Paper No. : 04 Genetic Engineering and Recombinant DNA Technology
Module : 14 Evolution In Enzymology: DNA Polymerases: Klenow, Taq and T7
Principal Investigator: Dr Vibha Dhawan, Distinguished Fellow and Sr. Director The Energy and Resouurces Institute (TERI), New Delhi
Co-Principal Investigator: Prof S K Jain, Professor, of Medical Biochemistry Jamia Hamdard University, New Delhi
Paper Coordinator: Dr Mohan Chandra Joshi, Assistant Professor, Jamia Millia Islamia, New Delhi
Content Writer: Dr. Bhaswati Banerjee, Assistant Professor, Gautam Buddha University, Greater Noida, UP
Content Reviwer: Dr Mohan Chandra Joshi, Assistant Professor, Jamia Millia Islamia, New Delhi
Description of Module Subject Name Biotechnology
Paper Name Genetic Engineering and Recombinant DNA Technology
Module Name/Title
Evolution In Enzymology: DNA Polymerases: Klenow, Taq and T7 Module Id 14
Pre-requisites DNA Polymerase structure and functions
Objectives Gain comprehensive information about gradual advancement of DNA polymerases in RDT
Keywords DNA pol, Klenow, Taq, KlenTaq, T7, Sequenase
INDEX
_____________________________________________________________________________________________________________________________
LEARNING OBJECTIVES ABOUT THIS MODULE I. INTRODUCTION
A. Enzymes used in RDT
B. DNA Polymerases: A Timeline Study
II. KLENOWPOLYMERASE-THE TRUNCATED DNA POL I A. Background
B. Structural Properties and Mode of action C. Functional Properties
D. Applications
III. TAQ POLYMERASE-THE THERMOSTABLE DNA POLYMERASE A. Background
B. Enzyme Properties and Mode of action C. KlenTaq
D. Applications
IV. T7 DNA POLYMERASE-THE SEQUENASE A. Background
B. Enzyme Properties and Mode of action C. Applications
V. SUMMARY VI. REFERENCES
LEARNING OBJECTIVES
Enzymes used in Molecular Biology
Klenow Polymerase: Functions and Applications
Taq Polymerase: Functions and Applications
T7 Polymerase: Functions and Applications
ABOUT THIS MODULE
The focus of discussion of the present module is “evolution in enzymology”
pertaining to enzymes being used in Recombinant DNA Technology and Genetic Engineering. This module begins with comprehensive overview routinely used enzymes as molecular reagents and attempts to give an outline of enzymes used to generate and manipulate nucleic acids in vitro. Thereafter we focus on three important types of DNA polymerases routinely used in RDT: the Klenow polymerase, the Taq polymerase and the T7 polymerase. The structure, function, mode of action and applications of these enzymes are discussed in detail. A separate panel focusses on the principle of Polymerase Chain reaction. Important general applications are highlighted to wrap up the module.
I. INTRODUCTION
We shall begin this module by addressing a very obvious question: “What is Recombinant DNA Technology”?
In the simplest terms, Recombinant DNA technology is the combined application of all of the techniques involved in the construction, study and use of recombinant DNA molecules (Khan 2010; Brown 1989). Recombinant DNA molecule is created in vitro by ligating together two different pieces of DNA that are normally not contiguous and usually obtained from two different naturally occurring (sometimes synthetic) sources. Although recombination of DNA was discovered as a naturally occurring genetic phenomenon, the first artificial or engineered recombinant DNA molecule was created in 1973 by Stanley Cohen, H. Boyer and co-workers (Stetten & DeWitt Stetten 1979; Cohen 1985; Brown 2015)
Basics of RDT: As the name suggests, the cornerstone of recombinant DNA technology is preparation of Recombinant DNA molecules and all the techniques involved therein. Recombinant DNA is mostly prepared by molecular cloning technique in order to characterize certain genes or proteins. A generalized protocol for preparation of recombinant DNA involves following fundamental steps, each of which can be manipulated or modified to small or large extent as per requirements of the experiment (Brown 2011; Murphy et al. 1984; Nicholl 2008; Primrose & Twyman 2013).
1) The primary step includes identification of the target gene or DNA sequence to be characterized i.e. “gene of interest”, considering the requirements of the experiment and feasibility of cloning, which then is known as the “insert”. The insert could be sourced from viral, prokaryotic or eukaryotic origin.
2) Selection of an appropriate DNA vehicle as carrier molecule, or the
“vector” (also known as “cloning vector”) and identifying compatible restriction sites or cloning site where the insert could be cloned into the vector comes next. The vector could be viral or prokaryotic or eukaryotic in origin
3) The insert is then introduced into the vector to prepare the vector-insert rec-DNA construct or the “clone” by a three step method: cleaving out the insert from the donor genome by restriction digestion; cutting the vector with the same set of restriction enzymes, ligating the insert into the clone by DNA ligase.
4) The rDNA molecule thus synthesized is then introduced into the host organism by a method known as “transformation” and as the host cell multiplies, it also amplifies the rDNA by producing its multiple copies autonomously thus amplifying the gene of interest.
5) Positive transformants are then selected and allowed to amplify in vitro to produce the bulk copies of the gene of interest which is then isolated by
standard protocols to be used for further research and experimental purpose.
This protocol, although gives desired results, yet, it is quite time consuming and the time involved in this protocol may be greatly reduced by used by using in vitro DNA amplification technique such as PCR which utilizes the DNA polymerase enzymes.
It is quite obvious that a wealth of DNA modifying enzymes is routinely used in RDT to create a desired engineered rDNA. The designing and synthesis of rDNA includes various types of manipulations and modifications of DNA catalyzed by several classes of enzymes such as polymerases, nucleases, ligases, kinases, phosphatases and methylases (Primrose & Twyman 2013; Fairbanks 2004; Kendrew 2009).
A. Enzymes used in RDT
Before discussing the different DNA polymerases used in RDT, we shall have a glimpse of the important enzymes-the molecular tools- used in RDT (Fairbanks 2004; Worthington 1988; Eun 1996). As we talk about cleaving, rejoining and amplification of DNA to create an engineered DNA molecule, the enzyme that we think of first and foremost is obviously the Restriction endonuclease.
1) Restriction Endonuclease: This happens to be one of the most widely used molecular tool in RDT. Since their discovery in early 1950s by Arber, Nathans and Smith many types of restriction endonuclease enzymes have been discovered. An endonuclease is an enzyme that cleaves nucleotides within the nucleic acid molecule. Restriction endonuclease is the enzyme that recognizes only specific nucleotide sequences within the DNA (recognition site) and cuts the DNA only at specific nucleotide sequences (restriction/cleavage site) within the DNA, by breaking the phosphodiester bonds between the nucleotides.
2) DNA polymerase: An enzyme that synthesizes DNA by polymerising deoxynucleotides on a DNA template (sometimes on a RNA template in special cases) in RDT we mostly use the DNA pol-I (or a fragment thereof) which is a DNA-dependent DNA polymerase.
3) DNA ligase: The enzyme which joins compatible ends of DNA fragments by synthesizing a phosphodiester bond between adjacent nucleotides using energy from ATP hydrolysis
4) RNAse A: Nuclease that digests RNA instead of DNA
5) Exonuclease/Exonuclease III: The enzyme that cleaves DNA from the free ends of the molecule by progressive digestion of nucleotides in 3’ to 5’
direction.
6) DNA Mtase: This enzyme adds a methyl group to a specific nucleotide in DNA.
7) Terminal Transferase: This enzyme catalyses the addition of one or more deoxyribonucleotides to the 3' terminus of the DNA molecule
8) Alkaline Phosphatase: An enzyme that removes phosphate group from the 5’ end of a DNA fragment, thus preventing non-specific and unwanted re- ligation of DNA fragments
9) Polynucleotide Kinase: This enzyme adds a phosphate group to the free 5’
terminus of a double-stranded or single-stranded DNA or RNA in an ATP dependent reaction.
10) RNA polymerase: This enzyme synthesizes new RNA in a DNA templated manner.
RDT
Restric- Endonu-tion
cleases DNA
Polymer- ases
LigaseDNA
RNAse A
Exonuc- lease III MtasesDNA
Terminal Transfe-
rases Alkaline
Phosph- atases Polynuc-
leotide kinases
RNA pol
DNA Polymerase: The key enzymes capable of catalyzing synthesis of long chain polymers of deoxyribonucleotides in 5’ to 3’ direction in a DNA templated manner are known as DNA polymerases. Since the discovery of the DNA pol I in E. coli by Arthur Kornberg (1956), several other types of DNA polymerases have been discovered, identified and characterized in prokaryotes as well as eukaryotes (Kornberg et al.
1974; Anon 2010; Hübscher 1983). Irrespective of the origin or type of the DNA polymerase, two characteristic features are known to be consistent and conserved in all DNA pols known so far
Lack of de novo synthesis ability, hence obligatory requirement of a primer to begin DNA synthesis
5’3’ Polymerization: Synthesis or polymerization of DNA bases only in the 5’ to 3 ’direction
Most of the DNA polymerases used in RDT are obtained from the bacteria or the bacteriophage. In this small section we focus briefly on the important types of DNA Polymerases that are routinely used in molecular cloning and RDT (Brown 2011;
Brown 2015; Stetten & DeWitt Stetten 1979; Eun 1996).
Frequently Used DNA Polymerases:
DNA Poly- merase
DNA pol I
Klenow Fragment
Thermostable DNA polyme-
rases Phage DNA
polymerases Reverse
Transcri- ptase
1) DNA Pol I: E. coli DNA polymerase I (Pol I) is the enzyme that has DNA- dependent DNA polymerase activity as well as DNA nuclease activity. It is employed for nick sealing, 5'-3' Polymerase, 3'-5' Exonuclease and 5'-3' Exonuclease activity.
2) Klenow Polymerase (large fragment of the E. coli Pol I): Klenow is the large fragment obtained by mild proteolysis of E. coli DNA polymerase I; it exhibits DNA polymerase and DNA proofreading action but lacks 5'-3' Exonuclease activity; it is used mostly in chain termination DNA sequencing reactions.
3) Taq DNA Polymerase: The thermostable DNA polymerase obtained from, the thermophilic bacteria Thermus aquaticus; mainly used for DNA synthesis by PCR.
4) Pfu Polymerase: Pfu DNA polymerase is a superior thermostable proofreading DNA polymerase enzyme isolated from the hyperthermophilic archaeon Pyrococcus furiosus; which is a high fidelity DNA polymerase used for in vitro DNA amplification by PCR.
5) Reverse Transcriptase: RNA dependent DNA polymerase used to synthesize cDNA from the mRNA template.
6) T7 DNA Polymerase: ssDNA templated DNA polymerase with unique properties; its recombinant form is commercially available as “Sequenase”.
B. DNA Polymerases: A Timeline Study
This is a comprehensive overview of the series of events from where we began to where we have reached so that we understand where we are heading to (Fig 1.1).
The enormous field of molecular biology, which forms the foundation of GE and RDT was unlocked by the pathbreaking discovery of DNA pol I in E. coli by Arthur Kornberg et. al. in 1956. Later, in 1959, Kornberg was awarded the Nobel Prize for his discovery of E. coli DNA polymerase I. The amino acid composition of Pol I was elucidated by Jovin et al. in 1969 (Worthington 1988; Stetten & DeWitt Stetten 1979). This discovery eventually changed the face of modern biology and biotechnology.
In this module we discuss a few important types of DNA polymerase that are routinely used for various RDT techniques
II. KLENOW POLYMERASE-THE TRUNCATED DNA POL I A. Background
Escherichia coli DNA polymerase I (Pol I), one of three DNA polymerases found in E. coli, coded by the PolA gene, is the prototype DNA-dependent DNA polymerase.
DNA Pol I was the first bacterial DNA polymerase to be discovered and characterized by Arthur Kornberg in 1956. ž Pol I present in E. coli cells at a concentration of 400 molecules per cell is also the most abundantly expressed DNA polymerase in E. coli. Pol I functions in a self-regulated autonomous manner. The inherent functional features of Pol I include the following:
5′3′ polymerase activity
3′5′ exonuclease activity (for proofreading)
5′3′ exonuclease activity (for nick translation, excision repair, and hydrolysis of the RNA primers during DNA replication)
Its primary functions include filling gaps between adjacent Okazaki fragments, primer removal and proofreading during repair and replication. Pol I is known to actively participate during, DNA replication, repair and recombination and is also required for rapid, cell proliferation in rich media (Anon n.d.; Anon 2015a).
Fig. 1.1: Timeline of discovery of DNA Polymerases
Source: https://www.dna-worldwide.com/resource/160/history-dna-timeline
Due to its characteristic inherent features, Pol I is put to several important use in RDT , which include:
Removing 3′ protruding DNA ends (in the absence of dNTPs)
Filling in the cohesive ends (in the presence of dNTPs) before addition of molecular linkers
But the 5’3’ exonuclease activity (involved in removal of primers) often interferes with the protocols that require only the 5’3’ polymerase activity such as copying the single-stranded DNA in dideoxy method for sequencing.
As a part of detailed structural and functional characterization of DNA pol I, Klenow and Henningsen (1970) discovered that upon mild proteolitic digestion by the protease
Subtilisin, E. coli DNA Pol I (103 kDa) produced two fragments (Fig. 2.2A, 2.2B) (Klenow & Henningsen 1970):
a large (68 kDa) fragment known as the Klenow fragment that contains both the 5’3’ DNA polymerase and 3′5′ exonuclease (proofreading) activities
a smaller fragment (35 kDa) that contains the 5′3′ exonuclease activity The Klenow fragment was
shown to have the capacity to synthesize new DNA strand directed by the template DNA strand but it lacked the primer removal and nick translation ability.
Thus Klenow became the enzyme of choice for the dideoxy sequencing method invented by Sanger et al (1977) and later on for several other RDT tools and techniques.
Fig. 2.1: DNA pol I-Klenow fragment Source: Biochemistry| 6th edition
Fig. 2.3A: Ribbon diagram showing crystal structure of Klenow fragment Source: http://www.pnas.org/content/95/7/3402/F1.expansion.html
B. Structural Properties and Mode of Action
Klenow fragment was the first polymerase fragment whose 3-D structure was illustrated by X-ray crystallography. Structural and functional characterization of Klenow fragment paved the way for elucidation of structural, functional and biochemical features of DNA pol I and its mode of action as a polymerase.
Structure: Crystallographic analysis of the domain structure of Klenow fragment revealed that it is the large fragment (604 AA, 68kDa) of E. coli Pol I which corresponds to the C-terminal and the central domain of the native enzyme.
Functionally, the Klenow fragment exhibits 5’3’ polymerase and 3’5’
exonuclease activity (Klenow & Overgaard-Hansen 1970; Freemont et al. 1986) Crystal structure analysis of the Klenow fragment further reveals that in its three dimensional functionally active form Klenow resembles the shape of a human right hand where the regions resembling the fingers, palm and the thumb can be clearly identified (Fig. 2.3A, 2.3B) (Patel & Loeb 2002; Brautigam & Steitz 1998).
Mode of action: During the process of DNA polymerization, the Klenow-DNA complex is known to shuttle between the “editing mode” and the “polymerizing mode” (Brown & LiCata 2011). As the Klenow polymerase slides over the nascent DNA strand, nearly 5-8 base pairs of DNA get shifted into a large positively charged cleft for editing during which, the primer is first detached from the DNA template and then shifted to the exonuclease domain for 3’5’ exonucleolytic degradation (Fig.
2.4) (Beese et al. 1993; Beese et al. 1994).
Fig 2.3B: Structure of Klenow fragment-DNA complex Source: http://slideplayer.com/slide/5098545/
The Klenow-DNA complex
then undergoes
intramolecular
conformational change to
“polymerizing mode” thus allowing continuation of DNA synthesis. The palm- shaped subdomain of Klenow is thought to host the catalytically active polymerization site that mediates phosphoryl transfer (Mekbib Astatke et al. 1998). Klenow polymerase fragment catalyzes the DNA
template directed addition of mononucleotides from deoxynucleoside-5′- triphosphates (dNTPs) to the free 3′-OH terminus of a primer/template DNA. Due to its inherent mechanism of action, Klenow is employed in synthesis of DNA complementary to single-stranded DNA templates (Beese et al. 1993; Wang &
Beese 2011).
Further studies have also established that a single side chain in the Klenow fragment prevents mis-incorporation of rNTPs in the nascent DNA strand (M. Astatke et al.
1998).
The “small fragment” released by mild proteolytic digestion of E. coli Pol I is a 323 aa, 35 kDa fragment that corresponds to the N-terminal domain of the native enzyme and exhibits 5’3’ Exonuclease activity. It is involved in primer removal and nick translation during DNA polymerization by the native enzyme and degradation of misincorporated DNA and RNA to monomers during DNA repair.
C. Functional Properties
Processivity: Klenow fragment shows moderate processivity. The average rate of incorporation of mono-nucleotides by Klenow fragment is 50 nucleotides per second (Beaussire & Pochet 1999; Spratt 1997; Benkovic & Cameron 1995)
Reverse Transcriptase activity: Klenow fragment has a unique functional feature.
It has been shown that Klenow polymerase may also acts as a reverse transcriptase by copying RNA templates into DNA in a distributive manner. During this RNA dependent DNA polymerization, it incorporates one to two nucleotides per binding event. However, the biological relevance of this activity is yet to be established (Ben- Mahrez et al. 1991; Bao & Cohen 2004).
Fidelity: Klenow polymerase is a DNA dependent DNA polymerase by function, which catalyzes the polymerization with fidelity approximately 1/50,000. In other
Fig: 2.4: Proofreading mechanism of Klenow fragment Source: https://www.slideshare.net/ar_shad/dna-replication-and-pcr
words, in the event of DNA polymerization by Klenow, there occurs one mis- incorporation per 50,000 nucleotides. It is believed that 35′ editing activity of Klenow contributes to its fidelity (Beaussire & Pochet 1999; Spratt 1997; Benkovic &
Cameron 1995; van Dierendonck n.d.; Kuchta et al. 1988).
D. Applications
Due to its inherent mechanism of 5’3’ polymerase action and lack of 5’3’
exonuclease activity, Klenow is widely employed in synthesis of DNA complementary to single-stranded DNA templates thus synthesizing double stranded DNA from single stranded template.
This forms the basis of Klenow being an enzyme of choice in DNA sequencing and is predominantly used in sequencing of DNA according to Sanger et al. by the dideoxy- chain terminator method.
Other important applications in recombinant DNA technology where Klenow fragment is used are as follows:
Synthesizing blunt DNA ends after restriction digestion: It accomplishes blunt ending of restricted DNA fragments by Removal of 3’ overhangs and fill-in of 5’ overhangs created by restriction enzymes
For partial or complete filling up of 3´recessed ends (after restriction enzyme digestion)
Digestion of protruding 3’ overhangs to produce blunt end
Incorporating radiolabeled nucleotides onto 3′ DNA ends
For random primed labeling
Synthesizing the second strand on cDNA
Fill-in reactions of gaps in double-stranded DNA (Nick translation)
Elongation of oligonucleotides during the site-directed mutagenesis technique
Conversion of 3’-recessed ends of restricted DNA fragments to blunt ends by fill-in
3’-end-labeling of DNA fragments with -32P deoxynucleotides
To end label the double-stranded oligonucleotides during electrophoretic mobility-shift assay (EMSA).
III. TAQ POLYMERASE-THE THERMOSTABLE DNA POLYMERASE The progress in the discipline of molecular biology and evolution of recombinant DNA Technology is starred by a number of landmark experiments and path-breaking discoveries. One such phenomenal invention is that of the PCR or the Polymerase Chain Reaction technique. A comprehensive discourse about this PCR technique is given at the end of this section of the present module.
A. Background
PCR is a technique used to rapidly amplify a gene or a segment of a gene using repeated cycles of in vitro DNA synthesis. When the polymerase chain reaction (PCR) was originally developed by an American scientist Kary Mulis and his coworkers in 1983, the only enzyme which was being used for in vitro DNA synthesis and sequence extension was the Klenow fragment derived from the E. coli DNA Polymerase I (Mullis et al. 2012). But, like most other known enzymes at that time, the Klenow was a thermolabile enzyme that got denatured at the elevated temperatures required for PCR . Hence there was a need to replenish the enzyme before each replication and extension cycle which made the process tedious and cumbersome (Bustin 2009).
This problem was resolved only with the knowledge and application of the thermostable DNA polymerase. The first major breakthrough in this direction had come earlier when Thomas Brock and Hudson Freeze (1969) discovered a thermophilic archaebacterium thriving at extremely elevated temperatures in the hot springs and thermal vents in the Yellowstone National Park, USA (Fig. 3.1) and named it Thermus aquaticus (T. aquaticus) (Brock 1978; Brock & Freeze n.d.). As such, it was presumed that the various enzymes present in such bacteria would be resistant to elevated temperatures (temperatures greater than 45 °C). In the subsequent years, several thermostable enzymes were purified from T. aquaticus.
Chien and his coworkers (1976) isolated and characterized one of the first thermostable DNA polymerase from the T. aquaticus which was shown to retain its functional activity at elevated temperatures they named as the Taq polymerase (Chien A n.d.).
Fig. 3.1: Original habitat and source of Thermus aquaticus Source: Life at High Temperatures by Thomas D. Brock
Fig. 3.2: Crystal structure of Taq Polymerase http://www.ncbi.nlm.nih.gov/pubmed/8717047
Eventually, Taq polymerase was one of the first thermostable DNA polymerase that was successfully used to eliminate the need to replenish enzyme after each denaturation cycle in PCR. Randall Saiki was the first scientist to employ the thermostable Taq polymerase for in vitro DNA synthesis by polymerase chain reaction. It was established that the Taq polymerase retained its functional activity at above average survival temperatures and that its halflife was nearly 40 minutes at 95C and nearly 5 minutes at 100C. This feature allowed the use of Taq polymerase as the in vitro DNA synthesizing enzyme for PCR protocols (Innis et al. 1995; Bartlett
& Stirling 2003).
B. Enzyme Properties and Mode of Action
The extensive use of Taq polymerase as the enzyme of choice for routine PCR technique may be attributed to its exceptional thermal stability and high resistance to denaturation at elevated temperatures. The structural and functional properties of Taq polymerase have been illustrated and documented through a series investigative studies including cloning, overexpression, crystallography etc. While Taq pol shows certain striking similarities with E. coli DNA pol I in terms of sequence and structure, yet, Taq has certain unique features in terms of enzyme functionality, character and dependencies which differentiate Taq pol from Pol I and make Taq the ideal polymerase for use in PCR and qPCR-based gene expression analysis. Here we summarize the important structural features of Taq polymerase and then proceed to discuss its domain structure (Urs et al. 1995; Anon 2015b; Jones 1995).
Domain Structure, Function and Fidelity:
Cloning and characterization of Taq pol has established close resemblance of Taq pol with E. coli Pol I. These two polymerases show similar domain structure and organization in both the primary and the tertiary structural forms (Lawyer FC n.d.). Taq pol is also a single subunit DNA dependent DNA polymerase enzyme that has a full length of 832 amino acids and molecular weight of 94 kDa.
Taq pol in its purified full length form shows a single subunit structure with two distinct domains exhibiting distinct functions. Similar to DNA pol I, the C- terminal of Taq is associated with the 5′3′ polymerase activity while the N- terminal domain of Taq exhibits 5′3′ exonuclease activity (Huang & Li 2009; Lawyer FC n.d.).
Taq polymerase altogether lacks the 3′5′ exonuclease (proofreading) function associated with the central domain of E. coli. DNA pol I. Thus Taq pol performs DNA polymerization with a relatively moderate fidelity as it is not equipped to excise polymerization errors incurred during synthesis (Lawyer FC n.d.)
Functionally, Taq polymerase can synthesize DNA in 5’3’ direction only and it lacks the ability of de novo DNA synthesis; thus it requires a DNA
strand extension and achieve DNA templated DNA synthesis (Lawyer FC n.d.).
Taq DNA polymerase exhibits a modest fidelity and is reported to polymerize dNTPs with an error rate between 1 × 10−4 and 2 × 10−5 errors per base pair, depending on experimental conditions. In routine PCR experiments, Taq is known to incur 1 error in 9000 nucleotides. It is estimated that during Taq mediated PCR, error rate of Taq pol is one error per 400 bases after 25 cycles (Lawyer FC n.d.; Eckert & Kunkel 1990; Tindall & Kunkel 1988)
Taq achieves its fidelity by the contribution of two main components:
o Discrimination against nucleotide misinsertion and o Discrimination against extension of the misinserted base
In effect, Taq shows selective preference for extending only properly matched primers and correctly paired polynucleotide sequences. The extension of incorrectly paired nucleotides by Taq occurs at a very low efficiency of 10-3 for a T-G mispair to 10-6 for A-A mispair. Further, it has also been reported that the error rate by Taq pol is approximately 10-5 for base substitution errors and 10-6 for frameshift errors (Tindall & Kunkel 1988; Elias & Andrés Cisneros 2014).
All these properties together account for the fact that Taq pol is the most obvious choice for in vitro analysis of DNA sequences and is frequently used to detect specific in vivo mutations (Eckert & Kunkel 1990).
Another distinguishing feature of Taq DNA polymerase is its high thermostability and resistance to thermal denaturation. Biochemical analyses have revealed that Taq pol loses only 10% of its activity upon 30 minutes of incubation at 72°C and it loses 50% activity after 30 minute incubation at 95°C. Owing to the temperature dependency of Taq, its optimal functional activity is observed at 80C, unlike other enzymes whose optimal activity occurs at 37C. Taq polymerase is reported to lose half of its enzyme activity after 40 minutes of incubation at 95°C and after 5 minutes of incubation at 100°C. However, this activity span is sufficient to allow Taq pol to be used for PCR reaction, where its activity is optimized by presence of Mg2+ and a temperature of 80°C (Eckert & Kunkel 1990; Lawyer FC n.d.)
Mode of Action: Taq polymerase has been well characterized and its crystal structure has been elucidated by X-ray crystallography. Taq polymerase, in its three dimensional tertiary structure reportedly resembles the conventional “human right hand cupped structure”, a property that is common and typical for all the known DNA polymerases characterized so far. Crystal structure analyses reveal that the site for binding the single stranded-template is located in the subdomains corresponding to the fingers, binding site for the incoming dNTP is associated with the subdomain corresponding to the palm like region and the thumb shaped subdomain hosts the binding site for the dsDNA (Fig. 3.3).
Further, it is also reported that the polymerase subdomains of Taq pol shows high degree of homology with that of the Klenow fragment and have 51% amino acid similarity. A significant deviation of the Taq from the Klenow fragment is in the fact that the 3’-5’ exonuclease domain of Taq is inactive and lacks four loops present in the Klenow fragment on one side of its 3′-5′ exonuclease site (Xu et al. 2014; Huang
& Li 2009; Lawyer FC n.d.) C. KlenTaq
Klentaq is the Klenow analog of Taq DNA polymerase. Taq pol is susceptible to proteolytic cleavage similar to DNA pol I. Upon mild protease digestion the native Taq DNA polymerase yields two distinct fragments. The smaller fragment corresponding to the N-terminal region contains the site for 5’3’ exonuclease activity.
The larger fragment corresponding to the C-terminal subdomain of Taq pol, that hosts the site for 5’3’ polymerase action is the Taq counterpart of the Klenow fragment derived from DNA pol I. Thus it is named as the “KlenTaq”or the Stoffel fragment (Marx et al. 2010; Marx et al. 2013; Wu 2015a).
Biochemical evidences indicate that KlenTaq is far more thermostable compared to the native Taq pol and it loses only 10% activity after 30 min incubation at 95 °C.
Further, KlenTaq polymerase is reported to replicate non-canonical base pairs by inducing a Watson-Crick geometry. Thus, KlenTaq proves to be a more efficient enzyme to achieve DNA amplification by polymerase chain reactions (PCR) and
Fig. 3.3: 3-D structure of Taq polymerase-DNA complex Source: https://hubpages.com/education/What-Is-Polymerase-Chain-
Reaction-PCR
incubations at elevated temperatures and incorporation of abnormal bases (Wu 2015b)
D. Applications
Taq is a thermostable DNA dependent DNA polymerase which finds extensive applications in the Polymerase Chain reactions (PCR) which happens to be a frequently used technique in RDT and Genetic Engineering. Most of the research and industrial applications of Taq polymerase are based on its thermostability and certain unique properties.
1) Taq pol applications in PCR: PCR is a technique for in vitro amplification of genetic material, by rapidly creating multiple copies of a gene or segment of DNA using repeated cycles of in vitro DNA synthesis. PCR includes three fundamental steps: denaturation of the dsDNA template which requires extremely elevated temperature, annealing of the primer to the exposed ssDNA template which occurs at lower temperature and strand extension by the DNA polymerase. Prior to characterization and use of Taq in PCR, the process was tedious and inefficient as the E. coli DNA pol I which was used for the PCR technique was thermolabile and needed to be replenished at the beginning of every replicative cycle.
However, with the advent of Taq pol, the PCR became much simplified and seamless process, as a result several advanced versions and diverse applications of PCR could be conceptualized (Mullis et al. 2012; Erlich 1989).
Fig. 3.3: Polymerase-DNA interactions
Source: http://www.licatalab.biology.lsu.edu/andy/DNABindingEDIT.htm
Denaturing - at 95C the H-bonds denature and dsDNA template is
opened up to provide single stranded
template
Annealing - from 45-68C the primers attach to
the homologous DNA sequence that they match Extension - Taq
polymerase begins adding
nucleotides around 72C and
extending the DNA sequence.
Fig. 3.4: Schematic representation of PCR steps Source: http://slideplayer.com.br/slide/10903472
Kary Mullis together with Michael Smith invented the path breaking technique of PCR in 1983 for which they later shared the 1993 Nobel Prize in Chemistry. But it was Randall Saiki who for the first time in 1986, employed the thermostable Taq DNA polymerase to amplify DNA segments, thus giving the PCR the mechanism that we know today. This invention revolutionized molecular biology and forensics. PCR allows amplification of trace quantities of DNA into significant amounts that can be used for experimentation or for forensic testing. Template used in PCR can be any form of DNA, and only a single molecule of DNA is needed to generate millions of copies.
PCR is based on two basic normal cellular activities:
Binding of complementary strands of DNA, and
Replication of DNA molecules by DNA polymerases
2) PCR (and the advanced,modified versions) itself finds numerous applications in molecular biology. PCR is employed both in research and clinical investigations and industrial applications for wide range of purposes including, DNA amplification, DNA sequencing, forensic diagnostics, disease diagnostics, detection of pathogen (specially virus), detection of in vivo mutations through PCR etc [Panel-1](Biolabs 2014) 3) Taq pol used in DNA sequencing: The native Taq polymerase
discriminates against extending a DNA sequence with a misincorporated base or non-canonical base. Thus native Taq pol could not be used in Sanger’s dideoxy method for DNA sequencing as the native Taq pol incorporates dideoxynucleotide (ddNTP) with much less efficiency compared to dNTP. But with synthesis of mutated form of Taq, known as Taq Sequenase (Thermo Sequenqse) by a single substitution of substitution of Phe667Tyr in Taq pol active site, efficiency of incorporation of ddNTP by Taq was increased many folds. Thermo Sequenase is known to make “cycle sequencing” possible. It also enabled producing accurate sequences that could be analyzed by using either radioactive or fluorescent sequencing technologies (Brow 1990)
4) Taq Pol is used in T/A Cloning: Taq polymerase exhibits a deoxynucleotidyl transferase activity by which it adds a few non-templated nucleotides (usually Adenine residues) onto the the blunt 3′-end of PCR products. This property of Taq pol accounts for frequent use of Taq pol in routine molecular cloning techniques to achieve specific ligation of DNA fragments by T/A cloning protocols (Landgraf & Wolfes n.d.; Biolabs 2014). The PCR amplification of the “insert” with Taq results in synthesis of large amount of synthetic DNA with oligo-A overhangs at the 3’
terminals of both the DNA strands. When this PCR product is allowed to ligate with a linearized vector bearing 3’-T overhangs, a more specific ligation is mediated by the DNA ligase, resulting in a circular vector-insert DNA construct (Gál & Kálmán n.d.; Guo & Bi n.d.)
IV. T7 DNA POLYMERASE
DNA synthesis catalyzed by DNA polymerases is a fundamental phenomenon in terms of both in vivo processes and in vitro techniques. This happens to be one of the most investigated biological processes and almost every component involved in DNA replication has been well characterized. DNA polymerase is the master enzyme driving this process in all organisms bearing a DNA genome; as such, DNA polymerase has been a favorite with early researchers. T7 DNA polymerase is the third member of the Family A DNA polymerases, the other two members being E.
coli DNA pol I and Taq DNA polymerase (Coen n.d.; Carroll & Benkovic 1990) which we have already discussed. It is well accepted that bacteriophage T7 provides an excellent biological system to be used as a model for studying DNA synthesis
under regulated experimental conditions, that has contributed substantially in elucidating individual steps. T7 DNA polymerase is considered to be a prototypical polymerase for characterization of the structure and function of DNA polymerase and deciphering its biochemical properties (Brown 2015; Anon 1989; Fuller 1992). Due to its flexibility in terms of incorporating desired manipulations and modifications, T7 DNA pol finds wide applications as a molecular reagent in DNA sequencing and other experiments in Recombinant DNA Technology and Genetic Engineering.
DNA sequencing enzyme|desired features
Sanger’s DNA sequencing method, also known as the Chain-terminator or Dideoxy sequencing method is by far the most frequently used method for DNA sequencing. The DNA polymerase to be used for chain-terminatior DNA sequencing, should essentially possess the following properties (Primrose & Twyman 2013):
Ability to use 2′,3′-dideoxynucleotides as substrates: The polymerase of choice must have the ability to incorporate dideoxy- and other modified nucleotides at an efficiency similar to that for the cognate deoxynucleotides;
High processivity polymerase
High fidelity in the absence of proofreading/exonuclease activity: The polymerase must be able to faithfully and completely synthesize a complementary copy of a single-stranded DNA template.
Ability to produce clear and uniform signals for detection
The DNA polymerase encoded by bacteriophage T7 is naturally endowed with or can be engineered to have all these characteristics (Richardson 1993; Fuller et al. n.d.) Most of the DNA polymerases characterized till date are known to possess the 5′→3′
exonuclease and/or the 3′→5′ exonuclease activities in addition to their polymerase activity. But both these properties are undesirable and detrimental to DNA sequencing by most of the prevalent methods. The polymerase originally used by Sanger’s dideoxy method was the Klenow fragment that was derived by proteolytically removing the 5’3’ exonuclease domain of E. coli Pol I. The major disadvantages were the presence of 3′→5′ exonuclease activity which would hydrolyze the single-stranded sequencing primers and its inherent property to discriminate against dideoxynucleotides (ddNTPs) for corresponding canonical nucleotides (dNTPs) in a sequence-dependent manner. The method was then rendered inefficient and quite expensive due to requirement of very high concentrations of ddNTPs.
Much of these problems were resolved with the use of the native Taq pol in place of Klenow fragment as Taq pol naturally lacked the 3′→5′ exonuclease activity or by the use of KlenTaq fragment which naturally lacked both the 5’3’ exonuclease and 3′→5′ exonuclease activities. However the problem of discrimination against the ddNTPs still existed.
All these obstacles were ruled out with the discovery and characterization of the DNA polymerase from the bacteriophage T7. T7 DNA polymerase provided two immediate advantages:
Absence of the complete 5’3’ exonuclease domain
Lack of inherent ability to discriminate against the ddNTPs for dNTPs.
Upon further characterization, it was illustrated that this feature of non-discrimination was a result of difference in a single amino acid, tyrosine against phenylalanine.
Klenow and Taq pol are known to have Phenylalanine in their active site while T7 DNA pol contains Tyrosine in its active site which greatly reduces discrimination against ddNTPs. Further, T7 DNA Pol offered a wide scope for manipulations and modifications to obtain the DNA polymerase of precise and desired properties.
With a series of technical refinements Sanger’s DNA sequencing method has been greatly improvised and in the present day, DNA sequencing with great precision, high quality and throughput is obtained by this technique, especially when the improvements derived from 35S-labeled precursors and T7 DNA polymerase are exploited
A. Background
In the year 1971, two researchers from the Harvard Medical School, Pasquale Grippo and Charles C. Richardson identified a novel DNA polymerase of phage origin, in a mutant E. coli infected with T7 bacteriophage (Grippo & Richardson 1971). Preliminary biochemical analyses established the T7 DNA pol as a DNA templated DNA polymerase which required single stranded DNA, dNTPs and Mg2+ to be activated (Richardson 1983; Masker & Richardson 1976; Kolodner &
Richardson 1977). It was also documented that similar to E. coli DNA pol, the T7 DNA pol possesses strong 5’3’ polymerase and 3’5’ exonuclease activity. But a striking difference between the two was conspicuous absence of 5’-3’
exonuclease activity in the T7 pol unlike E. coli DNA pol I.
With further characterization and detailed investigation, certain unique and intriguing properties were observed. It was revealed that the although the structural gene for the T7 DNA pol was mapped to the gene 5 of the T7 genome, but the individual product expressed from the gene 5, known as the gp5 (84kDa), visibly lacked any DNA polymerase activity but exhibited only 3’-5’ ssDNA exonuclease activity (Hori et al. 1979b; Lee et al. 2009; Tamanot et al. 1980). It was logically speculated that the T7 DNA pol secreted by the T7 bacteriophage required a “host-component”
to reconstitute the T7 DNA polymerase holoenzyme. This “host factor’ was identified as a small 12 kDa E. coli redox protein called “Thioredoxin” (Fig. 4.1) (Mark &
Richardson 1976) (Knippers et al. 1973; Richardson 1983; Mark & Richardson 1976). Another striking revelation was that the Thioredoxin does not participate in the catalytic function of DNA polymerase through its redox capacity. T7 gp5 contributes the catalytic function to the holoenzyme while the thioredoxin plays a largely
structural role. It essentially stabilizes the binding of gene 5 protein to a primer- template and thus increases the processivity of the polymerase more than 100- fold (Bedford et al. 1997; Tabor S n.d.).
Thus the chemically modified or the genetically engineered T7 DNA polymerase enzyme (Sequenase) revolutionized the process of DNA sequencing technology and heralded the
“Sequenase Era” (Fuller 1992; Venkitaraman 1989).
B. Enzyme Properties and Mode of Action
Enzyme Complex: T7 DNA polymerase also requires certain other accessory proteins for its DNA polymerizing action (Lee et al. 2009; Crampton & Richardson 2003). The important ones discovered so far are as follows (Fig. 4.2):
Host thioredoxin
Gp4: acts as the helicase and primase for T7 DNA polymerase
Gp2.5: functions as the single strand binding protein
Gp1.7: Nucleoside monophosphate kinase
Gp6:
Structural and Functional Properties:
Fig. 4.1: Crystal structure of T7 DNA replication complex Source:https://en.wikipedia.org/wiki/T7_DNA_polymerase
T7 DNA pol is equipped with extraordinary processivity for DNA polymerization as a holoenzyme i.e., after the T7 gp5 associates with the E.
coli Thioredoxin (Bedford et al. 1997)
T7 DNA pol exhibits strong 3’-5’ Exonuclease activity by which the incorrectly paired bases are removed from the newly synthesized DNA strand. This accounts for the fidelity of DNA synthesis by T7 DNA polymerase (Grippo &
Richardson 1971)
A notable functional property of this phage polymerase is the fact that it exhibits exonuclease activity on both single stranded and double stranded DNA. It is also known that presence of thioredoxin is essential for the double- stranded DNA exonuclease activity of T7 DNA pol (Hori et al. 1979a).
T7 DNA pol lacks the inherent 5′–3′ exonuclease activity found in E. coli DNA polymerase I (Grippo & Richardson 1971)
Mode of Action: Putting it all together, it was confirmed that T7 DNA polymerase is an extraordinary polymerase with certain distinct features desirable for distinct applications. On account of its unique structural and functional properties, T7 DNA pol offers promising options for incorporating targeted biochemical modifications.
The high processivity and high fidelity of T7 DNA pol owing to its 3’5’
exonuclease activity are desirable features for PCR. However, 3’5’ exonuclease activity is detrimental to DNA sequencing protocols, especially the “chain termination method” of DNA sequencing. On the other hand, the inherent absence of discrimination against ddNTPs by T7 and its ability to incorporate dNTP and ddNTP with similar efficiency make it the most obvious choice as the polymerase for DNA sequencing by Sanger’s dideoxy method (Fuller et al. n.d.; Dürre &
Geriscker n.d.; Fuller 1995).
Fig. 4.2: Phage T7-DNA replication machinery Source: https://en.wikipedia.org/wiki/T7_DNA_polymerase
With further biochemical and biophysical investigations, it was revealed that the exonuclease activity of T7 DNA polymerase could be specifically inactivated by an oxidation reaction in presence of ferrous ion and the biochemically modified T7 DNA pol (Sequenase) retained other desirable features of T7 DNA pol (Fuller 1992; Tabor
& Richardson 1987). This ruled out the only hindrance against using T7 DNA pol as the DNA sequencing polymerase, which otherwise is naturally equipped with high processivity and lack of discrimination against incorporating ddNTPs.
Thus the chemically modified or genetically engineered recombinant T7 DNA polymerase complexed with Thioredoxin, commercially available by the name
“Sequenase” with extraordinary processivity, revolutionized the progress of DNA sequencing technology and marked the onset of Sequenase Era (Fuller 1992; Fuller et al. n.d.)
C. Applications
The easily modifiable exonuclease and extraordinary processivity of T7 DNA polymerase piloted the Sequenase Era; a powerful tool in the DNA sequencing Important applications of T7 DNA polymerase are as follows:
1) Strand extensions in site directed mutagenesis
2) T7 DNA polymerase provides a biological model to study interactions within a replisome
3) The modified T7 DNA pol, the Sequenase is widely used for DNA sequencing by Sanger’s dideoxy method
4) As T7 DNA pol is a ssDNA templated DNA polymerase, it is employed for second strand synthesis of cDNA
5) It is possible to automate DNA sequencing by replacing radioactive labels with fluorescent labels
V. SUMMARY
DNA replication – the first step in “Central Dogma” is the single most essential process for growth and proliferation of any organism
DNA polymerases catalyze the biosynthesis of DNA, a vital process for all living organisms and a process central to multiple in vitro techniques used in GE and RDT
Family A DNA polymerases include E. coli DNA polymerase I, Taq DNA polymerase and T7 DNA polymerase
Identification and isolation of these enzymes have served as prototypes for biochemical and structural studies on DNA
polymerases which are being widely used as molecular reagents
These have a pivotal role in the very foundation of RDT and Genetic
engineering and have opened up many unexplored dimensions, such as
gene cloning, DNA sequencing, PCR technology, disease detection and
diagnostics and forensic diagnostics etc.
VI. REFERENCES
Anon, 1989. 4795699 T7 DNA polymerase. Biotechnology advances, 7(2), p.249.
Anon, 2015a. DNA polymerase I (Pol I; Kornberg enzyme, Kornberg polymerase, DNA-dependent DNA polymerase I; EC 2.7.7.7). In The Dictionary of Genomics, Transcriptomics and Proteomics. pp. 1–1.
Anon, 2010. DNA Polymerases in the Three Kingdoms of Life: Bacteria, Archaea and Eukaryotes. In DNA Polymerases. pp. 59–83.
Anon, DNA Replication, Prokaryotes. In SpringerReference.
Anon, 2015b. Thermus aquaticus(Taq) DNA polymerase (Taqpolymerase, TaquenaseTM; EC 2.7.7.7). In The Dictionary of Genomics, Transcriptomics and Proteomics. pp. 1–1.
Astatke, M. et al., 1998. A single side chain prevents Escherichia coli DNA polymerase I (Klenow fragment) from incorporating ribonucleotides. Proceedings of the National Academy of Sciences, 95(7),
pp.3402–3407.
Astatke, M., Grindley, N.D.F. & Joyce, C.M., 1998. How E. coli DNA polymerase I (klenow fragment) distinguishes between deoxy- and dideoxynucleotides. Journal of molecular biology, 278(1), pp.147–
165.
Bao, K. & Cohen, S.N., 2004. Reverse transcriptase activity innate to DNA polymerase I and DNA
topoisomerase I proteins of Streptomyces telomere complex. Proceedings of the National Academy of Sciences of the United States of America, 101(40), pp.14361–14366.
Bartlett, J.M.S. & Stirling, D., 2003. PCR Protocols, Springer Science & Business Media.
Beaussire, J.-J. & Pochet, S., 1999. Recognition of 2′-Deoxyisoinosine Triphosphate by the Klenow Fragment of DNA Polymerase I. Nucleosides and Nucleotides, 18(3), pp.403–410.
Bedford, E., Tabor, S. & Richardson, C.C., 1997. The thioredoxin binding domain of bacteriophage T7 DNA polymerase confers processivity on Escherichia coli DNA polymerase I. Proceedings of the National Academy of Sciences, 94(2), pp.479–484.
Beese, L., Derbyshire, V. & Steitz, T., 1993. Structure of DNA polymerase I Klenow fragment bound to duplex DNA. Science, 260(5106), pp.352–355.
Beese, L.S., Friedman, J.M. & Steitz, T.A., 1994. CRYSTAL STRUCTURES OF THE KLENOW
FRAGMENT OF DNA POLYMERASE I COMPLEXED WITH DEOXYNUCLEOSIDE TRIPHOSPHATE AND PYROPHOSPHATE. Available at: http://dx.doi.org/10.2210/pdb1kfd/pdb.
Benkovic, S.J. & Cameron, C.E., 1995. [20] Kinetic analysis of nucleotide incorporation and misincorporation by klenow fragment of Escherichia coli DNA polymerase I. In Methods in Enzymology. pp. 257–269.
Ben-Mahrez, K. et al., 1991. Reverse transcriptase in archaebacteria. Purification and characterization of a primase - reverse-transcriptase complex from Halobacterium halobium. European journal of
biochemistry / FEBS, 195(1), pp.157–162.
Biolabs, N.E., 2014. PCR with Taq DNA Polymerase (M0273) v1. protocols.io. Available at:
http://dx.doi.org/10.17504/protocols.io.ch7t9m.
Brautigam, C.A. & Steitz, T.A., 1998. DNA POLYMERASE I KLENOW FRAGMENT (E.C.2.7.7.7) MUTANT/DNA COMPLEX. Available at: http://dx.doi.org/10.2210/pdb1ksp/pdb.
Brock, T.D. & Freeze, H., Thermus aquaticus gen. n. and sp. n., a nonsporulating extreme thermophile. - PubMed - NCBI. Available at: https://www.ncbi.nlm.nih.gov/pubmed/5781580 [Accessed July 30, 2017].
Brow, M.A.D., 1990. SEQUENCING WITH Taq DNA POLYMERASE. In PCR Protocols. pp. 189–196.
Brown, H.S. & LiCata, V.J., 2011. Time Dependence of Switching Between Polymerization and Editing Mode DNA Binding by Klenow Polymerase. Biophysical journal, 100(3), p.74a.
Brown, T., 2011. Gene Cloning and Colony Picking. Genetic Engineering & Biotechnology News, 31(19), pp.22–23.
Brown, T.A., 2015. Gene Cloning and DNA Analysis: An Introduction, John Wiley & Sons.
Brown, T.A., 1989. Genetics: a molecular approach, American Book Company.
Bustin, S.A., 2009. The PCR Revolution: Basic Technologies and Applications, Cambridge University Press.
Carroll, S.S. & Benkovic, S.J., 1990. Mechanistic aspects of DNA polymerases: Escherichia coli DNA polymerase I (Klenow fragment) as a paradigm. Chemical reviews, 90(7), pp.1291–1307.
Chien A, E. al, Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. - PubMed - NCBI. Available at: https://www.ncbi.nlm.nih.gov/pubmed/8432 [Accessed July 30, 2017].
Coen, D.M., MODEL OF RB69 DNA POLYMERASE WITH T7 DNA POLYMERASE PRIMER/ TEMPLATE.
Available at: http://dx.doi.org/10.2210/pdb1b1f/pdb.
Cohen, A., 1985. Cloned DNA as a Substrate of Bacterial Recombination System. In Recombinant DNA Research and Viruses. pp. 59–72.
Crampton, D.J. & Richardson, C.C., 2003. Bacteriophage T7 gene 4 protein: A hexameric DNA helicase. In The Enzymes. pp. 277–302.
van Dierendonck, J.H., DNA Damage Detection Using DNA Polymerase I or its Klenow Fragment:
Applicability, Specificity, Limitations. In In Situ Detection of DNA Damage. pp. 81–108.
Dürre, P. & Geriscker, U., Dideoxy Sequencing Reactions Using T7 Polymerase. In DNA Sequencing Protocols. pp. 91–102.
Eckert, K.A. & Kunkel, T.A., 1990. High fidelity DNA synthesis by theThermus aquaticusDNA polymerase.
Nucleic acids research, 18(13), pp.3739–3744.
Elias, A.A. & Andrés Cisneros, G., 2014. Computational Study of Putative Residues Involved in DNA Synthesis Fidelity Checking in Thermus aquaticus DNA Polymerase I. In Advances in Protein Chemistry and Structural Biology. pp. 39–75.
Erlich, H.A., 1989. Polymerase Chain Reaction,
Eun, H.-M., 1996. Enzymology Primer for Recombinant DNA Technology, Academic Press.
Fairbanks, S.D., 2004. Cloning: Chronology, Abstracts and Guide to Books, Nova Publishers.
Freemont, P.S. et al., 1986. A domain of the klenow fragment ofEscherichia coli DNA polymerase I has polymerase but no exonuclease activity. Proteins: Structure, Function, and Genetics, 1(1), pp.66–73.
Fuller, C.W., 1992. [29] Modified T7 DNA polymerase for DNA sequencing. In Methods in Enzymology. pp.
329–354.
Fuller, C.W. et al., DNA Sequencing Using Sequenase Version 2.0 T7 DNA Polymerase. In Basic DNA and RNA Protocols. pp. 373–388.
Fuller, C.W., 1995. Modified T7 DNA Polymerase for DNA Sequencing. In Recombinant DNA Methodology II. pp. 93–117.
Gál, J. & Kálmán, M., Autosticky PCR: Directional Cloning of PCR Products with Preformed 5’ Overhangs.
In PCR Cloning Protocols. pp. 141–151.
Grippo, P. & Richardson, C.C., 1971. Deoxyribonucleic acid polymerase of bacteriophage T7. The Journal of biological chemistry, 246(22), pp.6867–6873.
Guo, B. & Bi, Y., Cloning PCR Products: An Overview. In PCR Cloning Protocols. pp. 111–119.
Hori, K., Mark, D.F. & Richardson, C.C., 1979a. Deoxyribonucleic acid polymerase of bacteriophage T7.
Characterization of the exonuclease activities of the gene 5 protein and the reconstituted polymerase.
The Journal of biological chemistry, 254(22), pp.11598–11604.
Hori, K., Mark, D.F. & Richardson, C.C., 1979b. Deoxyribonucleic acid polymerase of bacteriophage T7.
Purification and properties of the phage-encoded subunit, the gene 5 protein. The Journal of biological chemistry, 254(22), pp.11591–11597.
Huang, Q. & Li, Q., 2009. Characterization of the 5′ to 3′ nuclease activity of Thermus aquaticus DNA polymerase on fluorogenic double-stranded probes. Molecular and cellular probes, 23(3-4), pp.188–
194.
Hübscher, U., 1983. DNA polymerases in prokaryotes and eukaryotes: Mode of action and biological implications. Experientia, 39(1), pp.1–25.
Innis, M.A., Gelfand, D.H. & Sninsky, J.J., 1995. PCR Strategies, Academic Press.
Jones, M.D., 1995. Reverse Transcription of mRNA by Thermus aquaticus DNA Polymerase followed by Polymerase Chain Reaction Amplification. In Recombinant DNA Methodology II. pp. 707–713.
Kendrew, J., 2009. Encylopaedia of Molecular Biology, John Wiley & Sons.
Khan, R., 2010. A Textbook of Biotechnology Volume-I Genetics and Molecular Biology, Laxmi Publications.
Klenow, H. & Henningsen, I., 1970. Selective elimination of the exonuclease activity of the deoxyribonucleic acid polymerase from Escherichia coli B by limited proteolysis. Proceedings of the National Academy of Sciences of the United States of America, 65(1), pp.168–175.
Klenow, H. & Overgaard-Hansen, K., 1970. Proteolytic cleavage of DNA polymerase fromEscherichia ColiB into an exonuclease unit and a polymerase unit. FEBS letters, 6(1), pp.25–27.
Knippers, R., Strätling, W. & Krause, E., 1973. Some Biochemical Elements in Bacteriophage T7 DNA Replication in Vitro. In DNA Synthesis in Vitro. pp. 451–461.
Kolodner, R. & Richardson, C.C., 1977. Replication of duplex DNA by bacteriophage T7 DNA polymerase and gene 4 protein is accompanied by hydrolysis of nucleoside 5’-triphosphates. Proceedings of the National Academy of Sciences, 74(4), pp.1525–1529.
Kornberg, T., Gefter, M.L. & Molineux, I.J., 1974. Escherichia coli DNA Polymerases II and III. In Mechanism and Regulation of DNA Replication. pp. 1–12.
Kuchta, R.D., Benkovic, P. & Benkovic, S.J., 1988. Kinetic mechanism whereby DNA polymerase I
Landgraf, A. & Wolfes, H., Taq Polymerase (EC 2.7.7.7): With Particular Emphasis on Its Use in PCR Protocols. In Enzymes of Molecular Biology. pp. 31–58.
Lawyer FC, E. al, Isolation, characterization, and expression in Escherichia coli of the DNA polymerase gene from Thermus aquaticus. - PubMed - NCBI. Available at:
https://www.ncbi.nlm.nih.gov/pubmed/2649500 [Accessed July 30, 2017].
Lee, S.-J. et al., 2009. Rescue of Bacteriophage T7 DNA Polymerase of Low Processivity by Suppressor Mutations Affecting Gene 3 Endonuclease. Journal of virology, 83(17), pp.8418–8427.
Mark, D.F. & Richardson, C.C., 1976. Escherichia coli thioredoxin: a subunit of bacteriophage T7 DNA polymerase. Proceedings of the National Academy of Sciences, 73(3), pp.780–784.
Marx, A., Diederichs, K. & Obeid, S., 2013. Snapshot of the large fragment of DNA polymerase I from Thermus Aquaticus processing modified pyrimidines. Available at:
http://dx.doi.org/10.2210/pdb4elv/pdb.
Marx, A., Diederichs, K. & Obeid, S., 2010. Snapshots of the large fragment of DNA polymerase I from Thermus Aquaticus processing C5 modified thymidines. Available at:
http://dx.doi.org/10.2210/pdb3ojs/pdb.
Masker, W.E. & Richardson, C.C., 1976. Bacteriophage T7 deoxyribonucleic acid replication in vitro.
Journal of molecular biology, 100(4), pp.557–567.
Mullis, K.B., Ferre, F. & Gibbs, R.A., 2012. The Polymerase Chain Reaction, Springer Science & Business Media.
Murphy, F.A., Brown, F. & Obijeski, J.F., 1984. Genetic Engineering Technology in Vaccine Production and Control of Animal Virus Diseases. In Applied Virology. pp. 17–30.
Nicholl, D.S.T., 2008. An Introduction to Genetic Engineering, Cambridge University Press.
Patel, P.H. & Loeb, L.A., 2002. DNA Polymerase I and Klenow Fragment. In Encyclopedia of Molecular Biology.
Primrose, S.B. & Twyman, R., 2013. Principles of Gene Manipulation and Genomics, John Wiley & Sons.
Richardson, C.C., 1983. Bacteriophage T7: Minimal requirements for the replication of a duplex DNA molecule. Cell, 33(2), pp.315–317.
Richardson, C.C., 1993. Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing; Progress report, June 1, 1990--May 31, 1993, Available at:
http://dx.doi.org/10.2172/142503.
Spratt, T.E., 1997. Klenow Fragment−DNA Interaction Required for the Incorporation of Nucleotides Opposite Guanine andO6-Methylguanine†. Biochemistry, 36(43), pp.13292–13297.
Stetten, H.D. & DeWitt Stetten, H., 1979. THE EARLY HISTORY OF THE RECOMBINANT DNA MOLECULE PROGRAM ADVISORY COMMITTEE OF NIH. In Recombinant DNA and Genetic Experimentation. pp. 157–160.
Tabor S, E. al, Escherichia coli thioredoxin confers processivity on the DNA polymerase activity of the gene 5 protein of bacteriophage T7. - PubMed - NCBI. Available at:
https://www.ncbi.nlm.nih.gov/pubmed/3316214 [Accessed July 30, 2017].
Tabor, S. & Richardson, C.C., 1987. Selective oxidation of the exonuclease domain of bacteriophage T7 DNA polymerase. The Journal of biological chemistry, 262(32), pp.15330–15333.
Tamanot, F. et al., 1980. REPLICATION OF BACTERIOPHAGE T7 DNA. In Mechanistic Studies of DNA Replication and Genetic Recombination. pp. 411–428.
Tindall, K.R. & Kunkel, T.A., 1988. Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase.
Biochemistry, 27(16), pp.6008–6013.
Urs, U.K. et al., 1995. Characterization of crystals of the thermostable DNA polymerase I fromthermus aquaticus. Proteins: Structure, Function, and Genetics, 23(1), pp.111–114.
Venkitaraman, A.R., 1989. Use of modified T7 DNA polymerase (sequenase version 2.0TM) for oligonucleotide site-directed mutagenesis. Nucleic acids research, 17(8), pp.3314–3314.
Wang, W. & Beese, L.S., 2011. Crystal Structure of Bacillus DNA Polymerase I Large Fragment Bound to Duplex DNA with Cytosine-Adenine Mismatch at (n-1) Position. Available at:
http://dx.doi.org/10.2210/pdb3tan/pdb.
Worthington, C.C., 1988. Worthington enzyme manual: enzymes and related biochemicals,
Wu, E.Y., 2015a. Binary complex structure of Klenow fragment of Taq DNA polymerase I707L mutant (Cs3C KlenTaq) with DNA. Available at: http://dx.doi.org/10.2210/pdb4n56/pdb.
Wu, E.Y., 2015b. Ternary complex structure of Klenow fragment of Taq DNA polymerase I707L mutant (Cs3C KlenTaq) with DNA and ddCTP. Available at: http://dx.doi.org/10.2210/pdb4n5s/pdb.
Xu, C., Maxwell, B.A. & Suo, Z., 2014. Conformational dynamics of Thermus aquaticus DNA polymerase I during catalysis. Journal of molecular biology, 426(16), pp.2901–2917.
