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

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.

Ancient bacteria show evidence of DNA repair

“…In the 5-to 30-Kyr age range, low-GC Gram-positive bacteria with the capacity to form dormant endospores accumulated hydrolytic damage at the 99% confi- † † Stone, J., Sletten, R. S., Hallet, B., Caffee, M. *Both DNA concentration and the frequency of interstrand cross-links were assayed on these samples in (9,11). Consistent with the DNA degradation undergone in dead cells, DNA concentration decreases with increasing age, whereas the number of interstrand cross-links increases.…”

“…As a result, their genomes will degrade with time because of spontaneous chemical reactions like hydrolysis and oxidation (6) that finally become fatal, preventing the cell from germinating. Models suggest that unrepaired genomic DNA will be fragmented into small pieces Ͻ100 bp in size or will become severely crosslinked within at most 100,000 to 1 million years (100 Kyr-1 Ma) under optimal frozen conditions and much faster in warmer settings (6)(7)(8)(9)(10). Thus, the controversy of viable ancient bacteria is heightened by an absence of convincing evidence for mechanisms by which a cell can withstand damage to DNA and other unstable molecules such as ATP over geological timescales (11)(12)(13)(14).…”

Ancient DNA reveals late survival of mammoth and horse in interior Alaska

“…No evidence of DNA leaching been found under permafrost settings, despite several investigations (15,(18)(19)(20), and for mammoth and horse DNA to be recovered from sediments several millennia younger than the youngest macrofossil remains from mainland Alaska/Yukon would require the DNA to have migrated more than 8 m upward through frozen sediments, without leaving any traces behind in the intervening strata (Fig. 1).…”

“…This so-called ''sedimentary'' ancient DNA (sedaDNA) has been shown to be of local origin (12,13,17), requiring an animal to have been physically present at the site for its DNA to be deposited (13). Although leaching of DNA may occur between layers in nonfrozen depositional settings (13), several studies have demonstrated that this problem does not appear to affect either perennially frozen sediments (17)(18)(19)(20) or sediments frozen recently (15). Furthermore, in cases where strata have remained undisturbed, DNA extracted from modern surface sediments at localities in the Arctic and temperate regions has yielded the genetic signatures of extant fauna only (12,16), which suggests that DNA is not readily reworked from older deposits and incorporated into younger deposits.…”

Ancient DNA extraction from bones and teeth

“…6 for rare exceptions) and, owing to as yet uncharacterized chemical modifications, it may even be difficult to get the DNA into aqueous solution 7 . Second, ancient DNA is damaged in various ways [8][9][10][11] , so extraction methods also have to avoid overly aggressive treatments, such as high temperatures or use of strong detergents 12 . Although these treatments might increase DNA release, they would decrease overall DNA yield by inflicting further damage to the ancient DNA molecules.…”