditions are only poorly understood.

Even though it is difficult to predict the long-term survival of DNA, empirical claims of geologically ancient DNA in the order of 1,000-fold older than theoretical predictions for maximal DNA survival are of considerable concern. In general, models for long-term DNA preservation predict a maximum survival time of about 100,000 years for short pieces of amplifiable DNA (~100 bp) (Pââbo and Wilson 1988; Poinar et al. 1996; Smith et al. 2001). Together with the huge problems with contamination, it is very important that we evaluate and authenticate the claims of very old DNA (Hebsgaard et al. 2005).

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