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Ancient DNA Repair

Ancient DNA Repair

Team members involved: Nathalie Mouttham
Former team members involved: Carsten Schwarz
Collaboration with Michael Weinfeld (Cross Cancer Institute – University of Alberta) and Roger Woodgate (Laboratory of Genomic Integrity – National Institutes of Health, Bethesda, Maryland)
Funding provided by: NSERC (National Sciences and Engineering Research Council of Canada)

In contrast to modern material the DNA extracted from fossil remains like bones or coprolites show a variety of types of damage.

The most obvious type of damage is fragmentation caused by single stand breaks which lead to a reduced average molecule length of the extracted DNA. Depending on this length the molecular analysis via PCR of a given sample is either complicated due to a limited amplicon size or impossible at all.

In order to improve the integrity of the extracted DNA from ancient samples the status of the DNA and its damage is determined and an enzymatic approach is attempted to restore DNA by filling in gaps as well as 5’overhangs and sealing nicks.

We are exploring various enzymatic repair strategies for our DNA extracts.

DNA Damage

The DNA molecule is a polymer composed of 2′ deoxyribose sugar units linked to each other via phosphate ester links (see figure below). To every sugar, a purine (adenine or guanine) or a pyrimidine (cytosine or thymine) is attached at the 1′ position by a glycosidic bond. In vivo, the DNA molecule is supported by water molecules in the major grooves, thus the molecule is never really dry. In addition nuclear DNA (not mitochondrial DNA) is wound tightly around histone proteins, which presumably absorb some of the surrounding damage. Despite this the DNA molecule is labile and prone to many forms of damage, all of which presumably limit the molecules “half-life” in the geosphere.

DNA degradation and preservation

Unfortunately for the paleo-geneticist, comparatively speaking, the DNA molecule is one of the least stable molecules within our cells. It is the subject of rapid hydrolysis and oxidation over short time periods limiting its “life” in vivo and within the environment.

Once an organism dies, presumably the single most important factor in the long-term preservation of its DNA is the rate at which specific cellular enzymes, called nucleases, can be stopped. These endonucleases are efficient and can rapidly cleave DNA into small fragments. However as these are energy requiring functions, and a cell without oxygen will deplete its energy sources quickly, nuclease degradation may cease relatively soon post mortem. The organism must then face the bacterial, fungal and insect onslaught which can be quite effective but often incomplete. Once bacterial onslaught has slowed, the DNA molecule is still subject to chemical degradation via hydrolysis and oxidation. To understand the processes which degrade the DNA molecule in the fossil record and under what conditions these reactions are minimized, one needs to look briefly at the molecule itself and its susceptible sites.

The DNA molecule is particularly prone to hydrolytic damage.

The removal of the ribose sugars 2′ OH group, creating the deoxy-ribose sugar does afford the bonds joining two nucleotides (phosphodiester bond) increased strength, however it weakens the glycosidic bond joining the bases to the sugars. Diesters, like the bonds in the phosphate sugar backbone, are normally quite labile and subject to quick hydrolytic cleavage. It has been estimated that direct cleavage of the phosphate backbone is probably the most frequent type of hydrolytic damage the DNA molecule must cope with, generating single stranded nicks. In vivo, in a fully hydrated system this event takes place about once every 2.5 hours. It has been estimated that under dry conditions this rate drops some 20 fold.

 

The glycosidic bond is also subject to direct hydrolytic attack.

Base protonation, making the base a better leaving group, will cause the cleavage of the glycosidic bond, termed depurination, and form what is known as a baseless or an abasic site (AP site). Estimated rates of depurination in double stranded DNA suggest one event takes place about every 10 hours in vivo. The rate of depurination is increased and dependent upon the temperature, the ionic strength, the pH, and heavy metal ion chelation.  Once a nucleotide base is released from DNA the AP site can undergo cleavage, termed ß-elimination, and thus result in a single stranded nick. AP site hydrolysis occurs at a rate similar to or slightly slower than depurination probably taking place a few days after depurination. At these rates of hydrolytic damage, a small DNA fragment of a few hundred base pairs would not be expected to survive beyond 104 years in most temperate settings, 105 years in colder ones such as the permafrost and perhaps even 106 years in exceptional cases like fossil glacial ice cores. The fact that most DNA retrieved from fossil or even subfossil samples is consistently around 100 to 500 base pairs (bp) in length would suggest that hydrolytic DNA damage takes place relatively soon post mortem and thus is one of the more important pathways reducing its life in the geosphere.

Apart from the hydrolytic cleavage of the phosphodiester and the glycosidic bonds, DNA bases with secondary amino groups such as adenine, cytosine, 5-methylcytosine and guanine can undergo deamination, the hydrolytic cleavage of their amino groups, resulting in hypoxanthine, uracil, thymine, and xanthine respectively. Deamination has recently been shown to be a prominent component of some fossil DNA remains.

Oxidative DNA damage

Even when the remains of an organism “dry” (although there is nothing in nature that is 100% dry) the molecular components of a cell are still susceptible to oxidative attack. Free radicals such as peroxide radicals (.O2), hydrogen peroxide (H2O2), and hydroxy radicals (.OH) are all endogenously occurring products and hence are an important source of endogenous DNA damage, and are likely to play an important role in limiting the life of DNA in the fossil record. They may also derive from exogenous sources such as ionizing radiation, UV light and cellular processes during bacterial and fungal degradation. Recently DNA extractions from fossil remains were subject to gas chromotagrahpy mass spectrometry in the attempt to identify oxidative base damage. From all samples where no endogenous DNA could be retrieved higher levels of two oxidative forms of base damage, 5-Hydroxyhydantoin and 5-Hydroxy-5-methylhydantoin were detected. As these and many other oxidative lesions block the polymerase in PCR, and hence its ability to make copies, oxidative damage will also limit the successful retrieval of DNA from fossil remains.

Both hydrolysis and oxidation limit the survival of DNA in the fossil record and it is likely for these reasons that few fossil samples still contain authentic endogenous DNA.

As all DNA extraction methods to date are destructive methods and can require as much as a few grams of material, most museum curators are reasonably hesitant to release prized possessions, knowing that there is a small chance that something may have survived in the specimen. For these reasons it is important to screen samples prior to their extraction, in order to assess the state of molecular preservation.