Ab Initio Synthesis by DNA Polymerases

1. Authors
Nadezhda V. Zyrina, Valeriya N.
Antipova & Lyudmila A. Zheleznaya
Presented by:
Amna Jalil

2. Introduction
The polymerization of free nucleotides
into new genetic elements by DNA
polymerases in the absence of DNA,
called ab initio DNA synthesis
little known phenomenon

3. In general, template-independent
generation of genetic information by DNA
polymerases is a known process.
A number of error-prone DNA
polymerases efficiently incorporate
nucleotides in DNA lesions where
template information is absent.

4. Another instance of template-independent
nucleotide polymerization
is terminal deoxynucleotidyl transferase
like activity, achieved by adding dNTPs
to the 3′-OH terminus of a blunt-ended
duplex DNA substrate.

5. Ab initio synthesis takes place in the
absence of any added DNA.
Ab initio DNA synthesis, has been known
for 50 years, incontrovertible evidence was
only obtained in the last decade.

6. History of ab initio DNA synthesis
research
In a typical replication mode, initiation of
DNA synthesis requires:
 A template DNA strand and
 A primer
 A short oligonucleotide complementary to
the template DNA region with a free 3′-
OH terminus

7. • In the 1960 and 1970s it was shown that
some prokaryotic DNA polymerases are
capable of providing the de novo
synthesis of poly (dA-dT) and poly (dG)
poly (dC) without any added primer or
template DNA.

8. These studies were conducted with
partially purified preparations of
enzymes; the scientific community
assumed that this synthesis might be due
to contamination by DNA or other
enzymes and these data have therefore
not been given due attention.

9. 30 years later was it convincingly
demonstrated that highly purified
thermophilic DNA polymerases Tli and
Tth were able to synthesize about 50 kb
of DNA without any template and primer.
This phenomenon was called ‘creative’, or
ab initio DNA synthesis.

10. The possibility of DNA contamination in the
reaction mixture, which may serve as a primer
and/or template, was vigorously excluded.
The synthesized double-stranded DNAs had mainly:
 Short repetitive and palindromic sequences
 GC content was about 25%
The reaction conditions (temperature, ionic
strength, and pH) were extremely important for
this reaction

11. Based on these findings, Ogata
& Miura suggested that genetic
information might be created
directly by protein.

12. The primer/template-independent polymerization
appeared to proceed via two reactions
 Slow formation of 16–19-nt-long oligo(dA-T)
without primer/template
Rapid elongation of the oligo(d A-T) by self-priming

13. Ab initio synthesis in the presence
of restriction endonucleases
Ab initio DNA synthesis was extremely
enhanced if a thermostable restriction
endonuclease (Tsp509I, TspRI, etc.) was
added to the reaction with thermophilic
DNA polymerase (Vent, Bst and 9ºNm).

14. ◊ The high efficiency of this synthesis resulted
from the exponential amplification involving
digestion /elongation cycles.
◊ A longer DNA with numerous recognition sites
was digested to short fragments, and the short
fragments were used as seeds for elongation to
synthesize longer DNA.

15. Ab initio synthesis stimulated by
nicking endonucleases
Very intensive ab initio synthesis takes
place in the presence of nicking
endonuclease Nt.BspD6I
Similar to restriction endonucleases,
nicking endonucleases recognize a short
specific sequence in double stranded DNA
and cleave DNA at a fixed position relative
to the recognized sequence.

16. However, unlike restriction
endonucleases, nicking
endonucleases make a nick in
only one, predetermined DNA
strand.

17. • The macromolecular structure and the
characteristics of the sequence DNAs
synthesized in the presence of Nt.BspD6I
differed from those synthesized by DNA
polymerases alone or in the presence of
restriction endonucleases.
• Some of DNA molecules had a branched
structure.

18. The sequences of DNA were
represented mainly by non-palindromic
containing Nt.BspD6I
recognition site (GAGTC)

19. Ab initio synthesis stimulated by
DnaB helicase
New DNA molecules over 100 kbp long
can be synthesized without preexisting
matrices when helicase DnaB is added to a
reaction mixture with:
Thermophilic polymerases
Mesophilic polymerases

20. The synthesized double-stranded DNA had:
Single-stranded
stretches
A–T-rich
sequence
Highly repetitive
sequence

21. A hypothetical model of ab initio
DNA synthesis by DNA
polymerases
Initiation of ab initio DNA synthesis
remains a mystery.
The hypothetical mechanism of ab initio
DNA synthesis can be described with
several stages.

22. In the initial step, DNA polymerase
generates a pool of oligonucleotides with
random sequences.
At the following stage, oligonucleotides
with specific sequences, which can
‘facilitate’ their own replication, are
amplified.
Palindromic sequences are preferable
because they can form reversible hairpin
structures at their 3′-termini, thus priming
the DNA elongation

23. Very long DNA stretches may be
synthesized through multiple strand
displacement reactions on the 3′-termini
of formed hairpins
A restriction endonuclease may somehow
help DNA polymerase to select the
sequence to be synthesized.

25. However, these models do not entirely
explain how non-palindromic repeats
propagate.
The formation of hairpins in non
palindromic sequences is not possible
either in the middle or at the ends of the
molecule.

26. A possible functional role of ab
initio DNA synthesis
Tandem sequences consisting of short
repeats occur in all genomes.
A comparison of ab initio DNA sequences
with those of the known natural DNAs
revealed that very similar tandem repeats are
present in coding and non-coding regions.
This fact suggests that repeating sequences
were synthesized by DNA polymerase in a
template-independent manner.

27. Conclusions
The major problem of numerous nucleic
acid amplification methods is the
accumulation of non-specific products,
which hamper identification of specific
sequences.
This process may be a result of ab initio
DNA synthesisby thermophilic DNA
polymerases.

28. Besides PCR, some other nucleic acid
amplification techniques
Strand displacement amplification
Rolling circle amplification
Exponential amplification reaction
also hampered by non-specific synthesis

29.  However, the problem of nonspecific amplification still
remains unsolved.
 A new strategy was offered for isothermal DNA
amplification in the presence of nicking enzymes .
 It was based on the use of SSB proteins as inhibitors of
non-specific ab initio synthesis.
 One of the proteins, T4 gp32, almost completely
inhibited ab initio DNA synthesis.

30. The knowledge gained will increase our
understanding of how DNA polymerases
function and will also suggest future
research in molecular biology.
The results may be very useful to develop
techniques requiring fast and inexpensive
preparation of large amounts of DNA.