Dr.
Sanyucta Kumari
Assistant Professor (Chemistry)
MLT College, Saharsa
DNA Polymerization
A DNA polymerase is a member of a family of enzymes that catalyze the synthesis
of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes
are essential for DNA replication and usually work in groups to create two identical DNA duplexes from
a single original DNA duplex. During this process, DNA polymerase "reads" the existing DNA strands to
create two new strands that match the existing ones. These enzymes catalyze the chemical reactio
deoxynucleoside triphosphate + DNAn ⇌ pyrophosphate + DNAn+1
DNA polymerase adds nucleotides to the three prime (3')-end of a DNA strand, one nucleotide at a time.
Every time a cell divides, DNA polymerases are required to duplicate the cell's DNA, so that a copy of
the original DNA molecule can be passed to each daughter cell. In this way, genetic information is
passed down from generation to generation
Before replication can take place, an enzyme called helicase unwinds the DNA molecule from its tightly
woven form, in the process breaking the hydrogen bonds between the nucleotide bases. This opens up
or "unzips" the double-stranded DNA to give two single strands of DNA that can be used as templates
for replication in the above reaction
History
In 1956, Arthur Kornberg and colleagues discovered DNA polymerase I (Pol I), in Escherichia coli. They
described the DNA replication process by which DNA polymerase copies the base sequence of a
template DNA strand. Kornberg was later awarded the Nobel Prize in Physiology or Medicine in 1959
for this work. DNA polymerase II was discovered by Thomas Kornberg (the son of Arthur Kornberg) and
Malcolm E. Gefter in 1970 while further elucidating the role of Pol I in E. coli DNA replication. Three
more DNA polymerases have been found in E. coli, including DNA polymerase III (discovered in the
1970's) and DNA polymerases IV and V (discovered in 1999)
Function
DNA polymerase moves along the old strand in the 3'–5' direction, creating a new
strand having a 5'–3' direction.
DNA polymerase with proofreading abilit
The main function of DNA polymerase is to synthesize DNA from deoxyribonucleotides, the building
blocks of DNA. The DNA copies are created by the pairing of nucleotides to bases present on each
strand of the original DNA molecule. This pairing always occurs in speci c combinations,
with cytosine along with guanine, and thymine along with adenine, forming two separate pairs,
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respectively. By contrast, RNA polymerases synthesize RNA from ribonucleotides from either RNA or
DNA
When synthesizing new DNA, DNA polymerase can add free nucleotides only to the 3' end of the newly
forming strand. This results in elongation of the newly forming strand in a 5'–3' direction
It is important to note that the directionality of the newly forming strand (the daughter strand) is opposite
to the direction in which DNA polymerase moves along the template strand. Since DNA polymerase
requires a free 3' OH group for initiation of synthesis, it can synthesize in only one direction by
extending the 3' end of the preexisting nucleotide chain. Hence, DNA polymerase moves along the
template strand in a 3'–5' direction, and the daughter strand is formed in a 5'–3' direction. This
difference enables the resultant double-strand DNA formed to be composed of two DNA strands that
are antiparallel to each other
The function of DNA polymerase is not quite perfect, with the enzyme making about one mistake for
every billion base pairs copied. Error correction is a property of some, but not all DNA polymerases.
This process corrects mistakes in newly synthesized DNA. When an incorrect base pair is recognized,
DNA polymerase moves backwards by one base pair of DNA. The 3'–5' exonuclease activity of the
enzyme allows the incorrect base pair to be excised (this activity is known as proofreading). Following
base excision, the polymerase can re-insert the correct base and replication can continue forwards.
This preserves the integrity of the original DNA strand that is passed onto the daughter cells
Fidelity is very important in DNA replication. Mismatches in DNA base pairing can potentially result in
dysfunctional proteins and could lead to cancer. Many DNA polymerases contain an exonuclease
domain, which acts in detecting base pair mismatches and further performs in the removal of the
incorrect nucleotide to be replaced by the correct one. The shape and the interactions accommodating
the Watson and Crick base pair are what primarily contribute to the detection or error. Hydrogen bonds
play a key role in base pair binding and interaction. The loss of an interaction, which occurs at a
mismatch, is said to trigger a shift in the balance, for the binding of the template-primer, from the
polymerase, to the exonuclease domain. In addition, an incorporation of a wrong nucleotide causes a
retard in DNA polymerization. This delay gives time for the DNA to be switched from the polymerase
site to the exonuclease site. Different conformational changes and loss of interaction occur at different
mismatches. In a purine:pyrimidine mismatch there is a displacement of the pyrimidine towards the
major groove and the purine towards the minor groove. Relative to the shape of DNA polymerase's
binding pocket, steric clashes occur between the purine and residues in the minor groove, and
important van der Waals and electrostatic interactions are lost by the pyrimidine. Pyrimidine:pyrimidine
and purine:purine mismatches present less notable changes since the bases are displaced towards the
major groove, and less steric hindrance is experienced. However, although the different mismatches
result in different steric properties, DNA polymerase is still able to detect and differentiate them so
uniformly and maintain delity in DNA replication. DNA polymerization is also critical for many
mutagenesis processes and is widely employed in biotechnologies
Structur
The known DNA polymerases have highly conserved structure, which means that their overall
catalytic subunits vary very little from species to species, independent of their domain structures.
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Conserved structures usually indicate important, irreplaceable functions of the cell, the maintenance of
which provides evolutionary advantages. The shape can be described as resembling a right hand with
thumb, nger, and palm domains. The palm domain appears to function in catalyzing the transfer
of phosphoryl groups in the phosphoryl transfer reaction. DNA is bound to the palm when the enzyme is
active. This reaction is believed to be catalyzed by a two-metal-ion mechanism. The nger domain
functions to bind the nucleoside triphosphates with the template base. The thumb domain plays a
potential role in the processivity, translocation, and positioning of the DNA
Processivit
DNA polymerase's rapid catalysis is due to its processive nature. Processivity is a characteristic of
enzymes that function on polymeric substrates. In the case of DNA polymerase, the degree of
processivity refers to the average number of nucleotides added each time the enzyme binds a template.
The average DNA polymerase requires about one second locating and binding a primer/template
junction. Once it is bound, a nonprocessive DNA polymerase adds nucleotides at a rate of one
nucleotide per second. Processive DNA polymerases, however, add multiple nucleotides per second,
drastically increasing the rate of DNA synthesis. The degree of processivity is directly proportional to the
rate of DNA synthesis. The rate of DNA synthesis in a living cell was rst determined as the rate of
phage T4 DNA elongation in phage infected E. coli. During the period of exponential DNA increase at
37 °C, the rate was 749 nucleotides per second
DNA polymerase's ability to slide along the DNA template allows increased processivity. There is a
dramatic increase in processivity at the replication fork. This increase is facilitated by the DNA
polymerase's association with proteins known as the sliding DNA clamp. The clamps are multiple
protein subunits associated in the shape of a ring. Using the hydrolysis of ATP, a class of proteins
known as the sliding clamp loading proteins open up the ring structure of the sliding DNA clamps
allowing binding to and release from the DNA strand. Protein-protein interaction with the clamp prevents
DNA polymerase from diffusing from the DNA template, thereby ensuring that the enzyme binds the
same primer/template junction and continues replication. DNA polymerase changes conformation,
increasing af nity to the clamp when associated with it and decreasing af nity when it completes the
replication of a stretch of DNA to allow release from the clamp
Variation across species
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DNA polymerase family A
c:o6-
methyl-guanine pair in the polymerase-2
basepair position
Identi ers
Symbol DNA_pol_A
Pfam PF00476
InterPro IPR001098
SMART -
PROSITE PDOC00412
SCOP2 1dpi / SCOPe / SUPFAM
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crystal
structure of rb69 gp43 in complex with dna
containing thymine glycol
Identi ers
Symbol DNA_pol_B
Pfam PF00136
Pfam clan CL0194
InterPro IPR006134
PROSITE PDOC00107
SCOP2 1noy / SCOPe / SUPFAM
DNA polymerase type B,
organellar and viral
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phi29
dna polymerase, orthorhombic crystal form,
ssdna complex
Identi ers
Symbol DNA_pol_B_2
Pfam PF03175
Pfam clan CL0194
InterPro IPR004868
Based on sequence homology, DNA polymerases can be further subdivided into seven different
families: A, B, C, D, X, Y, and RT
Some viruses also encode special DNA polymerases, such as Hepatitis B virus DNA polymerase.
These may selectively replicate viral DNA through a variety of mechanisms. Retroviruses encode an
unusual DNA polymerase called reverse transcriptase, which is an RNA-dependent DNA polymerase
(RdDp). It polymerizes DNA from a template of RNA
Types of
Fami DNA
Taxa Examples Feature
ly[16] polymer
ase
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Replicati
ve and Eukaryotic T7 DNA Two exonuclease
A Repair and polymerase, Pol domains (3'-5' and
Polymer Prokaryotic I, Pol γ, θ, and ν 5'-3')
ases
Replicati
3'-5 exonuclease
ve and Eukaryotic
Pol II, Pol B, Pol (proofreading); viral
B Repair and
ζ, Pol α, δ, and ε ones use protein
Polymer Prokaryotic
primer
ases
Replicati
ve 3'-5 exonuclease
C Prokaryotic Pol III
Polymer (proofreading)
ases
No "hand" feature,
Replicati
double barrel RNA
ve Euryarchae PolD (DP1/DP2
D polymerase-like; 3'-5
Polymer ota heterodimer)[17]
exonuclease
ases
(proofreading)
Replicati Pol β, Pol σ, Pol
template optional; 5'
ve and λ, Pol μ,
phosphatase (only Pol
X Repair Eukaryotic and terminal
β); weak "hand"
Polymer deoxynucleotidyl
feature
ases transferase
Replicati
ve and Eukaryotic Pol ι, Pol κ, Pol
Translesion
Y Repair and η,[18] Pol IV, and
synthesis[19]
Polymer Prokaryotic Pol V
ases
Replicati
Viruses, Ret
ve and
roviruses, Telomerase,
RT Repair RNA-dependent
and Hepatitis B virus
Polymer
Eukaryotic
ases
Prokaryotic polymeras
Prokaryotic polymerases exist in two forms: core polymerase and holoenzyme. Core polymerase
synthesizes DNA from the DNA template but it cannot initiate the synthesis alone or accurately.
Holoenzyme accurately initiates synthesis
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Pol
Prokaryotic family A polymerases include the DNA polymerase I (Pol I) enzyme, which is encoded by
the polA gene and ubiquitous among prokaryotes. This repair polymerase is involved in excision repair
with both 3'–5' and 5'–3' exonuclease activity and processing of Okazaki fragments generated during
lagging strand synthesis. Pol I is the most abundant polymerase, accounting for >95% of polymerase
activity in E. coli; yet cells lacking Pol I have been found suggesting Pol I activity can be replaced by the
other four polymerases. Pol I adds ~15-20 nucleotides per second, thus showing poor processivity.
Instead, Pol I starts adding nucleotides at the RNA primer:template junction known as the origin of
replication (ori). Approximately 400 bp downstream from the origin, the Pol III holoenzyme is assembled
and takes over replication at a highly processive speed and nature
Taq polymerase is a heat-stable enzyme of this family that lacks proofreading ability.[22]
Pol I
DNA polymerase II is a family B polymerase encoded by the polB gene. Pol II has 3'–5' exonuclease
activity and participates in DNA repair, replication restart to bypass lesions, and its cell presence can
jump from ~30-50 copies per cell to ~200–300 during SOS induction. Pol II is also thought to be a
backup to Pol III as it can interact with holoenzyme proteins and assume a high level of processivity.
The main role of Pol II is thought to be the ability to direct polymerase activity at the replication fork and
help stalled Pol III bypass terminal mismatches
P f u D N A p o l y m e r a s e i s a h e a t - s t a b l e e n z y m e o f t h i s f a m i l y f o u n d i n t h e
hyperthermophilic archaeon Pyrococcus furiosus.[24] Detailed classi cation divides family B in archaea
into B1, B2, B3, in which B2 is a group of pseudoenzymes. Pfu belongs to family B3. Others PolBs
found in archaea are part of "Casposons", Cas1-dependent transposons. Some viruses (including Φ29
DNA polymerase) and mitochondrial plasmids carry polB as well
Pol II
DNA polymerase III holoenzyme is the primary enzyme involved in DNA replication in E. coli and
belongs to family C polymerases. It consists of three assemblies: the pol III core, the beta sliding
clamp processivity factor, and the clamp-loading complex. The core consists of three subunits: α, the
polymerase activity hub, ɛ, exonucleolytic proofreader, and θ, which may act as a stabilizer for ɛ. The
beta sliding clamp processivity factor is also present in duplicate, one for each core, to create a clamp
that encloses DNA allowing for high processivity. The third assembly is a seven-subunit (τ2γδδ′χψ)
clamp loader complex
The old textbook "trombone model" depicts an elongation complex with two equivalents of the core
enzyme at each replication fork (RF), one for each strand, the lagging and leading. However, recent
evidence from single-molecule studies indicates an average of three stoichiometric equivalents of core
enzyme at each RF for both Pol III and its counterpart in B. subtilis, PolC. In-cell uorescent microscopy
has revealed that leading strand synthesis may not be completely continuous, and Pol III* (i.e., the
holoenzyme α, ε, τ, δ and χ subunits without the ß2 sliding clamp) has a high frequency of dissociation
from active RFs. In these studies, the replication fork turnover rate was about 10s for Pol III*, 47s for
the ß2 sliding clamp, and 15m for the DnaB helicase. This suggests that the DnaB helicase may remain
stably associated at RFs and serve as a nucleation point for the competent holoenzyme. In vitro single-
molecule studies have shown that Pol III* has a high rate of RF turnover when in excess, but remains
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stably associated with replication forks when concentration is limiting. Another single-molecule study
showed that DnaB helicase activity and strand elongation can proceed with decoupled, stochastic
kinetics
Pol I
In E. coli, DNA polymerase IV (Pol IV) is an error-prone DNA polymerase involved in non-targeted
mutagenesis.[30] Pol IV is a Family Y polymerase expressed by the dinB gene that is switched on via
SOS induction caused by stalled polymerases at the replication fork. During SOS induction, Pol IV
production is increased tenfold and one of the functions during this time is to interfere with Pol III
holoenzyme processivity. This creates a checkpoint, stops replication, and allows time to repair DNA
lesions via the appropriate repair pathway. Another function of Pol IV is to perform translesion
synthesis at the stalled replication fork like, for example, bypassing N2-deoxyguanine adducts at a
faster rate than transversing undamaged DNA. Cells lacking dinB gene have a higher rate of
mutagenesis caused by DNA damaging agents
Pol
DNA polymerase V (Pol V) is a Y-family DNA polymerase that is involved in SOS
response and translesion synthesis DNA repair mechanisms. Transcription of Pol V via the umuDC
genes is highly regulated to produce only Pol V when damaged DNA is present in the cell generating an
SOS response. Stalled polymerases causes RecA to bind to the ssDNA, which causes the LexA protein
to autodigest. LexA then loses its ability to repress the transcription of the umuDC operon. The same
RecA-ssDNA nucleoprotein posttranslationally modi es the UmuD protein into UmuD' protein. UmuD
and UmuD' form a heterodimer that interacts with UmuC, which in turn activates umuC's polymerase
catalytic activity on damaged DNA. In E. coli, a polymerase “tool belt” model for switching pol III with pol
IV at a stalled replication fork, where both polymerases bind simultaneously to the β-clamp, has been
proposed. However, the involvement of more than one TLS polymerase working in succession to
bypass a lesion has not yet been shown in E. coli. Moreover, Pol IV can catalyze both insertion and
extension with high ef ciency, whereas pol V is considered the major SOS TLS polymerase. One
example is the bypass of intra strand guanine thymine cross-link where it was shown on the basis of the
difference in the mutational signatures of the two polymerases, that pol IV and pol V compete for TLS of
the intra-strand crosslink
Family
In 1998, the family D of DNA polymerase was discovered in Pyrococcus furiosus and Methanococcus
jannaschii. The PolD complex is a heterodimer of two chains, each encoded by DP1 (small
proofreading) and DP2 (large catalytic). Unlike other DNA polymerases, the structure and mechanism
of the DP2 catalytic core resemble that of multi-subunit RNA polymerases. The DP1-DP2 interface
resembles that of Eukaryotic Class B polymerase zinc nger and its small subunit. DP1, a Mre11-like
exonuclease, is likely the precursor of small subunit of Pol α and ε, providing proofreading capabilities
now lost in Eukaryotes. Its N-terminal HSH domain is similar to AAA proteins, especially Pol III subunit
δ and RuvB, in structure. DP2 has a Class II KH domain.[17] Pyrococcus abyssi polD is more heat-
stable and more accurate than Taq polymerase, but has not yet been commercialized. It has been
proposed that family D DNA polymerase was the rst to evolve in cellular organisms and that the
replicative polymerase of the Last Universal Cellular Ancestor (LUCA) belonged to family D
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Eukaryotic DNA polymeras
Polymerases β, λ, σ, μ (beta, lambda, sigma, mu) and Td
Family X polymerases contain the well-known eukaryotic polymerase pol β (beta), as well as other
eukaryotic polymerases such as Pol σ (sigma), Pol λ (lambda), Pol μ (mu), and Terminal
deoxynucleotidyl transferase (TdT). Family X polymerases are found mainly in vertebrates, and a few
are found in plants and fungi. These polymerases have highly conserved regions that include two helix-
hairpin-helix motifs that are imperative in the DNA-polymerase interactions. One motif is located in the 8
kDa domain that interacts with downstream DNA and one motif is located in the thumb domain that
interacts with the primer strand. Pol β, encoded by POLB gene, is required for short-patch base
excision repair, a DNA repair pathway that is essential for repairing alkylated or oxidized bases as well
as abasic sites. Pol λ and Pol μ, encoded by the POLL and POLM genes respectively, are involved
in non-homologous end-joining, a mechanism for rejoining DNA double-strand breaks due to hydrogen
peroxide and ionizing radiation, respectively. TdT is expressed only in lymphoid tissue, and adds "n
nucleotides" to double-strand breaks formed during V(D)J recombination to promote immunological
diversity
Polymerases α, δ and ε (alpha, delta, and epsilon
Pol α (alpha), Pol δ (delta), and Pol ε (epsilon) are members of Family B Polymerases and are the
main polymerases involved with nuclear DNA replication. Pol α complex (pol α-DNA primase complex)
consists of four subunits: the catalytic subunit POLA1, the regulatory subunit POLA2, and the small and
the large primase subunits PRIM1 and PRIM2 respectively. Once primase has created the RNA primer,
Pol α starts replication elongating the primer with ~20 nucleotides. Due to its high processivity, Pol δ
takes over the leading and lagging strand synthesis from Pol α. Pol δ is expressed by genes POLD1,
creating the catalytic subunit, POLD2, POLD3, and POLD4 creating the other subunits that interact
with Proliferating Cell Nuclear Antigen (PCNA), which is a DNA clamp that allows Pol δ to possess
processivity. Pol ε is encoded by the POLE1, the catalytic subunit, POLE2, and POLE3 gene. It has
been reported that the function of Pol ε is to extend the leading strand during replication, while Pol δ
primarily replicates the lagging strand; however, recent evidence suggested that Pol δ might have a role
in replicating the leading strand of DNA as well. Pol ε's C-terminus "polymerase relic" region, despite
being unnecessary for polymerase activity, is thought to be essential to cell vitality. The C-terminus
region is thought to provide a checkpoint before entering anaphase, provide stability to the holoenzyme,
and add proteins to the holoenzyme necessary for initiation of replication. Pol ε has a larger "palm"
domain that provides high processivity independently of PCNA
Compared to other Family B polymerases, the DEDD exonuclease family responsible for proofreading
is inactivated in Pol α. Pol ε is unique in that it has two zinc nger domains and an inactive copy of
another family B polymerase in its C-terminal. The presence of this zinc nger has implications in the
origins of Eukaryota, which in this case is placed into the Asgard group with archaeal B3 polymerase
Polymerases η, ι and κ (eta, iota, and kappa
Pol η (eta), Pol ι (iota), and Pol κ (kappa), are Family Y DNA polymerases involved in the DNA repair by
translation synthesis and encoded by genes POLH, POLI, and POLK respectively. Members of Family
Y have ve common motifs to aid in binding the substrate and primer terminus and they all include the
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typical right hand thumb, palm and nger domains with added domains like little nger (LF),
polymerase-associated domain (PAD), or wrist. The active site, however, differs between family
members due to the different lesions being repaired. Polymerases in Family Y are low- delity
polymerases, but have been proven to do more good than harm as mutations that affect the
polymerase can cause various diseases, such as skin cancer and Xeroderma Pigmentosum Variant
(XPS). The importance of these polymerases is evidenced by the fact that gene encoding DNA
polymerase η is referred as XPV, because loss of this gene results in the disease Xeroderma
Pigmentosum Variant. Pol η is particularly important for allowing accurate translesion synthesis of DNA
damage resulting from ultraviolet radiation. The functionality of Pol κ is not completely understood, but
researchers have found two probable functions. Pol κ is thought to act as an extender or an inserter of
a speci c base at certain DNA lesions. All three translesion synthesis polymerases, along with Rev1,
are recruited to damaged lesions via stalled replicative DNA polymerases. There are two pathways of
damage repair leading researchers to conclude that the chosen pathway depends on which strand
contains the damage, the leading or lagging strand
Polymerases Rev1 and ζ (zeta
Pol ζ another B family polymerase, is made of two subunits Rev3, the catalytic subunit, and Rev7
(MAD2L2), which increases the catalytic function of the polymerase, and is involved in translesion
synthesis. Pol ζ lacks 3' to 5' exonuclease activity, is unique in that it can extend primers with terminal
mismatches. Rev1 has three regions of interest in the BRCT domain, ubiquitin-binding domain, and C-
terminal domain and has dCMP transferase ability, which adds deoxycytidine opposite lesions that
would stall replicative polymerases Pol δ and Pol ε. These stalled polymerases activate ubiquitin
complexes that in turn disassociate replication polymerases and recruit Pol ζ and Rev1. Together Pol ζ
and Rev1 add deoxycytidine and Pol ζ extends past the lesion. Through a yet undetermined process,
Pol ζ disassociates and replication polymerases reassociate and continue replication. Pol ζ and Rev1
are not required for replication, but loss of REV3 gene in budding yeast can cause increased sensitivity
to DNA-damaging agents due to collapse of replication forks where replication polymerases have
stalled
Telomeras
Telomerase is a ribonucleoprotein which functions to replicate ends of linear chromosomes since
normal DNA polymerase cannot replicate the ends, or telomeres. The single-strand 3' overhang of the
double-strand chromosome with the sequence 5'-TTAGGG-3' recruits telomerase. Telomerase acts like
other DNA polymerases by extending the 3' end, but, unlike other DNA polymerases, telomerase does
not require a template. The TERT subunit, an example of a reverse transcriptase, uses the RNA subunit
to form the primer–template junction that allows telomerase to extend the 3' end of chromosome ends.
The gradual decrease in size of telomeres as the result of many replications over a lifetime are thought
to be associated with the effects of aging
Polymerases γ, θ and ν (gamma, theta and nu
Pol γ (gamma), Pol θ (theta), and Pol ν (nu) are Family A polymerases. Pol γ, encoded by
the POLG gene, was long thought to be the only mitochondrial polymerase. However, recent research
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shows that at least Pol β (beta), a Family X polymerase, is also present in mitochondria. Any mutation
that leads to limited or non-functioning Pol γ has a signi cant effect on mtDNA and is the most common
cause of autosomal inherited mitochondrial disorders. Pol γ contains a C-terminus polymerase domain
and an N-terminus 3'–5' exonuclease domain that are connected via the linker region, which binds the
accessory subunit. The accessory subunit binds DNA and is required for processivity of Pol γ. Point
mutation A467T in the linker region is responsible for more than one-third of all Pol γ-associated
mitochondrial disorders. While many homologs of Pol θ, encoded by the POLQ gene, are found in
eukaryotes, its function is not clearly understood. The sequence of amino acids in the C-terminus is
what classi es Pol θ as Family A polymerase, although the error rate for Pol θ is more closely related to
Family Y polymerases. Pol θ extends mismatched primer termini and can bypass abasic sites by
adding a nucleotide. It also has Deoxyribophosphodiesterase (dRPase) activity in the polymerase
domain and can show ATPase activity in close proximity to ssDNA. Pol ν (nu) is considered to be the
least effective of the polymerase enzymes. However, DNA polymerase nu plays an active role
in homology repair during cellular responses to crosslinks, ful lling its role in a complex with helicase
Plants use two Family A polymerases to copy both the mitochrondrial and plastid genomes. They are
more similar to bacterial Pol I than they are to mamallian Pol γ
Reverse transcriptas
Retroviruses encode an unusual DNA polymerase called reverse transcriptase, which is an RNA-
dependent DNA polymerase (RdDp) that synthesizes DNA from a template of RNA. The reverse
transcriptase family contain both DNA polymerase functionality and RNase H functionality, which
degrades RNA base-paired to DNA. An example of a retrovirus is HIV. Reverse transcriptase is
commonly employed in ampli cation of RNA for research purposes. Using an RNA template, PCR can
utilize reverse transcriptase, creating a DNA template. This new DNA template can then be used for
typical PCR ampli cation. The products of such an experiment are thus ampli ed PCR products from
RNA
Each HIV retrovirus particle contains two RNA genomes, but, after an infection, each virus generates
only one provirus. After infection, reverse transcription is accompanied by template switching between
the two genome copies (copy choice recombination). From 5 to 14 recombination events per genome
occur at each replication cycle. Template switching (recombination) appears to be necessary for
maintaining genome integrity and as a repair mechanism for salvaging damaged genomes
Bacteriophage T4 DNA polymeras
Bacteriophage (phage) T4 encodes a DNA polymerase that catalyzes DNA synthesis in a 5’ to 3’
direction.[62] The phage polymerase also has an exonuclease activity that acts in a 3’ to 5’ direction, and
this activity is employed in the proofreading and editing of newly inserted bases. A phage mutant with a
temperature sensitive DNA polymerase, when grown at permissive temperatures, was observed to
undergo recombination at frequencies that are about two-fold higher than that of wild-type phage
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It was proposed that a mutational alteration in the phage DNA polymerase can stimulate template
strand switching (copy choice recombination) during replication
