Paper No. : 04 Genetic engineering and recombinant DNA technology

Module : 07 DNA polymerases

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 Samer Singh, Assistant Professor, Panjab University, Chandigarh

Content Reviwer: Dr Mohan Chandra Joshi, Assistant Professor, Jamia Millia Islamia, New Delhi

Genetic engineering and recombinant DNA technology Biotechnology DNA polymerases

Description of Module

Subject Name Biotechnology

Paper Name Genetic engineering and recombinant DNA technology

Module Name/Title DNA polymerases

Module Id 07

Pre-requisites Basic DNA Structure and DNA replication

Objectives 1) WHAT ARE DNA POLYMERASES? - DISCOVERY 2) TYPES in Escherichia coli 3) WHAT THEY DO? 4) STRUCTURE - CONSERVATION & FUNCTION 5) DNA POLYMERIZATION REACTION and ITS ATTRIBUTES 6) DNA POLYMERASE'S FIDELITY AND PROCESSIVITY 7) DNA POLYMERASE FAMILY AND EXAMPLES 8) HOW SPECIAL FUNCTION DNA POLYMERASE 'TELOMERASE' CARRIES OUT LENGTHENING OF LINEAR DNA ENDS Keywords DNA polymerase, reverse transcriptase, DNA polymerization, Processivity, Fidelity, Telomerase

Genetic engineering and recombinant DNA technology Biotechnology DNA polymerases

Table of contents

A. Learning Objectives B. Keywords C. DNA POLYMERASES C1. General Overview -Discovery C2. Comparison of DNA polymerases of Escherichia coli C3. DNA POLYMERASE CORE STRUCTURE & FUNCTION C4. DNA POLYMERIZATION ACTIVITY and ITS ATTRIBUTES C5. DNA POLYMERASE FAMILIES C6. TELOMERASE- A special function DNA polymerase in eukaryotes D. Summary

A. LEARNING OBJECTIVES In this module, Students will learn following: 1. WHAT ARE DNA POLYMERASES? - DISCOVERY 2. TYPES in Escherichia coli 3. WHAT THEY DO? 4. STRUCTURE – CONSERVATION & FUNCTION 5. DNA POLYMERIZATION REACTION and ITS ATTRIBUTES 6. DNA POLYMERASE’S FIDELITY AND PROCESSIVITY 7. DNA POLYMERASE FAMILY AND EXAMPLES 8. HOW SPECIAL FUNCTION DNA POLYMERASE ‘TELOMERASE’ CARRIES OUT LENGTHENING OF LINEAR DNA ENDS

B. KEYWORDS DNA polymerase, reverse transcriptase, DNA polymerization, Processivity, Fidelity, Telomerase,

C. DNA POLYMERASES C1. General Overview -Discovery DNA polymerases are ubiquitous in living organism as they are required for the faithful transmission of genetic information from one generation to the next. They are primarily involved in the replication of the genomes and generally catalyze addition of deoxynucleotides at 3’ hydroxyl end of the pre-existing strand of polynucleotide chain, i.e., DNA (or RNA) using another strand of DNA (or RNA) as a template. DNA polymerases using one DNA (or RNA in case of reverse transcriptase) strand as a template make a complimentary copy, thus, allowing stable passage of ‘the information’.

A general DNA polymerase catalyzed reaction can be depicted as

DNA or (dNMP)n + dNTP  (dNMP)n+1+PPi

where (dNMP)n is a polynucleotide, dNTP is a deoxynucleotide triphosphate and PPi is pyrophosphate

Genetic engineering and recombinant DNA technology Biotechnology DNA polymerases

Different types of DNA polymerase enzymes are known and based upon their specificity for template type requirement, exonuclease activity, temperature tolerance etc they are classified into different groups. Most frequently encountered are DNA template dependent polymerases (e.g., DNA pol I, II, III, , , Taq, Pfu, Vent) and RNA template dependent polymerases or reverse transcriptase (e.g., Telomerase, M-MLV RT etc.). There are DNA polymerases that can add nucleotides in a template independent manner. These are usually referred to as terminal deoxynucleotidyl transferases (TdT).

They are responsible for accurately and efficiently replicating/copying the whole genome to ensure the maintenance of the genetic information during transmission through generations.

First evidence of the existence of an enzymatic activity capable of synthesizing DNA came from the work of Arthur Kornberg and colleagues (1955) who first purified and characterized the first DNA polymerase - a single-polypeptide enzyme from E. coli cells, which later came to be known as DNA polymerase I or simply ‘Pol I’. For this discovery, Arthur Kornberg was later awarded the Nobel Prize in Physiology / Medicine in year 1959. Initially, it was assumed that DNA polymerase I is the enzyme responsible for DNA polymerase activity in E.coli. Soon after the isolation of this enzyme in 1955, however, evidence began to accumulate that it is not suited for replication of the large E. coli chromosome and more DNA polymerases should exist such as, about 90% of the DNA polymerase activity observed in E. coli extracts can be accounted for by DNA polymerase I; the rate of nucleotides addition (600 nucleotides/min) is too slow (by a factor of 100 or more) to account for the rates at which the replication fork moves in the bacterial cell; it has relatively low processivity (nucleotides added before enzyme falls off the template); genetic studies demonstrated that many genes, and therefore many proteins, are involved in replication; John Cairns in 1969 isolated a bacterial strain with an altered gene for DNA polymerase I that produced an inactive enzyme- although this strain was abnormally sensitive to agents that damaged DNA, it was nevertheless viable! These observations led to search for other DNA polymerases leading to the discovery of E. coli DNA polymerase II and DNA polymerase III in the early 1970s. Later, investigations revealed that E. coli contains at least four other distinct DNA polymerases.

C2. Comparison of DNA polymerases of Escherichia coli There are atleast five DNA polymerases in E.coli with distinct activity. DNA pol I was the first and still remains one of the best studied DNA polymerase among the lot. Detailed studies of DNA polymerase I activity revealed features of the DNA synthesis process that are now known to be common to all DNA polymerases. It was found to play role in lagging strand synthesis and DNA repair. DNA polymerase II is an enzyme mostly involved in one type of DNA repair. DNA polymerase III is the principal replication enzyme in E. coli because of its high processivity. A comparative account of major DNA pol. from E. coli is provided below in a tabular form.

Genetic engineering and recombinant DNA technology Biotechnology DNA polymerases

DNA pol IV performs translesion synthesis. DNA pol V is involved in SOS response and translesion synthesis.

C3. DNA POLYMERASE CORE STRUCTURE & FUNCTION The structure of a DNA polymerase core that contains the catalytic site for nucleotide addition is most highly conserved component of the polymerase complex (whether DNA polymerase is a multiple or single subunit enzyme).

The DNA polymerase core primarily folds into 3 structural domains that roughly resemble a right hand with 3 distinct domains. The uncanny similarity in the appearance has led to the 3 domains being called - the palm, the fingers, and the thumb. The ‘palm’ domain among the others is the most

Figure: DNA polymerase I (pol I) from Escherichia coli exemplifies the structure of the core polymerase (a and b). It is an essential specialized polymerase that is required for finishing DNA replication and removing the RNA primers incorporated during initiation of replication. (From ‘Molecular Biology: Principles of Genome Function’ (2010) by Nancy L Craig, Orna Cohen- Fix, Rachel Green, Carol W Greider, Gisela Storz and Cynthia Wolbergery. p-202, Figure 6.5)

Genetic engineering and recombinant DNA technology Biotechnology DNA polymerases

conserved domain, which forms a cleft into which the growing double-stranded DNA fits and has polymerase active sit. The ‘fingers’ domain help warp the single-stranded template strand of the DNA and positions the incoming complimentary dNTP (as per Watson-Crick base pairing) in relation to the template DNA. The ‘thumb’ domain primarily helps to hold the elongating duplex DNA and maintains the contact with the template allowing processive synthesis. The 3′ to 5′ exonuclease function that removes incorrect bases incorporated in proof reading DNA polymerases, whenever present as is found as an additional domain near polymerase activity site on ‘palm’ domain (see site labeled as ‘exonuclease’ just opposite to thumb domain in the figure shown).

C4. DNA POLYMERIZATION ACTIVITY and ITS ATTRIBUTES

The fundamental reaction catalyzed by DNA polymerase is a phosphoryl group transfer in which the 3’-hydroxyl group of the nucleotide at the 3’ end of the growing strand acts as a nucleophile attacking at the phosphorus of the incoming deoxynucleoside 5’- triphosphate that is going to be added to the polynucleotide chain (see figures about polymerase reaction). An Inorganic pyrophosphate molecule is released in the reaction.

The general polymerization reaction can be summarized and visually depicted as under

Figure: Overview of DNA polymerization reaction catalyzed by DNA polymerases: The addition of dNTPs by DNA polymerase to a polynucleotide chain leads to extension of the polynucleotide chain and the release of a pyrophosphate molecule (Top). The bottom part is schematic simple depiction of the same in DNA molecule (Adapted from Molecular Biology of the Cell, 6th Ed. by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, and Peter Walter, p- 241 figure 5-4 (A))

DNA polymerization reaction facts  DNA polymerases MAKE A COMPLEMENTARY COPY of one DNA strand

 TEMPLATE DEPENDENCE & WATSON-CRICK BASE PAIRING RULES FOLLOWED. All DNA polymerases require a template (DNA / RNA). The polymerization reaction is guided by a template strand according to the base- pairing rules predicted by Watson & Crick.

Genetic engineering and recombinant DNA technology Biotechnology DNA polymerases

 PRIMER REQUIREMENT. All DNA polymerases require a primer (RNA or DNA). A primer is a strand segment (complementary to the template) with a free 3- hydroxyl group to which a nucleotide can be added; the free 3’ end of the primer is called the primer terminus. In other words, all DNA polymerases would need a small stretch of preexisting strand, i.e., primer, to add nucleotides to extend it. Specialized enzymes synthesize primers whenever they are required.

 After adding a nucleotide to a growing DNA strand, a DNA polymerase either dissociates or moves along the template and adds another nucleotide.

 The rate of dissociation and reassociation of the polymerase to DNA strand limits its overall polymerization rate, i.e., the process is faster when a polymerase adds more nucleotides without dissociating/falling-off from the template.

 The average number of nucleotides added before a polymerase dissociates defines its processivity. DNA polymerases vary greatly in processivity, e.g., a few to many thousands nt.

Figure. Details of polymerization of DNA chain. (a) DNA polymerases requires a single unpaired strand to act as template and a primer strand to provide a free -OH group at the 3’ end, to which a new nucleotide unit is added. Each incoming nucleotide (dNTP) is selected in part by base pairing to the complementary nucleotide in the template strand. The new free 3’ -OH, available on the reaction product 2+ allows the addition of another nucleotide. (b) The two Mg ions, coordinated to the phosphate groups of the incoming nucleotide triphosphate and to three Asp residues, two of which are highly conserved in all 2+ DNA polymerases plays essential role in the polymerization. The Mg ion towards exterior (on the right) facilitates attack of the 3’- hydroxyl group of the primer on the  phosphate of the incoming nucleotide 2+ 2+ triphosphate; the other Mg ion facilitates displacement of the pyrophosphate. Both Mg ions supposedly stabilize the structure of the pentacovalent transition state. (Adapted from Lehninger Principles of Biochemistry, Fifth Edition, David L. Nelson and Michael M. Cox and by p- 980 figure 25-5).

Genetic engineering and recombinant DNA technology Biotechnology DNA polymerases

DNA POLYMERASES: FIDELITY AND PROCESSIVITY . The processivity of replicative polymerases is very important for genome replication. It is usually achieved by extra domains or subunits that modulate the dissociation of DNA polymerase from DNA (refer to comparison of DNA polymerases of E.coli).

. The accuracy of DNA polymerization to make a complementary copy of the template DNA is known as fidelity.

. It is of utmost importance for replicative DNA polymerases and to the replication of genome.

. Errors in DNA synthesis needs to be minimized because changes in the genetic code will be passed to progeny cells and would alter the essential cellular functions.

5 6 . A replicative polymerase typically makes 1 uncorrected error for every 10 -10 nucleotides added.

DNA POLYMERASES: FIDELITY- SELECTION OF CORRECT NUCLEOTIDES

This remarkable fidelity of replicative DNA polymerases is achieved by activities ensuring the identity of nucleotides at two distinct stages: during polymerization and post polymerization

1) During the polymerization reaction- SELECTION OF CORRECT NUCLEOTIDES: The spatial geometry of active site allows selective accommodation of correct complementary nucleotides as they fit precisely into the active site when base-paired with the template strand, i.e., the pairing of A with T or C with G will be preferred over mismatches as the geometry of an adenine–thymine (A–T) pair and a cytosine–guanine (C–G) pair are similar to each other allowing it to fit neatly in the active site, whereas mismatched nucleotides will have a different geometry that does not fit as well. The correct geometry favors the catalytic reaction and the addition of the incoming nucleotide onto the new DNA strand.

Genetic engineering and recombinant DNA technology Biotechnology DNA polymerases

Figure. Correctly matched complementary base pairs are favored at active site. Structure of mismatched base pairs disfavors its accommodation in active site as the incorrectly base-paired nucleotides have a distinctly different molecular shape from that of a correct base pair (Pink vs Gray) allowing polymerase reaction to proceed readily for correctly paired nucleotides while gets delayed in case mismatches. Adapted from ‘Molecular Biology: Principles of Genome Function’ (2010) by Nancy L Craig, Orna Cohen-Fix, Rachel Green, Carol W Greider, Gisela Storz and Cynthia Wolbergery. p-204, Figure 6.7

2) Post-polymerization – PROOF READING: The selective removal of the last misincorporated base to growing polynucleotide chain at polymerase active site is achieved by spatially separated 3’ to 5’ exonuclease activity site. When correct bases are being added the polymerase keeps adding new nucleotides and relocates to next template position but if a mismatched base gets incorporated the polymerase activity is slowed down allowing the growing polynucleotide chain to be relocated to exonuclease site which removes the terminal nucleotide then the chain is shifted back to polymerase site to resume polymerization at polymerase site (refer to figure depicting structure of polymerase and DNA polymerase proof reading)

Genetic engineering and recombinant DNA technology Biotechnology DNA polymerases

Figure. DNA polymerase proofreading: The incorrect base pair incorporation at the growing strands (shown in pink) 3’ end leads to mispairing at the 3′ end which is detected and the 3′ end is moved to the 3′ to 5′ exonuclease site on the polymerase. This involves breaking of several base pairs between the nascent strand and the template to allow relocation of the 3′ end to editing site where the 3′-end nucleotide is removed followed by allowing the 3′ end to return to the polymerase active site in the palm domain. Now the polymerase reaction continues again at active site till new misincorporation occurs.

From ‘Molecular Biology: Principles of Genome Function’ (2010) by Nancy L Craig, Orna Cohen-Fix, Rachel Green, Carol W Greider, Gisela Storz and Cynthia Wolbergery. p-205, Figure 6.8

C5. DNA POLYMERASE FAMILIES There are multiple DNA polymerase families. Cells have different DNA polymerases to perform distinct specialized functions - repair of DNA, continue replication across damaged DNA, replication of telomeres etc. besides the well known replicative DNA polymerases – DNA polymerase III in bacteria or DNA polymerases δ and ε in eukaryotes that copy the bulk of the DNA during replication. Polymerases may comprise just a single chain protein performing all range of functions, e.g., nucleotide addition, proofreading and substrate specificity or multiple subunits, with each subunit having a distinct function.

Genetic engineering and recombinant DNA technology Biotechnology DNA polymerases

Based on amino acid sequence similarity and structure, DNA polymerases have been classified into 7 families - A, B, C, D, X, Y and reverse transcriptase (RT) (See Table below)  A family – besides polymerase activity, generally contains a 5′ to 3′ exonuclease domain that allows it to remove DNA or RNA ahead of it and also features the 3′ to 5′ proofreading exonuclease activity (e.g., DNA polymerase I from E. coli).  B, C, and D families are replicative polymerases of eukaryotes, bacteria, and archaea, respectively. These polymerases have high fidelity and possess a 3′ to 5′ proofreading exonuclease.  X family polymerases: specialized for DNA repair, filling in the gaps generated in the DNA during repair.  Y family polymerases: specialized to replicate past bulky adducts present in damaged template DNA; other polymerases will stop polymerization whenever they encounter such lesions.  Reverse transcriptases (RT): specialized DNA polymerases that use single-stranded RNA as template to synthesize a complementary DNA strand. The structure of RT is very much like that of other polymerases-has thumb and finger domains and a palm domain. The active site of reverse transcriptase is similar to that of other DNA polymerases and uses the same catalytic mechanism for linking nucleotides together; encoded by viruses as well as DNA elements called retrotransposons found in eukaryotic genomes. Table. DNA polymerase Families

Genetic engineering and recombinant DNA technology Biotechnology DNA polymerases

C6. TELOMERASE- A special function DNA polymerase in eukaryotes.

Although the existence of this enzyme may not be surprising, the mechanism by which it acts is remarkable and unprecedented. Telomerase contain RNA - about 150 nucleotides long that contains about 1.5 copies of an appropriate CyAx telomere repeat and protein components. The region of the RNA acts as a template for synthesis of the complimentary TxGy strand of the telomere. Telomerase thereby acts as a cellular reverse transcriptase that provides the active site for RNA- dependent DNA synthesis. Telomere synthesis requires the 3’ end of a chromosome as primer and proceeds in the usual 5’ to 3’ direction. Having synthesized one copy of the repeat, the enzyme repositions to resume extension of the telomere. The complementary CyAx strand is synthesized by cellular DNA polymerases, starting with an RNA primer.

DNA terminus of linear DNA in eukaryotes is lengthened by special purpose reverse transcriptase ‘Telomerases’ that uses an internal RNA template strand to synthesize/ add complimentary sequence in 5’ to 3’ direction to the existing DNA end. Remember unlike the retroviral reverse transcriptases, telomerase copies only a small segment of RNA that it carries within itself. Figure. Mechanism of Telomere extension by Telomerase. Adapted from Lehninger Principles of Biochemistry, Fifth Edition, David L. Nelson and Michael M. Cox. p- 1055; Figure 26-39).

Genetic engineering and recombinant DNA technology Biotechnology DNA polymerases

D. SUMMARY  DNA polymerases catalyze polymerization of dNTPs on a preexisting polynucleotide tail with free 3’ -OH group while using a strand of DNA or RNA as a template  DNA polymerase structures are conserved (esp. the polymerase domain when present with other structural and functional domains and features)  Based upon the sequence similarity and function there are 7 families of enzymes namely, A, B, C, D, X, Y and RT  The polymerase or polymerization activity is 5’ to 3’ but exonuclease activity when present could be 5’ to 3’ and/ or 3’ to 5’.  The polymerases involved in replication of genomes (replicative polymerases) generally have high processivity and high fidelity due to inherent selectivity for the correct complementary nucleotide in catalysis site and presence of 3’ to 5’ proof reading (editing) exonuclease activity.

Disclaimer: The content of this module is created for teaching and learning purpose only. Any commercial use or exploitation is prohibited. Every effort has been made to ensure that the figure/tables/illustration adapted from different sources are correctly referenced / cited. The copyrights to the images/ figure/tables/illustration and associated text belong to the indicated sources. The Content writer don’t claim any copyright on them. In case of any error, omission, misinformation or unintentional copyright infringement that may have resulted from any reason including but not limited to error, accident or negligence, the author(s) do not assume and hereby disclaim any liability to any party for any loss, damage, or disruption caused by its usage. However, any such instances may be reported immediately to the content provider for taking the corrective measures possible.

Genetic engineering and recombinant DNA technology Biotechnology DNA polymerases