MINIREVIEW
Ab initio synthesis by DNA polymerases
Nadezhda V. Zyrina, Valeriya N. Antipova & Lyudmila A. Zheleznaya
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, Russia
Correspondence: Nadezhda V. Zyrina,
Institute of Theoretical and Experimental
Biophysics, RAS, 142290 Pushchino, Moscow
Region, Russia. Tel.: +7 4967 739421;
fax: +7 4967 330553;
e-mail: [email protected]
Received 14 August 2013; revised 29
September 2013; accepted 1 November
2013. Final version published online 27
November 2013.
DOI: 10.1111/1574-6968.12326
Editor: Olga Ozoline
MICROBIOLOGY LETTERS
Keywords
template/primer-independent DNA synthesis;
repetitive sequences; non-specific DNA
amplification.
Abstract
The polymerization of free nucleotides into new genetic elements by DNA
polymerases in the absence of DNA, called ab initio DNA synthesis, is a little
known phenomenon. DNA polymerases from prokaryotes can effectively synthesize long stretches of linear double-stranded DNA in the complete absence
of added primer and template DNAs. Ab initio DNA synthesis is extremely
enhanced if a restriction endonuclease or nicking endonuclease is added to the
reaction with DNA polymerase. The synthesized ab initio DNA have various
tandem repeats. Sequences similar to those of ab initio DNA products are
found in many natural genes. The significance of ab initio DNA synthesis is
that genetic information can be created directly by protein. The ab initio DNA
synthesis is considered a non-specific synthesis in various DNA amplification
techniques. In this review, we present the main studies devoted to this
phenomenon and introduce possible mechanisms of this synthesis from our
current knowledge.
Introduction
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 (Goodman, 2002). Interestingly,
some high-fidelity DNA polymerases are also able to
bypass DNA lesions in vitro (Hsu et al., 2004).
Another instance of template-independent nucleotide
polymerization is terminal deoxynucleotidyl transferaselike activity, achieved by adding dNTPs to the 3′-OH terminus of a blunt-ended duplex DNA substrate (Clark,
1988). Template-independent nucleotide polymerization
occurs upon template switching when DNA polymerases
synthesize a nascent strand of DNA from two discontinuous template strands (Clark, 1991; Garcia et al., 2004). In
this process, polymerases use the overhangs to juxtapose
two unlinked templates. The formation of these overhangs may be the result of template-independent nucleotide addition by DNA polymerases.
However, the highly efficient DNA synthesis discussed
in this review differs remarkably from the above described
examples. It takes place in the absence of any added
FEMS Microbiol Lett 351 (2014) 1–6
DNA. Although this phenomenon, called ab initio DNA
synthesis, has been known for 50 years, incontrovertible
evidence was only obtained in the last decade.
History of ab initio DNA synthesis
research
Initiation of DNA synthesis in a typical replication mode
requires a template DNA strand and a primer, a short oligonucleotide complementary to the template DNA region
with a free 3′-OH terminus. However, 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 (Schachman et al., 1960;
Okazaki & Kornberg, 1964; Burd & Wells, 1970). 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 (Nazarenko et al., 1979) and these data have
therefore not been given due attention. Only 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
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
2
primer (Ogata & Miura, 1997, 1998a). This phenomenon
was called ‘creative’, or ab initio DNA synthesis. 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,
and GC content was about 25%. The reaction conditions
(temperature, ionic strength, and pH) were extremely
important for this reaction (Ogata & Miura, 1998b). Based
on these findings, Ogata & Miura suggested that genetic
information might be created directly by protein.
At the same time, another group described the primer/
template-independent polymerization of dATP and dTTP
into poly(dA-T) by highly purified thermophilic DNA
polymerases Taq and Tth (Hanaki et al., 1997, 1998). The
5′–3′ exonuclease activity and the terminal deoxynucleotidyl transferase (TdT)-like activity seemed to be essential
for this synthesis. The primer/template-independent polymerization appeared to proceed via two reactions, the
slow formation of 16–19-nt-long oligo(dA-T) without
primer/template and the rapid elongation of the oligo(d
A-T) by self-priming. When the substrates were depleted,
DNA polymerases degraded the high-molecular-weight
polymer from the oligomers by their own exonuclease
activity. The authors proposed that the elongation and
the degradation reactions proceed simultaneously. More
detailed studies revealed that the majority of the ab initio
synthesized DNAs represented both repeated sequences
and short homologous blocks or randomly synthesized
sequences (Cheng & Calderon-Urrea, 2011).
Ab initio synthesis in the presence of
restriction endonucleases
The new type of ab initio DNA synthesis was found by
the Frank–Kamenetskii group (Liang et al., 2004). They
showed that 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).
The high efficiency of this synthesis resulted from the
exponential amplification involving digestion/elongation
cycles: a longer DNA with numerous recognition sites for
the restriction endonuclease was digested to short fragments, and the short fragments were used as seeds for
elongation to synthesize longer DNA. DNA was synthesized with a short lag period of 4 min and the synthesis
was almost complete in 1 h. More than 10 lg of DNA
was synthesized by 1 unit of DNA polymerase and the
yield of synthesized DNA was more than 90%! This reaction was faster than the ab initio DNA synthesis reported
by Ogata & Miura (with the yield of synthesized DNA of
2.2% after a lag period of 1 h) (Ogata & Miura, 1997,
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
N.V. Zyrina et al.
1998b). A clear relationship between the length of the
DNA molecules synthesized and the activity of endonuclease was observed (Liang et al., 2004). The synthesized
double-stranded DNA had a highly repetitive palindromic
sequence. Every repeating unit (motif) consisted of one
or two recognition sites for the restriction enzyme, separated by an additional small random sequence.
Later it was found that the ab initio synthesis could be
carried out at lower temperatures (4–37 °C) by combining non-thermophilic restriction endonuclease or nonspecific endonuclease DNAse I with the thermophilic
DNA polymerases (Liang et al., 2006). Moreover, the ab
initio synthesis by thermophilic DNA polymerases alone
[Vent, Vent (exo ) and Bst] was more efficient at lower
temperatures than at the optimal high temperatures
(Liang et al., 2007; N.V. Zyrina, unpublished data).
Ab initio synthesis stimulated by
nicking endonucleases
At approximately the same time we found that very
intensive ab initio synthesis takes place in the presence of
nicking endonuclease Nt.BspD6I (Fig. 1) (Zyrina et al.,
2007). Similar to restriction endonucleases, nicking endonucleases recognize a short specific sequence in doublestranded DNA and cleave DNA at a fixed position relative
to the recognized sequence. However, unlike restriction
endonucleases, nicking endonucleases make a nick in only
one, predetermined DNA strand. When nicking endonuclease Nt.BspD6I was added to a reaction mixture with
the large fragment of Bst DNA polymerase, DNA products of over 40 kb in an amount of about 10 lg were
synthesized, with the synthesis reaching a maximum
already after 1.5 h. Bst polymerase was used to carry out
template-independent DNA synthesis without nickase.
However, the amount of the product became clearly
detectable only after a reaction time of about 18 h.
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. The sequences of DNA were represented mainly by
non-palindromic, differently oriented tandem repeats
containing Nt.BspD6I recognition site (GAGTC) with the
only additional nucleotide being nucleotide A or T
(Fig. 2).
Efficient synthesis was also observed in the presence of
nicking endonucleases Nt.AlwI, Nb.BbvCI, and Nb.BsmI
(V.N. Antipova & N.V. Zyrina, unpublished data).
A surprising consistency became obvious: both restriction and nicking endonucleases strongly stimulate ab initio DNA synthesis, not only giving seeds for elongation
FEMS Microbiol Lett 351 (2014) 1–6
Ab initio synthesis by DNA polymerases
3
(a)
(b)
Fig. 1. Ab initio DNA synthesis stimulated by
Nt.BspD6I, 1-h incubation (adapted from
Zyrina et al., 2007). (a) Reaction products in
1% agarose gel. (b) The same products in
12% denaturing polyacrylamide gel. Top
numbers – activity of Nt.BspD6I; N – heatinactivated Nt.BspD6I (1 U); N1 – incubation
without Bst polymerase in the presence of 1 U
of Nt.BspD6I. The DNA size marker is shown
in bp.
Fig. 2. The sequences of DNA synthesized in the presence of
Nt.BspD6I (adapted from Zyrina et al., 2007). Color arrow, motif
orientation; circle, insertion; triangle, deletion.
but also somehow determining the sequence of a synthesized product.
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 (Bst, Tth,
Pfu) or mesophilic polymerases (T7 and Escherichia coli
polI Klenow fragment) (Kaboev & Luchkina, 2004). The
synthesized double-stranded DNA had single-stranded
stretches. The sequence was A–T-rich and highly repetitive.
Taken together, the data presented demonstrate convincingly that prokaryotic DNA polymerases are able to
synthesize repetitive and palindromic (or quasipalindromic) DNA sequences without preexisting matrices.
A hypothetical model of ab initio DNA
synthesis by DNA polymerases
The use of highly purified components by different
groups supports the hypothesis that endogenous priming
oligonucleotides are generated by DNA polymerase per se
FEMS Microbiol Lett 351 (2014) 1–6
without added primer and template DNAs (Ogata &
Miura, 1997; Liang et al., 2004; Cheng & Calderon-Urrea,
2011). However, the initiation of ab initio DNA synthesis
remains a mystery. Ogata & Miura (1997) proposed a
model in which amino-acid side chains of DNA polymerase, which normally interact with the single-stranded
region of a template and a primer, bind and utilize
dNTPs in a specific order to form discrete DNA
sequences. Ramadan and coworkers showed that the condensation of dNTPs to short oligonucleotides by DNA
polymerases does actually occur (Ramadan et al., 2004).
The hypothetical mechanism of ab initio DNA synthesis can be described with several stages. 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 (Fig. 3a, 1–4) (Ogata & Miura, 2000;
Ogata & Morino, 2000). The following stage is the DNA
elongation. Subsequent rounds of hairpin formation,
elongation and slippage lead to the propagation of the
palindromic sequences in the form of extended tandem
repeats (Fig. 3a, 3–3b). Very long DNA stretches may be
synthesized through multiple strand displacement
reactions on the 3′-termini of formed hairpins (Fig. 3a,
4–4a) (Liang et al., 2004). A restriction endonuclease
may somehow help DNA polymerase to select the
sequence to be synthesized. A partial digestion of the
repeats, which include palindromic sites recognized by
the endonuclease, leads to the formation of a pool of
shorter molecules serving as primers for DNA synthesis
(Fig. 3b).
These models are based on hairpin formation within 3′termini due to their palindromic nature. However, these
models do not entirely explain how non-palindromic
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
N.V. Zyrina et al.
4
(a)
(b)
(c)
Fig. 3. The hypothetical mechanisms of ab initio DNA synthesis. (a) Hairpin formation and elongation. (b) Digestion of one motif by a restriction
endonuclease. (c) DNA elongation and branching during ab initio DNA synthesis stimulated by nicking endonuclease. Numerals – processes;
arrow – motif orientation, color lines – complementary DNA strands; dashed line – elongating strand; black arrow –hydrolysis. Bold type indicates
a recognition site for restriction or nicking endonuclease.
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
FEMS Microbiol Lett 351 (2014) 1–6
Ab initio synthesis by DNA polymerases
repeats propagate. The formation of hairpins in nonpalindromic sequences is not possible either in the middle
or at the ends of the molecule. DNA elongation in this
case may result from the slippage synthesis in which a
repeat containing a loop forms and moves through the
whole DNA strand (Schlotterer & Tautz, 1992). However,
molecules need to remain linear after the slippage synthesis. The evidence of branched DNA molecules and the
presence of oppositely oriented motifs within a single
DNA molecule suggest an additional mechanism of DNA
elongation (Fig. 3c) (Zyrina et al., 2007). Oppositely
oriented motifs may result from the joining of the complementary ends of the displaced parental and nascent
strands (Fig. 3c, 1). Complementary ends are formed by
the TdT-like activity of polymerase. Differently oriented
motifs allow 3′-OH hairpin formation (Fig. 3c, 2).
Branched molecules appear as a result of intermolecular
hybridization when single-stranded molecules with oppositely oriented motifs accumulate (Fig. 3c, 3).
Of note, all these models consider ab initio DNA synthesis from the stage at which certain priming oligonucleotides for DNA synthesis are already present. They show
effective amplification of these oligonucleotides and the
formation of long DNA molecules. How priming oligonucleotides are formed remains unclear.
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 (Ogata & Miura, 1997; Liang et al., 2004; Cheng
& Calderon-Urrea, 2011). This fact suggests that repeating
sequences were synthesized by DNA polymerase in a template-independent manner.
Ab initio DNA synthesis by different enzymes polymerizing nucleic acids suggests that similar synthesis by primordial enzymes probably occurred early on Earth. It was
proposed that the pool of coding sequences that emerged
in the prebiotic world represented repeats of nucleotide
oligomers (Ohno, 1987). This hypothesis was further
developed in work concerned with ab initio DNA synthesis. Probably, primitive polypeptides with polymerase-like
activity synthesized DNAs consisting of simple repetitive
sequences (Ogata & Miura, 1998a). These molecules gradually ‘evolved’ into degenerate sequences during errorprone replication by primordial enzymes. Presumably, the
digestion of nucleic acids played an important role in
the early evolution of genetic material (Liang et al.,
2004). Apart from increasing the amplification efficiency,
restriction enzymes could serve as a factor in selectivity
FEMS Microbiol Lett 351 (2014) 1–6
5
and diversity of sequences. The polymerization and digestion reactions could be carried out by either a protein or
another functional molecule, thereby being providing the
foundations of life.
Conclusions
The works discussed in the review convincingly establish
ab initio DNA synthesis by DNA polymerases as the existing phenomenon. But what techniques can we apply these
data to?
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 synthesis
by thermophilic DNA polymerases. Ogata & Miura
(1997) found some DNA-like material during a PCR
experiment in a control reaction without added primer
and template DNAs. The utility of other nucleic acid
amplification techniques (strand displacement amplification, rolling circle amplification, exponential amplification
reaction, etc.) is also hampered by non-specific synthesis
(Chan et al., 2004; Ehses et al., 2005; Inoue et al., 2006;
Zyrina et al., 2007; Tan et al., 2008). One strategy for
suppressing or eliminating non-specific amplification is
based on improving the reaction conditions. However,
the problem of nonspecific amplification still remains
unsolved. Because a variety of amplification methods have
proven to be very useful for diagnostics, a new strategy
for eliminating non-specific amplification has yet to be
developed.
Such a new strategy was offered for isothermal DNA
amplification in the presence of nicking enzymes (Zyrina
et al., 2012). 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 by Bst polymerase alone or Bst polymerase
with nicking endonuclease and did not suppress the synthesis of a specific product.
The investigation of ab initio DNA synthesis raises
quite a number of interesting questions. 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.
Acknowledgements
The authors acknowledge Prof. Dr O.N. Ozoline for helpful
discussions on this paper and Mrs S.V. Sidorova for technical assistance. The work was supported by the Russian
Foundation for Basic Research, grant no. 12-04-01399_a.
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
6
References
Burd JF & Wells RD (1970) Effect of incubation conditions on
the nucleotide sequence of DNA products of unprimed
DNA polymerase reactions. J Mol Biol 53: 435–459.
Chan SH, Zhu Z, Van Etten JL & Xu SY (2004) Cloning of
CviPII nicking and modification system from chlorella virus
NYs-1 and application of Nt.CviPII in random DNA
amplification. Nucleic Acids Res 32: 6187–6199.
Cheng DW & Calderon-Urrea A (2011) Nontemplate
polymerization of free nucleotides into genetic elements by
thermophilic DNA polymerase in vitro. Nucleosides,
Nucleotides Nucleic Acids 30: 979–990.
Clark JM (1988) Novel non-templated nucleotide addition
reactions catalyzed by procaryotic and eucaryotic DNA
polymerases. Nucleic Acids Res 16: 9677–9686.
Clark JM (1991) DNA synthesis on discontinuous templates by
DNA polymerase I of Escherichia coli. Gene 104: 75–80.
Ehses S, Ackermann J & McCaskill JS (2005) Optimization and
design of oligonucleotide setup for strand displacement
amplification. J Biochem Biophys Methods 63: 170–186.
Garcia PB, Robledo NL & Islas AL (2004) Analysis of
non-template-directed nucleotide addition and template
switching by DNA polymerase. Biochemistry 43: 16515–16524.
Goodman MF (2002) Eror-Prone repair DNA polymerases in
prokaryotes and eukaryotes. Annu Rev Biochem 71: 17–50.
Hanaki K, Odawara T, Muramatsu T et al. (1997) Primer/
template-independent synthesis of poly d(A-T) by Taq
polymerase. Biochem Biophys Res Commun 238: 113–118.
Hanaki K, Odawara T, Nakajima N et al. (1998) Two different
reactions involved in the primer/template-independent
polymerization of dATP and dTTP by Taq DNA
polymerase. Biochem Biophys Res Commun 244: 210–219.
Hsu GW, Ober M, Carell T & Beese LS (2004) Error-prone
replication of oxidatively damaged DNA by a high-fidelity
DNA polymerase. Nature 431: 217–221.
Inoue J, Shigemori Y & Mikawa T (2006) Improvements of
rolling circle amplification (RCA) efficiency and accuracy
using Thermus thermophilus SSB mutant protein. Nucleic
Acids Res 34: e69.
Kaboev OK & Luchkina LA (2004) Template-free
primer-independent DNA synthesis by bacterial DNA
polymerases I using the DnaB protein from Escherichia coli.
Dokl Biochem Biophys 398: 265–267.
Liang X, Jensen K & Frank-Kamenetskii MD (2004) Very
efficient template/primer-independent DNA synthesis by
thermophilic DNA polymerase in the presence of a
thermophilic restriction endonuclease. Biochemistry 43:
13459–13466.
Liang X, Li BC, Jensen K & Frank-Kamenetskii MD (2006) Ab
initio DNA synthesis accelerated by endonuclease. Nucleic
Acids Symp Ser (Oxf) 50: 95–96.
Liang X, Kato T & Asanuma H (2007) Unexpected efficient ab
initio DNA synthesis at low temperature by using
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
N.V. Zyrina et al.
thermophilic DNA polymerase. Nucleic Acids Symp Ser
(Oxf) 51: 351–352.
Nazarenko IA, Bobko LE, Romashchenko AG, Khripin YL &
Salganik RI (1979) A study on the unprimed poly (dA-dT)
synthesis catalyzed by preparations of E. coli DNA
polymerase I. Nucleic Acids Res 6: 2545–2560.
Ogata N & Miura T (1997) Genetic information ‘created’ by
archaebacterial DNA polymerase. Biochem J 324(Pt 2):
667–671.
Ogata N & Miura T (1998a) a Creation of genetic information
by DNA polymerase of the thermophilic bacterium Thermus
thermophilus. Nucleic Acids Res 26: 4657–4661.
Ogata N & Miura T (1998b) Creation of genetic information
by DNA polymerase of the archaeon Thermococcus litoralis:
influences of temperature and ionic strength. Nucleic Acids
Res 26: 4652–4656.
Ogata N & Miura T (2000) Elongation of tandem repetitive
DNA by the DNA polymerase of the hyperthermophilic
Archaeon Thermococcus litoralis at a hairpin-coil transitional
state: a model of amplification of a primordial simple DNA
sequence. Biochemistry 39: 13993–14001.
Ogata N & Morino H (2000) Elongation of repetitive DNA
by DNA polymerase from a hyperthermophilic
bacterium Thermus thermophilus. Nucleic Acids Res 28:
3999–4004.
Ohno S (1987) Evolution from primordial oligomeric repeats
to modern coding sequences. J Mol Evol 25: 325–329.
Okazaki T & Kornberg A (1964) Enzymatic synthesis of
deoxyribonucleic acid. Xv. Purification and properties of a
polymerase from Bacillus subtilis. J Biol Chem 239:
259–268.
Ramadan K, Shevelev IV, Maga G & Hubscher U (2004) De
novo DNA synthesis by human DNA polymerase lambda,
DNA polymerase mu and terminal deoxyribonucleotidyl
transferase. J Mol Biol 339: 395–404.
Schachman HK, Adler J, Radding CM, Lehman IR & Kornberg
A (1960) Enzymatic synthesis of deoxyribonucleic acid.
J Biol Chem 235: 3242–3249.
Schlotterer C & Tautz D (1992) Slippage synthesis of simple
sequence DNA. Nucleic Acids Res 20: 211–215.
Tan E, Erwin B, Dames S et al. (2008) Specific versus
nonspecific isothermal DNA amplification through
thermophilic polymerase and nicking enzyme activities.
Biochemistry 47: 9987–9999.
Zyrina NV, Zheleznaya LA, Dvoretsky EV, Vasiliev VD,
Chernov A & Matvienko NI (2007) N.BspD6I DNA nickase
strongly stimulates template-independent synthesis of
non-palindromic repetitive DNA by Bst DNA polymerase.
Biol Chem 388: 367–372.
Zyrina NV, Artyukh RI, Svad’bina IV, Zheleznaya LA &
Matvienko NI (2012) The effect of single-stranded DNA
binding proteins on template/primer-independent DNA
synthesis in the presence of nicking endonuclease
Nt.BspD6I. Russ J Bioorg Chem 38: 171–176.
FEMS Microbiol Lett 351 (2014) 1–6