Human DNA Polymerases

Biology, Medicine and Biotechnology

Giovanni Maga, Silvio Spadari;Giuseppe Villani;Ulrich Hübscher

• Publisher: WSPC
• Year: 2017
• ISBN:
  9789813226425
• Topic: Biological Sciences
• Subtopic: Biochemistry
• Index: Biological Sciences

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Maintenance of the information embedded in the genomic DNA sequence is essential for life. DNA polymerases play pivotal roles in the complex processes that maintain genetic integrity. Besides their tasks in vivo, DNA polymerases are the workhorses in numerous biotechnology applications such as the polymerase chain reaction (PCR), cDNA cloning, next generation sequencing, nucleic acids based diagnostics and in techniques to analyze ancient and otherwise damaged DNA (e.g. for forensic applications). Moreover, some diseases are related to DNA polymerase defects and chemotherapy through inhibition of DNA polymerases is used to fight HIV, Herpes and Hepatitis B and C infections. This book focuses on (i) biology of DNA polymerases, (ii) medical aspects of DNA polymerases and (iii) biotechnological applications of DNA polymerases. It is intended for a wide audience from basic scientists, to diagnostic laboratories, to companies and to clinicians, who seek a better understanding and the practical use of these fascinating enzymes.

--> Contents:

• Preface
• About the Authors
• History of DNA Polymerases
• DNA Polymerases: General Aspects
• Human DNA Polymerases: From Structure to Function
• Human DNA Polymerases in Different DNA Transactions
• DNA Polymerases and Human Diseases
• Human DNA Polymerases and Chemotherapy
• Polymerases Chain Reaction and Heat-Stable DNA Polymerases: The History and the Potential of Evolved DNA Polymerases
• Synthetic Evolution of DNA Polymerases for Novel Properties
• Market for Evolved DNA Polymerases in Routine and Medical Applications

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--> Readership: Academic and industry research scientists, from PhD students to senior professors, as well as R&D specialists and marketing experts working in biotech and pharmaceutical companies. -->
Keywords:DNA Polymerase;DNA Replication;DNA Repair;DNA Recombination, PCR;Cancer;Neurological Diseases;Medicine;Biology;Chemotherapy;Structural Biology;Enzymology;BiotechnologyReview: Key Features:

• The only book to merge basic science, biotechnological applications and marketing opportunities of DNA polymerases
• The most extensive literature coverage of the field, with more than 1,000 cited references and updated with the most recent contributions received by scientists all over the world
• Written by four leading experts in DNA polymerases, it gives the most complete overview of the field from its historical origins to the latest developments

Chapter 1

History of DNA Polymerases

1.1Imaging an enzyme that assembles the nucleotides into DNA

The beauty of the DNA double helix model¹ proposed by James Watson and Francis Crick (1916–2004) was that the structure immediately suggested its function. As they hinted in their Nature paper²: “It has not escaped our notice that the specific pairing we have postulated suggests a possible copying mechanism for the genetic material.” The chemical complexity of the molecule was thought to be sufficient to store the genetic information and definitely settled a decades-long controversy over whether DNA or protein was the “life molecule”. The authors had from the beginning the idea that the structure of DNA should allow the molecule to copy itself during cell division, so that an exact copy could pass into each new cell. Indeed the DNA molecule was self-replicating following the unwinding of the two complementary and antiparallel strands. “Each chain would act as a template for the formation on to itself of a new companion chain so that eventually we shall have two pairs of chains, where we only had one before.” Moreover, the sequence of the pairs of bases will have been duplicated exactly because each base would attract its complementary one, by hydrogen bonding, so that two new double helices are assembled. Watson and Crick also proposed that one of the strand of each daughter molecule was newly synthesized and the other one was derived from the parental molecule. This semiconservative mechanism of DNA replication was soon (1958) demonstrated by Mathew Meselson and Franklin Stahl.³

The discovery of DNA structure started a new era in biology and the following years led to the complete elucidation of the genetic code, as reviewed in many articles and books such as those by Martynas Ycas (1917–2014)⁴ and Carl Richard Woese (1928–2012),⁵ and to the realisation that DNA also directs the synthesis of proteins. This was enunciated by Crick in the so called “central dogma of molecular biology” (DNA→RNA→Protein) that implies a one-way flow of genetic information between macromolecules through DNA→DNA (DNA replication), DNA→mRNA (transcription) and mRNA→Protein (translation). Three kinds of RNA, synthesized by RNA polymerase, were found: that is messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA). The mRNA transcript of DNA was indeed found to be the template for protein synthesis; tRNAs carried amino acids to the ribosomes and sequentially read on mRNA codons of three bases that specify an amino acid, and rRNA is the major component of ribosomes.

Robert W. Holley (1922–1993), Gobind Khorana (1922–1988) and Marshall Nirenberg (1927–2010) shared the Nobel Prize in Physiology or Medicine in 1968 for their interpretation of the genetic code and its function in protein synthesis. However, other Nobel laureates such as Crick and Sidney Brenner, who found that amino acids are coded by groups of three bases, Severo Ochoa (1905–1993), who isolated the polynucleotide phosphorylase, the enzyme that, under test-tube conditions, allowed the synthesis of high molecular weigth polyribo-nucleotides from nucleoside diphosphates, Francois Jacob (1920–2013) and Jacques Monod (1910–1976), with their proposal of the mRNA hypothesis, contributed significantly to this outstanding accomplishment.

Going back to the DNA structure, the simplicity of the model for DNA replication, thanks to the complementarity of A to T and of G to C, even induced Watson and Crick to think that nucleotides, paired to the DNA template, might be zippered together without any enzyme action. Perhaps it was sufficient for the nucleotides to align spontaneously, because of their reciprocal affinity, along each of the two separated complementary strands, and the DNA was duplicated! But to a biochemist such as Arthur Kornberg (1918–2007) this must have sounded like a heresy. And indeed only a scientist who knew how nucleotides were synthesized and activated in the cell could not only imagine an enzyme that would assemble nucleotides into DNA but also try to do it outside a living cell.

Kornberg, born in Brooklyn, N.Y., in 1918 and laureate in Medicine in 1941 from Rochester University School of Medicine, in his discovery of DNA polymerase was undoubtely guided by “his unremitting fascination with enzymes, molecules which give the cell its life and personality and make things happen.” He learned his love for enzymes from some of the best enzymologists of his time. He worked with Ochoa in 1946 at New York University Medical School “learning the philosophy and practice of enzyme purification.” He then spent six months in 1947 with Carl F. (1896–1984) and Gerty (1896–1957) Cori at Washington University Medical School in St. Louis, and was fascinated and inspired by their discovery of glycogen phosphorylase. They all contributed toward his scientific formation, made him fall in love with enzymes and convinced him that enzymes were the key, the most effective way to understanding intracellular biochemical processes.^6,7

Thus, in the late 1940s–early 1950s, when it was becoming clear that pathways of degradation/energy production and biosynthesis were distinct, Kornberg returned to the NIH and started an “Enzymes and Metabolism Section” that included Leon Heppel (1912–2010) and Bernard Horecker (1914–2010). He started to purify and study enzymes that assembled various coenzymes such as NAD⁺, NADP, FAD, thus greatly contributing to focusing the interest of biochemists in biosynthetic pathways. He first purified nucleotide pyrophosphatase from potatoes and found that the enzyme cleaved the pyrophosphate bond of NAD (nicotinamide adenin dinucleotide) releasing NMN (nicotinamide mononucleotide) and AMP. This led Kornberg to discover the enzyme NAD synthetase that utilized ATP to transform NMN into NAD with the release of pyrophosphate.

Then Kornberg discovered that FAD (flavine adenin dinucleotide) was synthesized by a similar mechanism as well as were the nucleotides. Thus, the mechanism involving nucleotidyl transfer from ATP (or any other nucleoside triphosphate such as UTP and CTP) and the release of pyrophosphate turned out to be a general mechanism for the synthesis of the precursors of each class of macromolecules in cells and tissues, proteins, lipids, carbohydrates, and, somehow, the nucleic acids. The building blocks, aminoacids, acetic acid, sugar, nucleotides first react with ATP (or UTP or CTP) to become activated precursors before being added to the corresponding molecule through this mechanism that involves the nucleotidyl transfer from a nucleoside triphosphate with the release of pyrophosphate. The pyrophosphate is then hydrolyzed to inorganic orthophosphate by an efficient inorganic pyrophosphatase present in all cells, thus driving the reaction in the direction of the synthesis.

The synthesis of DNA and RNA, in fact, also resembled that of the coenzymes NAD and FAD, each nucleotide being incorporated as the monophosphate with release of pyrophosphate. This research earned Kornberg the Annual Paul–Lewis award in enzymology (now the Pfizer Award) and started attracting applications from young and promising biochemists. But the mechanism of the synthesis of nucleic acids was still far away.

Then in the late 1940s–early 1950’s, with John Buchanan (1918–2007) at the University of Pennsylvania and MIT, and Robert Greenberg (1918–2005) at the University of Michigan, who were investigating the biosynthesis of purine nucleotides A and G, Kornberg focused on pyrimidine biosynthesis. He studied orotic acid and showed first how a liver enzyme could produce an activated form of ribose, phosphoribosyl pyrophosphate (PRPP), from ribose phosphate and ATP, and then how two yeast enzymes could transfer the activated ribose to orotic acid and then decarboxylate orotic ribose-phosphate to uracil ribose phosphate (UMP). UMP and other nucleoside monophosphates were then converted to diand triphosphates by nucleoside monophosphate kinases that utilize ATP as the phosphoryl donor. Thus, UMP could be converted to UDP and UTP, and CTP could be formed by amination of UTP. Rather than uracil, DNA contains thymine, the 5′-methyl derivative of uracil; thus uracil deoxyribose phosphate (deoxyuridylate, dUMP) is methylated to thymine deoxyribose phosphate (deoxythymidylate, dTMP). Contrary to the biosynthesis of pyrimidine nucleotide, where ribose phosphate from PRPP is added to the pyrimidine ring, for example orotic acid, in the byosinthesis of purine nucleotides such as AMP Buchanan et al. found that PRPP reacts with smaller molecules, such as aminoacids, in building the purine structure.

But soon Kornberg et al. found enzymes that could condense preformed adenine and guanine, such as those obtained from the breakdown of RNA and DNA by enzymes that degrade these molecules, with PRPP to make nucleotides directly. It thus became clear that there were two pathways for the biosynthesis of nucleotides: the de novo pathway in which the activated nucleotides are built from simpler molecules (aminoacids, carbon dioxide, sugar phosphate etc.) and a salvage pathway in which A, G, C, U and T obtained from the hydrolysis of RNA and DNA are phosphorylated and recycled to produce activated nucleotides.

1.1.1DNA polymerase activity in extracts of Escherichia coli

In 1953, when he was only 35 years old, Kornberg resigned from NIH to become Professor and Chairman of the Department of Microbiology, Washington University, St. Louis. Having elucidated the biosynthesis of several coenzymes and learned how nucleotides are synthesized and activated in the cells, he was ready to try the synthesis of DNA in a brokencell extract and to search for enzyme(s) that assemble the nucleotides into DNA. Of course Watson and Crick had just proposed in 1953 a mechanical model for DNA replication that did not exclude a spontaneous assembly of nucleotides in the synthesis of a DNA chain directed by base pairing with each strand of the parental duplex, and anyhow to scientists the chemical mechanism of DNA synthesis in the cell a...

Table of contents

• Cover Page
• Title
• Copyright
• Preface
• About the Authors
• Contents
• Chapter 1: History of DNA Polymerases
• Chapter 2: DNA Polymerases: General Aspects
• Chapter 3: Human DNA Polymerases: From Structure to Function
• Chapter 4: Human DNA Polymerases in Different DNA Transactions
• Chapter 5: DNA Polymerases and Human Diseases
• Chapter 6: Human DNA Polymerases and Chemotherapy
• Chapter 7: Polymerase Chain Reaction and Heat-Stable DNA Polymerases: The History and the Potential of Evolved DNA Polymerases
• Chapter 8: Synthetic Evolution of DNA Polymerases for Novel Properties
• Chapter 9: Market for Evolved DNA Polymerases in Routine and Medical Applications
• Subject Index
• Author Index