Originally published as Genetics Published Articles Ahead of Print on April 2, 2006.

Genetics, Vol. 173, 1175-1179, June 2006, Copyright © 2006
doi:10.1534/genetics.106.057349

Crosslinks Rather Than Strand Breaks Determine Access to Ancient DNA Sequences From Frozen Sediments

Anders J. Hansen*,1, David L. Mitchell{dagger}, Carsten Wiuf{ddagger}, Lakshmi Paniker{dagger}, Tina B. Brand*, Jonas Binladen*, David A. Gilichinsky§, Regin Rønn** and Eske Willerslev*,1,2

* Centre for Ancient Genetics, Niels Bohr Institute and Biological Institute, University of Copenhagen, DK-2100 Copenhagen Ø, Denmark, {dagger} Department of Carcinogenesis, University of Texas, M. D. Anderson Cancer Center, Science Park/Research Division, Smithville, Texas 7895, {ddagger} BiRC—Bioinformatics Research Center, University of Aarhus, 8000 Aarhus C, Denmark, § Soil Cryology Laboratory, Institute for PhysicoChemical and Biological Problems in Soil Science, Russian Academy of Sciences, 142290, Pushchino, Moscow Region, Russia and ** Department of Terrestrial Ecology, Biological Institute, University of Copenhagen, DK-1353 Copenhagen K, Denmark

2 Corresponding author: University of Copenhagen, Juliane Maries vej 30, DK-2100 Copenhagen Ø, Denmark.
E-mail: ewillerslev{at}bi.ku.dk

ABSTRACT

Diagenesis was studied in DNA obtained from Siberian permafrost (permanently frozen soil) ranging from 10,000 to 400,000 years in age. Despite optimal preservation conditions, we found the sedimentary DNA to be severely modified by interstrand crosslinks; single- and double-stranded breaks; and freely exposed sugar, phosphate, and hydroxyl groups. Intriguingly, interstrand crosslinks were found to accumulate ~100 times faster than single-stranded breaks, suggesting that crosslinking rather than depurination is the primary limiting factor for ancient DNA amplification under frozen conditions. The results question the reliability of the commonly used models relying on depurination kinetics for predicting the long-term survival of DNA under permafrost conditions and suggest that new strategies for repair of ancient DNA must be considered if the yield of amplifiable DNA from permafrost sediments is to be significantly increased. Using the obtained rate constant for interstrand crosslinks the maximal survival time of amplifiable 120-bp fragments of bacterial 16S ribosomal DNA was estimated to be ~400,000 years. Additionally, a clear relationship was found between DNA damage and sample age, contradicting previously raised concerns about the possible leaching of free DNA molecules between permafrost layers.




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