DNA Polymerases: Discovery, Characterization and Functions in Cellular DNA Transactions

338 Pages·2010·5.54 MB·English

by  Ulrich Hübscher

DNA
POLYMERASES
Discovery, Characterization and Functions
in Cellular DNA Transactions
7667 tp.indd 1 3/5/10 12:19:08 PM
TThhiiss ppaaggee iinntteennttiioonnaallllyy lleefftt bbllaannkk
DNA
POLYMERASES
Discovery, Characterization and Functions
in Cellular DNA Transactions
Ulrich Hübscher
Institute of Veterinary Biochemistry and Molecular Biology,
University of Zurich, Switzerland
Silvio Spadari
Institute of Molecular Genetics IGM-CNR,
National Research Council, Pavia, Italy
Giuseppe Villani
Institute de Pharmacologie et de Biologie Structurale,
CNRS-Université Paul Sabatier, Toulouse, France
Giovanni Maga
Institute of Molecular Genetics IGM-CNR,
National Research Council, Pavia, Italy
World Scientific
NEW JERSEY • LONDON • SINGAPORE • BEIJING • SHANGHAI • HONG KONG • TAIPEI • CHENNAI
7667 tp.indd 2 3/5/10 12:19:08 PM
Published by
World Scientific Publishing Co. Pte. Ltd.
5 Toh Tuck Link, Singapore 596224
USA office: 27 Warren Street, Suite 401-402, Hackensack, NJ 07601
UK office: 57 Shelton Street, Covent Garden, London WC2H 9HE
British Library Cataloguing-in-Publication Data
A catalogue record for this book is available from the British Library.
DNA POLYMERASES
Discovery, Characterization and Functions in Cellular DNA Transactions
Copyright © 2010 by World Scientific Publishing Co. Pte. Ltd.
All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means, electronic or
mechanical, including photocopying, recording or any information storage and retrieval system now known or to
be invented, without written permission from the Publisher.
For photocopying of material in this volume, please pay a copying fee through the Copyright Clearance Center,
Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. In this case permission to photocopy is not required from
the publisher.
ISBN-13 978-981-4299-16-9
ISBN-10 981-4299-16-2
Typeset by Stallion Press
Email: [email protected]
Printed in Singapore.
XiaoLing - DNA Polymerases.pmd 1 5/6/2010, 3:55 PM
March16,2010 16:27 SPI-B932 9.75inx6.5in b932-fm
Preface
MaintenanceoftheinformationembeddedinthegenomicDNAsequenceiscrucial
for the survival of any living species. DNA polymerases play pivotal roles in this
complex process since they are involved in all DNA synthesis events occurring
in nature. Besides their essential tasks in vivo, DNA polymerases are now the
workhorsesinnumerousimportantmolecularbiologicalandmedicalcoretechnolo-
giessuchasthewidelyappliedpolymerasechainreaction(PCR),cDNAcloning,
genomesequencing,nucleicacids–baseddiagnosticsandintechniquestoanalyze
ancientandotherwisedamagedDNA.
The history of DNA polymerases goes back over 53 years to the mid-1950s.
With the discovery of a DNA polymerase (now known as DNA polymerase I)
in the bacterium Escherichia coli byArthur Kornberg and his colleagues, it was
for the first time possible to synthesize the genetic material, the DNA, in the test
tube.AmanuscriptreportingthediscoverysubmittedtotheJournalofBiological
Chemistrywasrejectedwithcommentssuchas“theresearcherswereincompetent”
andDNApolymerasewas“apoorname”forthisenzyme.Whataterriblejudgment
by an incompetent editor! The observation by Kornberg and collaborators that a
DNA polymerase synthesizes DNA according to theWatson–Crick base pair rule
(A–TandG–C),needsactivatedbases(dNTPs),atemplate,aprimerandMgCl is
2
stilltruetodayformostDNApolymerasesinallorganismstested.
BruceAlbertsinafeaturearticleinNaturestatedin20031:“Knowledgeofthe
structureofDNAenabledscientiststoundertakethedifficulttaskofdecipheringthe
detailed molecular mechanisms of two dynamic processes that are central to life:
thecopyingofthegeneticinformationbyDNAreplication,anditsreassortmentand
repair by DNA recombination. Despite dramatic advances towards this goal over
thepastfivedecades,manychallengesremainforthenextgenerationofmolecular
biologists.”
A comprehensive book focusing on DNA polymerases appeared in 1986 and
coveredexclusivelyanimalDNApolymerases.2AtthattimetheanimalDNApoly-
merase family was still small. Only three DNA polymerases, α, β and γ were
v
March16,2010 16:27 SPI-B932 9.75inx6.5in b932-fm
vi DNAPolymerases
known in detail and the scientific community just started to believe in a fourth
DNApolymerase,calledDNApolymeraseδ.Inthelast23years,duetotheanal-
ysis of various genomes, including the one from humans, we have witnessed the
discoveryofanabundanceofnovelDNApolymerasesinthethreekingdomsoflife
(bacteria,archaeaandeukaryotes)withspecializedpropertieswhosephysiological
functionsareonlybeginningtobeunderstood.Forseveraldecadesthedogmawas
widelyacceptedthatveryaccurateDNApolymerasesguaranteethefaithfuldupli-
cation of DNA, while their limited capacity of making mistakes might be one of
thedriversforevolutionandacauseofdisease.Whenin1999thehumangenome
was sequenced, nine newly discovered DNA polymerases appeared within three
years in the literature (1999–2002). Many of these DNA polymerases appear to
have distinct functions in translesion synthesis, in different DNA repair events or
inimmunoglobulinV(D)Jrecombination.
This book starts by presenting the history of the discovery of DNA and DNA
polymerases,includingthepolymerasechainreaction(PCR)(Chapter1),followed
bythepresentationofDNApolymerasesfromthethreekingdomsoflife:bacteria,
archaeaandeukaryotes(Chapter2).Next,thestructuralandfunctionalaspectsofthe
differentDNApolymerasefamiliesaredescribedinprokaryotes(Chapter3)andin
eukaryotes(Chapter4).Preventinggeneticinstabilityisofgreatimportanceinlife.
Temporal and spatial regulation of DNA polymerases is of paramount interest for
theorganism.Thismightoccurviaregulationoftheirexpression,theirstabilityand
theirlocalization.Manyposttranslationalmodificationscontributetotheseproper-
ties.DNApolymerasesarenotautisticenzymesbutratherworkinabroadercontext
withinacellandtheycanreplaceoneanotherundercertainphysiologicalandeven
pathologicalsituations(Chapter5:GlobalfunctionsofDNApolymerases).Many
fundamental mechanistic properties have been elucidated, thanks to the study of
DNApolymerasesfrombacterialandanimalcellsviruses.Themostrelevantones
willbedescribedinChapter6.ArecentfieldofChemicalBiologyhasdeveloped
techniques that allow the evolution of DNA polymerases in the test tube and thus
a variety of novel applications may be ahead of us in the near future (Chapter 7).
ManydiseaseshavebeencorrelatedwithmalregulationsandmalfunctionsofDNA
polymerases (Chapter 8) and DNA polymerase inhibitors are used as chemother-
apeutic agents (Chapter 9). The chapters are written so that they can be read and
understood in their own and this will necessarily bring a certain redundancy. We
have also made cross-references where appropriate. In summary, this book pro-
videstheargumentsandevidencethatcharacterizeDNApolymerasesasenzymes
essential to life and also place them among the most important tools in modern
chemistry,biologyandmedicine.
March16,2010 16:27 SPI-B932 9.75inx6.5in b932-fm
Preface vii
WewouldliketothankUrsulaHübscher,RalphImhof,NicolasTanguyLeGac
andFabioCobianchiforartworkandthefollowingcolleagueswhorevisedchapters
ofthisbook:PaulBoehmer,BillCopeland,MyronGoodman,GhyslaineHenneke,
Jean-SébastienHoffmann,NeilJohnson,EnniMarkkanen,AndreasMarx,Kristijan
Ramadan,ArthurWeissbachandGeorgeWright.
Zurich(Switzerland),November2009
References
1. AlbertsB.2003.Nature421:431–5
2. FryM,LoebLA.1986.AnimalCellDNApolymerases.CRCPress,Inc.BatonRouge,Florida.
UlrichHübscher
InstituteofVeterinaryBiochemistryandMolecularBiology,
UniversityofZurich,Switzerland
SilvioSpadari
InstituteofMolecularGeneticsIGM-CNR,
NationalResearchCouncil,Pavia,Italy
GiuseppeVillani
InstitutedePharmacologieetdeBiologieStructurale,
CNRS-UniversitéPaulSabatier,Toulouse,France
GiovanniMaga
InstituteofMolecularGeneticsIGM-CNR,
NationalResearchCouncil,Pavia,Italy
March16,2010 16:27 SPI-B932 9.75inx6.5in b932-fm
TThhiiss ppaaggee iinntteennttiioonnaallllyy lleefftt bbllaannkk
March16,2010 16:27 SPI-B932 9.75inx6.5in b932-fm
Contents
1. HistoryandDiscoveryofDNAPolymerases 1
1.1 DiscoveringDNA:AFirstStepTowardsUnderstanding
theBasisofLife. . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1.1 Nuclein . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.1.2 NucleicAcid . . . . . . . . . . . . . . . . . . . . . . . 5
1.1.3 NucleicAcidsAreComposedofNucleotides . . . . . . 6
1.1.4 DNAIstheGeneticMaterial . . . . . . . . . . . . . . 7
1.1.5 StructureofDNA:TheWatson–CrickDNADouble
HelixandMechanismofDNAReplication . . . . . . . 10
1.2 ImaginganEnzymethatAssemblestheNucleotides
intoDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.2.1 DNAPolymeraseActivityinExtracts
ofEscherichiacoli . . . . . . . . . . . . . . . . . . . . 15
1.2.2 EscherichiacoliDNAPolymeraseCanSynthesize
DNAwithGeneticActivity:CreatingLife
intheTestTube . . . . . . . . . . . . . . . . . . . . . 18
1.2.3 BacteriaContainManyDNAPolymerases . . . . . . . 19
1.2.4 HowIsaNewDNAChainStarted?Discontinuous
DNASynthesisandtheNeedforanRNAPrimer. . . . 21
1.2.5 RNAPrimingasaMechanismforInitiation:
DNAPrimase . . . . . . . . . . . . . . . . . . . . . . 22
1.3 Late1960stoEarly1970s:DNAReplicationShows
ItsComplexity . . . . . . . . . . . . . . . . . . . . . . . . . . 23
1.3.1 DNAStructureIsMuchMoreComplex,Rich
ofConformationalFlexibilityandthusFull
ofFunctionalPotentialitiesthantheOne
ProposedbyWatsonandCrick . . . . . . . . . . . . . 23
1.3.2 DNABindingProteins,DNA
Helicases,DNATopoisomerases . . . . . . . . . . . . 24
ix