Biochemistry. DNA Polymerase. Structure and Function of Biomolecules II. Principal Investigator
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1 Paper : 03 Module: 14 Principal Investigator Paper Coordinator and Content Writer Dr. Sunil Kumar Khare, Professor, Department of Chemistry, IIT-Delhi Dr. Sunil Kumar Khare, Professor, Department of Chemistry, IIT-Delhi Content Reviewer Prof. Arun Goyal, Professor, Department of Biochemical Engineering and Biotechnology, IIT-Guwahati 1
2 DESCRIPTION OF MODULE Subject Name Paper Name Module Name/Title Dr. Vijaya Khader Dr. MC Varadaraj 2
3 1. Objectives Function of DNA polymerase in DNA replication Mechanism of DNA polymerase catalysis Different types of Bacterial and Eukaryotic DNA polymerases Unique DNA polymerases 2. Concept Map 3
4 3. Description 3.1 Overview Deoxyribonucleic acid (DNA) is the genetic material in most of the organisms. To pass on the genetic information during cell division, DNA needs to be copied. The primary enzyme involved in synthesizing identical copy of DNA is DNA polymerase. DNA polymerase I a bacterial polymerase was first discovered by Arthur Kornberg in 1956 and for this significant achievement he was conferred Nobel Prize in DNA polymerase use single strand DNA as template and catalyzes DNA dependent DNA synthesis. This enzyme adds complementary nucleotide to 3 -OH end of newly synthesized DNA strand. The 3 -OH group of growing DNA strand act as nucleophile and attack α- phosphoryl group of incoming nucleotide. The reaction results in addition of new base by formation of phosphodiester bond and release of pyrophosphate. Hydrolysis of released pyrophosphate provides additional free energy to make this reaction irreversible with a ΔG of -7 kcal/ mole. The addition of new nucleotide is directed by template strand as the nucleotide that can form Watson-Crick base pair with template stand is added. Figure 1 depicts the mechanism of addition of base by DNA polymerase during DNA synthesis. 4
5 Figure 1. Diagrammatic representation of addition of nucleotide during DNA replication 3.2 Function of DNA polymerase DNA polymerase performs two basic functions during DNA synthesis viz. DNA polymerization and proofreading. Polymerization function is carried out by virtue of 5-3 polymerase activity. Newly incorporated nucleotide can only be added to 3 -OH group and because of this new strand is always synthesized in 5-3 direction. DNA replication is a bidirectional and semidiscontinuous process. The recently synthesized strand that moves in the direction of replication fork opening is synthesized continuously and is called leading strand. 5
6 Other strand is synthesized in small stretches as the direction of DNA polymerization is opposite to the movement of replication fork. This strand is called lagging strand and the small stretches of DNA synthesized is called Okazaki fragments (Figure 2). Figure 2. Bidirectional DNA polymerization in semidiscontinuous manner DNA polymerase has not only the role to synthesize DNA but also to make it correctly. Impaired synthesis of DNA can lead to altered metabolic functioning of cell and even death. DNA polymerase has two types of exonuclease activity i.e. 3-5 and 5-3 exonuclease activity. As wrongly paired nucleotide is incorporated during DNA synthesis, the polymerase activity is repressed and template strand moves from polymerase active site to 3-5 exonuclease active site. The mismatched base is excised and DNA synthesis resumes again. The function of DNA polymerase to remove mismatched base pair by virtue of 3-5 exonuclease activity is called proofreading. Proofreading function is performed without template strand getting dissociated from 6
7 DNA polymerase as polymerase and 3-5 exonuclease active sites are part of same polypeptide. Proofreading machinery helps in maintaining high fidelity during DNA replication. Generally DNA polymerase incorporates one wrong nucleotide for every 10 5 nucleotides added. Proofreading function improves this rate by a factor of 100 to Overall error rate of one in nucleotide added is achieved further by post replication repair mechanism. 5-3 exonuclease activity of DNA polymerase helps in nick translation process. DNA polymerase binds to single stranded nick and removes nucleotide from 5 end. New nucleotides are added by polymerase activity and the nick is sealed by DNA ligase. This machinery helps in joining lagging strands during DNA replication. 3.3 Mechanism of DNA polymerase DNA polymerases share common structural features. Structure of DNA polymerase resembles to right hand with three distinct domains designated as palm, finger and thumb domains (Figure 3A). Palm domain contains the active site for polymerase activity. Two metal ions usually Mg +2 bound to conserved aspartate catalyze nucleophilic attack of 3 OH on phosphate group. Metal ion A binds to 3 OH and decreasing its affinity for H group. This result in weakening of O-H bond and assist nucleophilic attack of 3 OH on α-phosphate group. Metal ion B interacts with phosphate groups and help release of pyrophosphate (Figure 3B). Palm domain also aids in maintaining correct base pairing. It forms extensive hydrogen bonds with 7
8 minor groove of newly synthesized DNA, which is not base pair specific. Addition of mismatched base pair results in reduced affinity of palm domain with primer-template junction as well as reduced rate of catalysis. This effects release of primer-template junction from polymerase active site to 3-5 exonuclease active site for removal of incorrectly paired base. A 8
9 B Figure 3. A. Different domains of DNA polymerase; B. Metal ions involved in catalysis during DNA synthesis. Finger domain interacts with incoming deoxynucleotide triphosphate (dntp). Once correctly paired base is added then finger domain moves to enclose the newly formed base pair. This closed conformation increases the proximity of DNA template with active site metal ions and in turn activates catalysis of DNA synthesis. Finger domain also interacts with template and induces 90⁰ turn in primer template junction to expose first unpaired base for catalysis. This avoids confusion for the site of new base addition. Thumb domain though not involved directly in catalysis, binds with newly synthesized DNA. This interaction helps in correct positioning of primer to active site. Thumb domain also increases the association of template with DNA polymerase. This close association increases the processivity of DNA polymerase i.e. number 9
10 of base pair added each time DNA polymerase binds to DNA template. In short closed association and coordination of palm, finger and thumb domain results in enhanced catalysis coupled with accurate paired base addition. 3.4 Types of DNA polymerases DNA polymerase has role to synthesize DNA with high fidelity matching the speed of cell division. To accomplish this task cell requires different types of s. Prokaryotic and eukaryotic DNA polymerase though undertakes same function but differ in types Bacterial DNA polymerases Five different types of polymerases designated as Pol I, II, III, IV and V has been reported in bacteria. They have been numbered in sequence of their discovery. I (Pol I) first discovered in Escherichia coli is 928 amino acids long single polypeptide, endowed with three activities i.e. 5-3 polymerase, 3-5 exonuclease and 5-3 exonuclease. 3-5 exonuclease activity of Pol I supports in proofreading activity to synthesize DNA with high fidelity. 5-3 polymerase and 5-3 exonuclease activity helps in combining and completing the synthesis of lagging strand. 5-3 exonuclease activity aids in removing DNA/ RNA from nick region particularly from DNA-RNA junction of Okazaki fragments. The gap created by this is filled by 5-3 polymerase activity of Pol I. Pol I is not a highly processive enzyme and adds nucleotides each time it binds to template but this is ideal for filling short gaps. Pol I is involved in nick translation and other DNA repair mechanism of cells. DNA polymerase I of E. coli can be proteolytically cleaved into a large fragment called Klenow fragment. This 10
11 fragment has largely been studied as model DNA polymerase. This fragment is devoid of 5-3 exonuclease activity and contains polymerase and 3-5 exonuclease catalytic sites. II (Pol II) has function in DNA repair and cells deficient in it can propagate usually. This is also involved in initiating replication at replication fork when it is stopped because of DNA damage. III (Pol III) is the primary polymerase involved in bacterial DNA replication. This is a highly processive enzyme with polymerase and 3-5 exonuclease catalytic site. These two activities help in DNA synthesis with high speed and fidelity. The DNA polymerase III core enzyme contains three subunits viz. α, ε and θ. Polymerase activity is associated with α, 3-5 exonuclease activity is found in ε and θ subunit stimulates exonuclease activity. Pol III lacks 5-3 exonuclease activity and cannot perform function of nick translation. The properties and functions of above three bacterial polymerases is enlisted in Table 1. Table 1. Properties and function of E. coli DNA polymerases Pol I Pol II Pol III Mass (kd) Molecules/cell 400 Not defined Turnover number Number of Subunit 1 1 Core(3) Holoenzyme (10) Structural gene pola polb polc Lethal mutant Yes No Yes 5' - 3' Polymerization activity Yes Yes Yes 3' - 5' Exonuclease activity Yes Yes Yes 5'- 3' Exonuclease activity Yes No No Function RNA primer removal; DNA repair DNA Replication DNA repair 11
12 IV and V are mainly involved in DNA repair. They also permit replication to escape certain DNA damage and are called error-prone polymerases Assembly of DNA polymerase III holoenzyme increases processivity DNA polymerase III being primary polymerase of bacteria need to have high processivity to do so it forms holoenzyme an association of closely linked 10 proteins. E. coli DNA Pol III holoenzyme is a 900 kd complex organized in four subcomplexes. Figure 4 depicts the steps and subunits involved in holoenzyme association. The β ring a homodimer of two β subunit form a clamp around template strand and can slide freely on it. Liaison of β ring to core complex increases its processivity (>5000 bases) as β ring does not allow the core complex to diffuse away from template DNA. The γ complex a cluster of seven proteins helps in placing β ring on template strand by using ATP. Because of this function γ complex is also called clamp loader. Once the β clamp is bound to DNA, DNA Pol III core enzyme associates with it. A τ dimer binds to core complex and initiates linking of another core complex already bound to β clamp. Core enzyme complex is bound to stretchy C-terminal domain of τ complex which provides it flexibility of movement. 12
13 Figure 4. Steps involved in III holoenzyme association 13
14 DNA polymerase III needs to synthesize DNA on both leading and lagging strand in close coordination. This task is accomplished by assembly of two DNA Pol III holoenzymes to form replisome (Figure 5). While one core synthesizes DNA continuously on leading strand, other core after completing one Okazaki fragment, dissociates and bind β clamp recently added on newly exposed template. Two core complexes need to have different processivity as lagging strand core need to dissociate each time it completes synthesis of one Okazaki fragment. The single clamp loader of replisome is associated with core polymerase of lagging strand. Clamp loader is also required for removing β clamp from DNA template and it has important function of loading and unloading of β clamp after each Okazaki fragment synthesis. Figure 5. Pictorial representation of E. coli replisome 14
15 3.4.2 Eukaryotic DNA polymerases Eukaryotic genetic makeup is complex as compared to prokaryotes. The size of genome is big and DNA replication starts at multiple origin points. In addition to this organelles DNA of mitochondria and chloroplast also needs to be replicated. Eukaryotes require more number of proteins for DNA replication. Eukaryotic cells need various DNA polymerases and on an average more than 15 polymerases are present. Eukaryotic polymerases are designated by Greek letters according to their sequence of finding. A newer classification based on sequence homology places both eukaryotic and prokaryotic polymerases into six families i.e. A, B, C, D, X and Y. Three main eukaryotic DNA polymerases α, δ and ε fall in B-family. Pol α/ primase is a four subunit complex consisting of two subunit each of Pol α and primase. Pol α/ primase initiates DNA replication in eukaryotes. Primase unit synthesizes 7-10 nucleotide RNA primer and Pol α adds dntp to it. Pol α lacks proofreading (exonuclease) activity but this is not a problem as stretch of initially synthesized DNA along with RNA primer is removed during processing. Pol α is a not a processive enzyme and get replaced with polymerase δ and ε, this process is called polymerase switching. DNA polymerase δ is a highly processive enzyme and contains 3-5 exonuclease activity. High processivity of this polymerase is due to association with eukaryotic sliding clamp protein called proliferating cell nuclear antigen (PCNA). John Kuriyan determined the X-ray structure of PCNA and it showed similarity with E. coli β clamp. 15
16 Though PCNA share common structure and function with β clamp but their sequence identity is not similar. The eukaryotic clamp loader replication factor C (RFC) loads PCNA to primer-template junction by hydrolysis of ATP. Pol δ is required for lagging strand synthesis however can take part in leading strand synthesis. DNA polymerase ε is also a highly processive polymerase having 3-5 exonuclease activity. Pol ε is needed for leading strand synthesis but it can help in lagging strand synthesis. The properties and function of above three main eukaryotic DNA polymerase is listed in Table 2. Table 2. Properties and function of primary eukaryotic DNA polymerases Pol α/ primase Pol δ Pol ε Mass (kd) Number of Subunits ' - 5' Exonuclease activity No Yes Yes Processivity Moderate High High Association with PCNA No Yes No Function Primer synthesis DNA replication; Proofreading DNA replication; Proofreading DNA polymerase γ a three subunit A-family enzyme is found in mitochondria. Pol γ is solely involved in mitochondrial DNA replication including DNA repair and recombination. Bulk of additional eukaryotic DNA polymerases function in DNA repair. DNA polymerase β a 39 kd monomeric protein is high fidelity repair polymerase. Fidelity of β matches with primary DNA polymerases. Other eukaryotic 16
17 DNA polymerase involved in DNA repair has much inaccuracy and are called errorprone polymerase Unique DNA polymerases Some of DNA polymerases have exceptional properties and it will be worth to mention them. One among them is Taq DNA polymerase isolated from thermophilic bacteria Thermus aquaticus. This enzyme revolutionized the world of molecular biology by being used in polymerase chain reaction (PCR). Taq Pol can amplify a template DNA into several copies at high temperature. PCR the process in which this enzyme is used forms the backbone of recombinant DNA technology. Taq pol lacks proofreading activity and its error rate is high. Other thermotolerant DNA polymerases such as Pfu polymerase from Archaea Pyrococcus furiosus synthesizes DNA with high fidelity and has replaced Taq Pol in PCR reaction. One more distinctive DNA polymerase is reverse transcriptase found in retroviruses. This catalyzes RNA dependent DNA synthesis. Using a RNA template it can synthesize complementary DNA in 5-3 direction. It was first discovered by Howard Temin and David Baltimore in Reverse transcriptase has been a useful tool of genetic engineering for synthesizing complementary DNA (cdna) from mrna (Figure 6). As mrna does not contain introns so, cdna can be used for expressing eukaryotic proteins in E. coli which lacks splicing machinery. 17
18 Figure 6. Illustration of cdna synthesis from eukaryotic mrna by use of reverse transcriptase Telomerase is a DNA polymerase used exclusively to synthesize DNA at ends of linear eukaryotic chromosomes called telomere. Lagging strand synthesis in eukaryotes pose a problem as 5 end cannot be synthesized and each cycle will lead to shortening of chromosome equivalent to the length of RNA primer. Shortening of end of chromosome after each cycle can cause cell death. This problem is overcome by ribonucleoprotein telomerase. Telomeres contain a >1000 tandem repeats of G-rich sequence i.e. TTGGGG in Tetrahymena and TTAGGG in human. RNA component of telomerase contain sequence similar to telomeres and act as template for addition of dntp at 3 end of DNA. Figure 7 illustrates telomerase action to synthesize ends of linear eukaryotic chromosome. 18
19 Figure 7. Graphic representation of telomere DNA synthesis by Tetrahymena telomerase By multiple round of replication telomerase synthesizes the end of chromosome leaving a G- rich overhang. Enhanced telomerase activity can cause uncontrolled cell growth and can result to cancer. Function of telomerase is related to reverse transcriptase and its highly conserved catalytic subunit TERT is homologous to reverse transcriptase. 4. Summary In this lecture we learnt about: 19
20 Functions of DNA polymerase i.e. Polymerase, proofreading and nick translation Mechanism of catalysis by DNA polymerase; role of palm, finger and thumb domain; function of active site metal ions. Different types of Bacterial polymerases III holoenzyme Eukaryotic DNA polymerases Special : Taq polymerase; Reverse transcriptase; Telomerase 20
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