New polymerases for old DNA
Phil Holliger
MRC LMB
Slide1:
Top left
In 79 A.D., Mount Vesuvius erupted and buried two towns. One of these was
Pompeii, the other was Herculaneum, a seaside resort for wealthy Romans.
Top right
Among the villas uncovered by excavation was the summer residence of
Julius Caesar's father-in-law, Lucius Calpurnius Piso. The picture shows a
reconstruction of the villa forming part of the Getty museum in LA.
Bottom left
Inside the villa a large number of what appeared to be sticks of charcoal were
discovered, some of them bundled together.
Bottom Middle
These turned out to be ancient papyrus scrolls carbonized by the volcanic
heat. The “Viilla dei Papyrii” as it became known, contained the only known
library of antiquity.
Bottom right
While some part of the text remained legible, large parts of the scrolls were
initially uninterpretable. However, the information encoded in this ancient
library was not lost but had to wait for better technology. Today, multi-spectral
imaging promises a breakthrough in deciphering the fragile scrolls.
Slide 2:
By analogy, many specimens paleontological, archaeological, or forensic
interest contain a wealth of information written in their DNA.
Ancient DNA sequences have been isolated from a wide variety of sources 1
and have provided information about human migration, animal and crop
domestication and the genetic relationship between modern Homo sapiens
and its closest extinct relative H. neandertalensis.(left panel)
Such ancient DNA can provide a window to the past but even under optimal
burial conditions, DNA damage is progressive, as the multitude of DNA repair
pathways, which maintain the integrity of the genome in living organisms no
longer function.
The right panel shows a summary of the multitude of types of damamge found
in ancient DNA.
This damage either limits the length of continuous sequence that can be
recovered or renders even well-preserved specimens unproductive despite
the demonstrable presence of DNA (by hybridization)
We reasoned that genetic information encoded in such samples may not be
lost but simply inaccessible due to the fact that the DNA polymerases
commonly used for PCR stall at sites of damage.
Polymerases capable of replicating across DNA damage should therefore be
able to allow the deciphering of previously unreadable ancient DNA
sequences just like modern imaging is helping decipher the burnt scrolls of
Herculaneum.
However, no such polymerases suitable for ancient DNA recovery existed in
nature. We therefore turned to evolution to generate such polymerases in the
laboratory using nature’s tricks.
Slide 3
Darwinian evolution can be applied not just to organisms but to molecules too.
Thus, molecular properties can be improved by iterative cycles of mutation,
selection and amplification.
Slide 4
We devised a strategy for the selection of polymerase function, which we call
“compartmentalized self-replication” or CSR.
CSR is based on a simple feedback loop, whereby a polymerase replicates its
own encoding gene.
Compartmentalization serves to isolate individual self-replication reactions
from each other.
Compartmentalization is a crucial aspect of life. All living organisms are made
from cells, which encase the genome and the proteins it encodes within a lipid
membrane.
We use a different approach to nature. In our case, polymerase genes and
the polymerase they encode are encapsulated in water droplets dispersed in
an oil phase, i.e. a water-in-oil emulsion. These “artificial cells” ensure
genotype-phenotype linkage, i.e. they ensure that a polymerase only
replicates only their own encoding gene and no other.
In such a system adaptive gains by a polymerase directly (and proportionally)
translate into more “offspring”.
In other words, if a polymerase (purple spheres) is well-adapted to the
selection conditions, it can replicate its own encoding gene and produce many
copies of itself, i.e. “offspring”. However, if a polymerase (yellow hexagon) is
poorly adapted, it cannot self-replicate and its gene will disappear from the
gene pool.
Slide 5
Left panel
When random mutagenesis of the polymerase genes proved unproductive we
turned to a method called molecular breeding, whereby the homologous
genes from different organisms (orthologues) are recombined to yield a library
of chimeras comprising segments of the different orthologues. Molecular
breeding samples only functional diversity and therefore a large number of
chimeras are active. We recombined three thermophilic polymerase (DNA pol
I) genes from the genus Thermus: Taq polymerase from
(Thermus
aquaticus), the standard polymerase used for PCR amplification of ancient
DNA, Tth (T. thermophilus) and Tfl (T. flavus)) to create a polymerase library
for selection.
Right panel
This shows a three dimensional model of Taq polymerase. Residues deriving
from Tth or Tfl that we find in our selected polymerases are shown in different
shades of blue. The darker the blue the more often they occur. This just to
illustrate how the offspring of an evolution experiment can comprise a
patchwork of elements of the parental genes.
Slide 6
Evolving polymerases, which combine the processivity and selectivity required
for PCR amplification with a high tolerance for template damage is
challenging. Furthermore, damage tolerance should be generic as detailed
information about the forms of DNA damage in ancient samples is lacking
(and damage may vary depending on burial conditions).
Top
Many lesions (red X) abrogate base-pairing and yield distorted, non-cognate
3’ structures, similar to mismatches.
While natural polymerases readily extend of matched primer terminus (ending
in a cognate GC base-pair), they stall at mismatches or sites of damage (G.X
mispair). Extension is significantly slowed down not just at the 3’ end but up to
four bases downstream (highlighted in red).
In order to maximize tolerance to such distorted primer-template structures,
we decided to select for polymerases capable of extending a primer 3’
terminus preceded by up to four mismatched bases.
Bottom left
This shows the selection scheme. Two independent aqueous compartments
of the water-in-oil emulsion are shown. Polymerases (such as Pol1 (left
compartment)) that are capable of utilizing quadruple mismatch primers
(AGGGAGGG, GGTGGGTG) to replicate their one encoding gene (pol1)
produce “offspring” i.e. increase their copy number in the post-selection
population, while polymerases like Pol2 (right compartment) that are unable to
utilize quadruple mismatch primers disappear from the gene pool
Bottom right
After three rounds of CSR selection, we recovered a diverse set of
polymerases with novel properties including the generic ability to utilize single,
double and quadruple mismatches (as seen in this picture for the polymerases
called 3D1). They could also process unusual DNA structures and bypass
template lesions such as abasic sites or hydantoins, as will be shown in the
following two slides.
Slide 7
Top left
We examined primer extension reactions using a radiolabelled primer and
resolved products on a polyacrylamide gel. We examined extension of three
different
quadruple
mismatches:
(M1:
GGTGGGTG,
M2:
AGGGAGGG)(used for selection), and the unrelated (M3: TTTTTTTT) and
compared it to extension of matched primer (M0). While Taq was unable to
extend any of the mismatches , theselected polymerases 3A10 and 3D1 yield
extension products with M1-3 but extension products are predominately
shorter than M0.
Top right and bottom
Possible primer template configurations and expected main product lengths
(N+1) are illustrated. Matched primer-template sequences (M0) at primer 3’
end are shown in blue, mismatched and misaligned structures are shown in
red.
Slide 8
Again we measured polymerase activity by examining the ability to extend a
radiolabelled primer.
Top left
On an undamaged template all three polymerases Taq and the selected
polymerases 3A10, 3D1 display approximately the same activity. To the left,
the chemical structure of the undamaged base T is shown.
Top right
This template contains an abasic site at the + 1 position (marked by a red
AP). To the right, the chemical structure of an abasic site is shown. Abasic
sites are among the most frequent forms of DNA damage and are generated
by spontaneous depurination or depyrimidination and as the end product of a
number of oxidation-induced DNA damage pathways.
As can be seen, while Taq polymerase can insert a nucleotide opposite the
abasic site (see arrow), it cannot bypass and remains stuck in the +1 position.
In contrast, the selected polymerases 3A10(and to a lesser extent 3D1) can
efficiently bypass the site of damage, inserting mostly an A opposite the absic
site.
Bottom left and right
These template contains hydantoins at the + 1 position (marked by a red 5H,
5M). Hydantoins are oxidative degradation products of the pyrimidine bases.
5-hydroxy-hydantoin derives from C and 5-methyl-5-hydroxy-hydantoin
derives from T. High levels of hydantoins have been found in some ancient
samples and associated with PCR failure. Their chemical structures are
shown to either side of the gel pictures.
These lesions show the same general picture. Taq polymerase can insert a
nucleotide opposite the hydantoins (see arrow), but bypasses them poorly 5hydroxy-hydantoin very poorly. The selected polymerases 3A10 also have
problems in bypassing the lesions but 3A10 is significantly better than Taq on
either lesion, inserting mostly an A in both cases.
Slide 9
The ability of 3A10 to bypass template damage is reflected in PCR
amplification. This shows a gel picture of the PCR amplification of DNA
containing 2 abasic sites. As can be seen while all natural polymerases such
as Taq but also Tth and Tfl fail to yield a PCR amplification product, 3A10
yields a strong amplification band. The other weaker bands are from other
selected polymerases.
Slide 10
Left panel
The ability of selected polymerases to efficiently bypass template lesions in
PCR encouraged us test their activity for the recovery of ancient DNA. We
performed subsequent experiments using a blend of Taq with the most
promising selected polymerases (3A10, 3D1 and others) (rather than testing
individual combinations) in order to minimize wastage of precious ancient
samples and maximize the chances of success. We first performed 56 PCR
amplifications at limiting dilutions of ancient DNA (aDNA) derived from a
47,000 year-old cave bear (Ursus spelaeus) bone and scored successful
amplifications for blend and Taq alone. We found that the blend yielded
amplification products at between 2 - 5-fold lower concentrations of aDNA
than Taq and indeed did yield amplification products at DNA concentrations,
where Taq no longer generated any
Right panel
Normalizing PCR activity on a dilution series of “modern” DNA showed that
this was not due to higher PCR efficiency of the blend. On the contrary, Taq
appeared to be more than an order of magnitude more active at low template
concentrations (of “modern” DNA), suggesting that the blend requires more
template than Taq to produce an equivalent PCR signal.
The increased template DNA requirement of the blend suggests that the
increased ability of the blend to amplify ancient DNA represents an
underestimate of the blend’s potential. Moreover, it implies that the blend can
tap into a pool of DNA molecules that are inaccessible to Taq, presumably
because they are damaged.
Slide 11
To stringently exclude sample heterogeneity and stochastic variation as the
source of the above effect, we performed a further 608 independent PCR
amplifications from two different samples of cave bear bone (~47,000 and
~60,000 years-old respectively), and scored the number of PCR amplicons at
limiting dilution. The blend yielded a larger number of amplicons (8-150%)
than Taq in all but one experiment, confirming previous results.
In conclusion, molecular breeding and directed evolution by CSR have
allowed the isolation of polymerases, which enhance the recovery of genetic
material from Pleistocene specimens, presumably due to their ability to
amplify damaged DNA.
Ice age genomics is upon us. Largely, thanks to novel sequencing methods,
such as the Roche /454 sequencer, which also utilize emulsion PCR,
Polymerases such as those described here should benefit the recovery of
ancient DNA and may speed up sequencing as they are pre-adapted to
emulsion PCR.
Polymerases capable of amplifying damaged DNA may also reduce bias
towards modern DNA contamination and enable novel applications in
palaeobiology, molecular archaeology and historic and forensic medicine.
The novel polymerases described here are really just a step in a direction, but
they show that we can use evolution to improve our molecular tools. Further
improvements should be within reach and hopefully will render ever more
ancient sequences readable to us.