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Biotechnology DNA Polymerases 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
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