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Bacterial DNA Polymerase IYing Li, Washington University, St Louis, Missouri, USA

Gabriel Waksman, Washington University, St Louis, Missouri, USA

Bacterial DNA polymerase I is a family of enzymes that are involved in bacterial DNA lesionrepair,as well as DNAreplication. These enzymeshave a multidomain structure containing

a polymerase activity,a proofreading 3’ – 5’ exonuclease activity and/ora 5’ – 3’ exonuclease

activity within one polypeptide chain.

Introduction

Bacterial DNA polymerase I (pol I) enzymes play animportant role in deoxyribonucleic acid (DNA) replicationand the repair of DNA lesions in prokaryotic organisms.These enzymes are multidomain proteins with eachdomain corresponding to a DNA polymerase activity, a

proofreading 3’ –5’ exonuclease activity and/or a 5’ –3’exonuclease activity respectively. The basic reactioncatalysed by the polymerase domain is the template-directed addition of a deoxyribonucleotide onto the 3’ OHgroup of a DNA primer strand. The 3’ –5’ exonucleasedomain catalyses the cleavage of a mismatched nucleotidefrom the 3’ end ofthe DNA. The 5’ –3’ exonuclease domainhas not only the 5’ exonuclease activity, which is thecleavage of a nucleotide from the 5’ end of the DNA, butalso a flap endonuclease activity, which recognizesspecifically 5’ flap single-stranded DNA structures andcleaves the flap at the single-stranded–double-strandedDNA (ssDNA–dsDNA) junction. Based on sequence

comparison and structural studies, all the known DNApolymerases can be grouped into five families: the pol I (orA) family, the pol a (B) family, the pol b (X) family, the polIII (C) family, and the reverse transcriptases (RT) family.Bacterial DNA polymerase I, as well as DNA polymerasefrom bacteriophage T7, belong to the pol I (A) family.

Basic Features of DNA Polymerization:The Enzyme and Substrates

DNA polymerases catalyse the template-directed DNApolymerization reaction, which is the addition of deoxyr-ibonucleoside 5’ triphosphate (dNTP) onto the 3’ end of aDNA primer strand, as outlined below:

ðdNMPÞnþ

DNA

dNTP $ðdNMPÞnþ1

DNA

þPPi

The enzyme has two substrates: one is the (dNMP)n, which

is the primer/template DNA with n residues on the primerstrand; the other is dNTP. The products of the DNA

polymerization reaction are a new primer/template DNA

with an elongated primer strand ((dNMP)n1 1) and ainorganic pyrophosphate (PPi). The reaction consists of nucleophilic attack by the 3’ OH group on the templatstrand to the a-phosphate group of the incoming dNTPThe overall direction of the DNA elongation is from 5’ t3’.

The addition of each dNTP to the 3’ end of the primestrand is directed by correct base pairing of the incomindNTP with the bases on the template strand. DNApolymerases have a broad substrate range, which enablethem to incorporate different kinds of dNTPs. Howeverduring each cycle of the polymerization reaction, thpolymerases have to select the right substrate against

pool of structurally similar dNTPs. This is accomplisheby altering substrate specificity at each step of the catalyticycle. During DNA synthesis by DNA polymerases, thDNA translocates along the polymerase with each cycle opolymerization; thus a new base on the template strand presented to the polymerase active site. The incorporatioof a nucleotide takes place when it forms a correct base pawith this template base.

The rate of DNA polymerization by DNA polymeraseas well as the processivity of these enzymes vary widelyDNA pol I from Escherichia coli (E. coli pol I) is moderately processive enzyme which extends a primechain by about 10–100 nucleotides before dissociatin

from the DNA. Its rate of polymerization is about 5nucleotides per second. In contrast, the DNA pol I enzymfor bacteriophageT7 extendsDNA primerstrands at a ratof 300 nucleotides per second and dissociates from thDNA only after incorporating 1500 bases (Johnson1993).

Article Contents

Secondary article

. Introduction

. Basic Features of DNA Polymerization: The Enzyme

and Substrates

. Escherichia coli DNA Polymerase I as the ArchetypePolymerase

. Assays

. Mechanism

. Multiple Activity (Polymerase, Nucleases, Reversal)

. Structural Information

. Biological Function

. Applications: Polymerase Chain Reaction, etc.

. Current Research Topics/Unanswered Questions

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net



Escherichia coli DNA Polymerase I as theArchetype Polymerase

E. coli pol I was the first DNA polymerase identifiedKornberg and Baker, 1956). As deduced from the sequenceof its encoding polA gene, E. coli pol I is a single

polypeptide chain containing 928 residues with calculatedmolecular weight of 103 kDa. E. coli pol I has at least threedistinct functions: (1) a DNA polymerase activity, which isresponsible for the 5’ –3’ DNA synthesis by this enzyme; (2)a 3’ –5’ exonuclease activity, which cleaves a mismatchedbase from the primer terminus, and thus supports theproofreading activity; (3) a 5’ –3’ exonuclease activity,which degrades DNA from its 5’ end and generates mono-or oligonucleotides.

Limited proteolytic cleavage of pol I generates twoactive components (Kornberg and Baker, 1992): a large C-terminal fragment ( 600 residues), also known as theKlenowfragment, contains the polymeraseactivity andthe

3’ –5’ exonuclease activity; and a small N-terminal frag-ment ( 300 residues) contains only the 5’ –3’ exonucleaseactivity. The large fragment carries out DNA synthesis onthe3’OHside ofa nickin a dsDNA.It alsocarriesout 3’ –5’exonucleaseactivity on both ssDNA or unpaired regions indsDNA. The small fragment degrades DNA from the 5’end and is capable of excising mismatched regions induplex DNA.

The 3.3 A ˚ crystal structure of the Klenow fragment of E.coli pol I complexed with a deoxythymidine monopho-sphate (dTMP) was the first structure solved for a DNApolymerase (Ollis et al ., 1985). This structure (Figure 1)shows that the enzyme is folded into two distinct domains.

The smaller domain (approximately the first 200 N-terminal residues) consists of a central parallel b sheetflanked bya helices. The larger domain (about 400 residuesat the C-terminus) is mainly a-helical. This domain has ashape reminiscent of a right hand, with a large cleftbetween the thumb, palm and fingers subdomains. Site-directed mutagenesis studies have identified the separatefunctions of the large and small domains: the 3’ –5’exonuclease active site is located in the small domain andthe polymerase active site is located in the cleft of the largedomain. There is a large distance ( 30 A ˚ ) between thepolymerase active site and the 3’ –5’ exonuclease active sitein the Klenow structure.

Assays

The mechanism by which the pol I enzymes achieve theirhigh fidelity DNA synthesis has been the subject of extensive studies. Chemical, kinetic and structural ap-proaches have been used to investigate the mechanisticdetails of polymerization and proofreading activities. Theuse of transient kinetic methods, especially the introduc-

tion of chemical quench flow techniques, has beeinstrumental in capturing the various steps in the pathwaof nucleotide incorporation. These experiments caachieve high sensitivity by using radiolabelled substratewhile the amount of enzyme required is small. It enablemeasurements of single enzyme turnover reactions withimillisecond time scales. Identification and quantificatio

of individual steps along the reaction sequence is madpossible by these methods.

Most of the early work on the DNA polymerasmechanism was performed using E. coli pol I or thKlenowfragment of this enzyme.Bacteriophage T7 andTDNA polymerases have also been used as model systemfor this kind of study. Although there are some functionaand kinetic differences between them, these enzymes showremarkably similar reaction pathways.

Mechanism

A minimal kinetic scheme (Johnson, 1993) of the DNApolymerization reaction by pol I enzymes is summarizebelow:

1. Binding of the primer/template DNA to the enzympositions the 3’ end of the primer strand near thpolymerase active site in the cleft.

2. Binding of a dNTP to the enzyme–DNA compleforms an enzyme–DNA–dNTP ternary complex.

3. A rate-limiting conformational change of the enzymleads to the formation of a productive ternarcomplex, which is poised for chemical reaction onucleotide addition.

4. The chemical reaction is a fast, kinetically nondetecable step. In the productive ternary complex, nucleophilic attack of the 3’ OH to the a-phosphate of thincoming dNTP forms the phosphodiester bond anthe products: the elongated DNA and a pyrophosphate. Divalent metal ions are required for thphosphoryl transfer reaction to happen.

5. A second rate-limiting conformational change returnthe enzyme to its original conformation and allowrelease of the pyrophosphate and translocation of thDNA to present the next template nucleotide to thpolymerase active site. The enzyme at this stage iready to enter the next cycle of nucleotide incorpora

tion (step 1).

During each cycle of nucleotide incorporation, thenzyme–DNA complex adopts two alternating conformations, referred to as ‘open’ and ‘closed’ (Figure 2). In thopen conformation, the dNTP binds to the enzyme–DNAcomplex at the dNTP-binding site. This binding eveninduces a rate-limiting conformational change in thenzyme from open to closed, which brings the dNTwithin the enzyme’s active site. In the closed conformation

Bacterial DNA Polymerase I

2 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net



Figure 1 Structure of the Klenow fragment of Escherichia coli DNA polymerase I showing the large polymerase domain and the small 3’ – 5’ exonucleadomain. Helices arelabelled with letters from A to R,whilestrandsarelabelledwithnumbersfrom1 to14. Thedivision betweenthe twodomainsis theloo

between helices F and G (Ollis et al ., 1985). This figure was produced using the coordinates with PDB entry code 1 dpi.

Figure 2 The open binary (a) and closed ternary (b) complexes of Klentaq with primer/template DNA and ddCTP. The N-terminal small domain of

Klentaq is shown in yellow; the thumb, palm and fingers sub-domain of the large C-terminal polymerase domain is shown in blue, magenta and greenrespectively, with theO-helix in thefingers domainshown in red; theprimer strandof theDNA is shown in silverand thetemplatestrandis shown in

cyan. The dCTP (shown in dark grey) in (a) is drawn to indicate the hypothetical dNTP binding site in the open complex; however, such an openternary complex has not been captured in a crystal structure. Modified from Li et al . (1998).





the enzyme and the substrates forma tight ternary complexin which they are positioned properly for the chemicalreaction to occur. Once the nucleotide is incorporated, theenzyme switches to the open conformation: DNA translo-cation and product release then occur. Comparison of studies on DNA pol I with those on HIV (Humanimmunodeficiency virus) I reverse transcriptase and mam-

malian DNA polymerase b suggests that this open-to-closed conformational switch may be a common mechan-ism for all polymerases (Doublie ´ et al ., 1999).

The fidelity of DNA pol I enzymes is about onemismatch per 108 to 1010 bases (Johnson, 1993). This ismuch higher than that predicted by the free energydifference between the correct and incorrect base pairs,which is about 1–3 kcalmol2 1 and gives an errorfrequency of about one mismatch per 5–150 bases. Thetwo-conformational state mechanism described aboveexplains how the enzyme achieves high fidelity (Johnson,1993). It has indeed been shown that the rate-limitingconformational change before the chemical reaction is very

sensitive to proper base pairing geometry. For T7 DNApolymerase, the conformational change is 2000–4000-foldslower when incorporating a mismatched nucleotide,contributing a factor of 2000–4000 to the fidelity of theenzyme. Furthermore, as suggested by kinetic andstructural studies, the rate-limiting conformational changeis not only sensitive to the base pairing between theincoming nucleotide and the single-stranded base on thetemplate, but also sensitive to the proper pairing of the baseincorporated in the previous cycle. When a mismatch isincorporated, the incorporation of the next nucleotide isstalled, allowing sufficient time for the 3’ –5’ exonuclease toremove the mismatched nucleotide.

The Klenow structure shows that the 3’ –5’ exonucleaseactive site is about 30 A ˚ away from the polymerase activesite. This suggests that at least 8–9 base pairs have to bemelted for the mismatched 3’ end of the primer strand tomove from the polymerase site into the exonuclease site.The kinetic mechanism for the exonuclease function hasbeen established based on studies on T7 DNA pol I(Johnson, 1993). Kinetic partitioning between the poly-merase site and the exonuclease site determines whether thepolymerase undergoes the polymerization reaction or theexonuclease reaction. Under normal conditions, thepolymerase favours primarily the polymerization reaction.However, when a mismatch is incorporated, the rate of

incorporation of the next correct nucleotide is greatlyreduced, while the rate of exonuclease activity is increased.These effects cumulate to increase overall fidelity by afactor of 200.

A two-metal ion mechanism for the catalysis at thepolymerase active site was first proposed by T. A. Steitz(Figure 3), based on comparison with the well-studied 3’ –5’exonuclease site (Beese and Steitz, 1991; Steitz, 1999). Thismechanism is supported by mutagenesis and structuralstudies. According to this mechanism, two divalent metal

ions are required for the catalysis of the phosphorytransfer reaction at the polymerase active site. One metaion, metal A, promotes the deprotonation of the 3’ OH anfacilitates the 3’ O2 attackontothea-phosphate; the othemetal ion, metal B, facilitates the leaving of the pyrophosphate group. Both metal ions help stabilize the pentacovalent transition state formed at the a-phosphate b

facilitating the formation of a 908 O-P-O angle. The twmetal ions are coordinated by three acidic residues lying athe bottom of the polymerase cleft, which are highlconserved among all polymerases. In the case of thexonuclease active site, the mechanism is similar excepthat the attacking group comes from a water molecule anthe leaving group is the 3’ OH of a primer strand. There aralso three highly conserved acidic residues coordinatinthe two metal ions at the exonuclease site.

Multiple Activity (Polymerase,

Nucleases, Reversal)As described above, E. coli pol I has three distincenzymatic activities in one polypeptide chain. The largfragment (Klenow fragment) has a polymerase activitthat is capable of synthesizing DNA on the 3 ’ OH side of nick in a dsDNA. It also has 3’ –5’ exonuclease activity oboth ssDNA and unpaired regions in dsDNA.

The small fragment contains the 5’ –3’ exonuclease/flap endonuclease activity. It degrades DNAinto mono- ooligonucleotides from the 5’ end. It also cleaves ribonucleic acid (RNA) primers at an RNA–DNA junction. Biochemical and structural studies suggest tha

this fragment has very similar structure and functions aeukaryotic flap endonuclease (FEN-1) enzymes annucleotide excision repair enzymes such as XP-G anRAD2 (Lieber, 1997). These enzymes recognize 5’ enbranched DNA structures and cleave the DNA at thbranch junction. The product of the cleavage can be monoor polynucleotides. Under some circumstances, the 5’ –3exonuclease domain can make endonucleolytic incision oa mismatched or distorted DNA region, in the absence othe 5’ terminus. The structure and mechanism of the 5’ –3exonuclease is less well understood compared with that othe large fragment and will not be discussed in detail in threview.

The pol I enzymes also catalyse pyrophosphorolysiwhich is the reversal of the polymerization reactionBecause of the high concentration of pyrophosphatrequired for this reaction and the relatively low concentration of inorganic pyrophosphate in the cells, the reversal opolymerization reaction may happen with very lowfrequencies in vivo and the possible biological significancfor this reaction has not been investigated.





Structural Information

Structural studies on the Klenow fragment of E. coli andBacillus stearothermophilus pol I enzymes, T7 DNApolymerase, as well as on the full-length and Klenowfragment of DNA pol I from Thermus aquaticus (Taq)show that DNA binding to these polymerases sharessignificant similarities (reviewed in Doublie ´ et al ., 1999).The primer/template DNA binds to the palm domain of the polymerasewith its 3’ endclose to the polymerase activesite, which is formed by a cluster of three highly conservedacidic side-chains (D705, D882 and E883 in Klenow) at thebottom of the palm domain. The thumb domain grips theDNA and holds it in position. The wrapping of the tip of the thumb domain around the duplex DNA may con-

tribute partially to the processivity of pol I enzymes. Theinteractions between the DNA and the protein areprimarily sequence-independent interactions with thephosphodiester backbone. The DNA is predominantly Bform with a kink in the single-stranded 5’ end of thetemplate strand. In none of these structures does the DNAgo through the cleft formed by the palm, fingers and thumbdomains (Figure2). About two base pairs near the active siteare partially unwound with decreased helical twist andwidened minor groove, which are characteristics of A form

DNA. The N3 position of purine basesand theO2 positioof pyrimidine bases on the minor groove side of the firsbase pair at the 3’ terminus form hydrogen bonds with twhighly conserved side-chains (corresponding to R668 anQ849 in E. coli pol I). Since only the N3 and O2 groupprovide the same, twofold symmetric hydrogen-acceptopatterns in both G.C and A.T base pairs, the ‘mino

groove recognition’ of these universal hydrogen-bondinacceptors by conserved protein side-chains providesequence-independent selectivity for correct base pairs.

A large conformational change in the fingers domaiwas revealed by the structure of a quaternary complex oT7 polymerase with primer/template DNA, incomindideoxyribonucleoside 5’ triphosphate (ddNTP) and processivity factor, thioredoxin, as well as by the ternarcomplexes of the Klenow fragment of Taq polymeras(Klentaq) with primer/template DNA and incominddNTP (Figure 2) (Doublie ´ et al ., 1999). The conformational change involves an 468 inward rotation of the tiof the fingers domain, resulting in a partial closing of th

cleft. This closed conformation positions the 3’ OH anincoming dNTP at the critical positions, allowing transienimmobilization of DNA and formation of a tight ternarcomplex ready for catalysis. In the closed form of thenzyme, a narrow pocket is formed around the incominnucleotide base pair, allowing only the correct base pair tfit in. This open-to-closed conformational change probably corresponds to the rate-limiting conformationachange identified by kinetic studies. In the closed complestructures, two divalent metal ions are observed at thpolymerase active site. Their positions relative to thvarious elements of the active site and the substrates arconsistent with the proposed two-metal ion mechanism fo

polymerase catalysis (Steitz, 1999; Figure 3).A cocrystal structure of the Klenow fragment of E. co

pol I with duplex DNA containing a 3’ overhang (Beesetal ., 1993) shows that theduplex part of the DNA binds ia similar way to that seen in other polymerase complexeHowever, the 3’ end overhang binds to the 3’ –5exonuclease site, in contrast to that seen in othepolymerase complexes, in which the 3’ primer end is parof the duplex DNA and binds to the polymerase site. Ashuttle mechanism whereby the exonuclease domain exertits function was proposed (see Mechanism above).

Biological Function

DNA pol I enzymes play an important role in DNAdamage repair in bacteria. Some physical or chemicaagents (ultraviolet light (UV), X-rays, alkylating agentsetc.) produce distorting lesions in DNA. In E. coli , excisioof UV-induced pyrimidine dimers and bulky lesions initiated by a protein complex, the UvrABC nucleaswhich makes endonucleolytic cleavages near the lesion

Figure3 Thetwometalionmechanism forthecatalysis atthe polymeraseactive site. Modified from Steitz (1999).





and generates free 5’ phosphate groups. The pyrimidinedimer is then cleaved by the 5’ –3’ exonuclease of pol I andthe gap is filled out by the polymerase, leaving only a nickonthe DNA.Thecoupling ofthe 5’ –3’ exonuclease activityto the polymerase activity in a single enzyme enables lesionexcision and gap filling at the same time, thus preventingthe 3’ end from being exposed to nuclease cleavage. Pol I is

remarkable in that it can promote replication from a nick,which involves degrading the DNA chain from the 5’ endand displacing it with a newly synthesized chain from the 3’end, resulting in a translated nick (nick translation). Therepair is completed by dissociation of pol I and ligation of the nick.

The 5’ –3’ exonuclease domain of DNA pol I alsorecognizes branched RNA structures and is able to cleavean RNA branch from an RNA–DNA junction. Thisfunction is essential in Okazaki fragment processingduringbacterial DNA replication. Okazaki fragments are oligo-nucleotide fragments of 1000–2000 residues. They aregenerated as intermediates of lagging strand synthesis

during DNA replication. Each Okazaki fragment isinitiated from a short RNA primer which is subsequentlyexcised by the 5’ –3’ exonuclease activity of DNA pol I andRNAase H: the resulting gaps are filled in by DNA pol Iand DNA fragments are ligated together to become onestrand.

Applications: Polymerase ChainReaction, etc.

The most important applications of DNA pol I enzymes

are in the polymerase chain reaction (PCR) and in DNAsequencing. PCR is a method of producing large quantitiesof a DNA fragment (Saiki et al ., 1988). It requires twooligonucleotides that flank the target DNA sequence toserve as primers. After annealing to the target DNAsequence, these primers are extended by DNA poly-merases. After denaturing and reannealing to primers,these newly synthesized DNA strands can serve astemplates for the next cycle of primer extension. Thus, intheory, n cycles of heat denaturing, annealing and primerextension result in exponential accumulation (2n) of thetarget DNA. Since its invention, the PCR method hasproved a powerful technique in molecular biology for

DNA cloning, mutagenesis and DNA sequence analysis.The method is not limited to DNA amplification: usingreverse transcriptases, PCR is also used to amplify RNAsequences.

Amplification of target DNA sequences by PCR can beaccomplished by DNA polymerases from many sources.These polymerases differ from each other in efficiency andfidelity. The introduction of the thermostable Taq DNApolymerase has greatly simplified the PCR method (Saikiet al ., 1988). With the ability to survive extended

incubation at 958C, Taq DNA polymerase remains activduring the heat denaturation step. Thus, it is no longenecessary to add fresh DNA polymerases after this stepAlthough lacking the 3’ –5’ exonuclease proofreadinactivity, the Taq DNA polymerase catalyses DNA synthesis in vitro with high accuracy. Furthermore, due to thhigh temperature optimum of the Taq polymerase, th

primer annealing and extension steps can be performed aelevated temperatures (558C instead of 378C), whicsignificantly improves the specificity, the homogeneity oproduct size and the yield of product.

DNA sequencing is also an important application oDNA polymerases in molecular biology. Current DNAsequencing protocols rely on the incorporation of ddNTPin order to terminate chain extension and generatsequence ladders (Sanger et al ., 1977). Because of iability to incorporate ddNTPs with high efficiency and tgenerate sequencing patterns with even band intensity anpeak height, T7 DNA pol I has been extensively used iDNA sequencing. However, because of the advantages o

thermocycling mentioned above, thermostable DNApolymerases are also commonly used in DNA sequencingIn particular, due to its high turnover number, lack of proofreading activity and ability to incorporate dyelabelled nucleotide analogue with high efficiency, TaDNA polymerase and its variants are the most useenzymes in automated sequencing methods.

Current Research Topics/UnansweredQuestions

Although considerable progress has been made towardefining the molecular principles of template-directeDNA polymerization, much remains unclear. A fundamental question is that of the determinant of fidelitySeveral reports have emphasized the observation of snuandclose fit of thepolymerase structure aroundthe nascenbase pair as the basis for fidelity. However, this argumentonly valid if one accepts the conventional view that mismatched base pair (wobble A.C o r G.T base pairs) haa configuration widely different from that of a matchebase pair (A.T or G.C). However, several reports havchallenged this view and shown that classical WatsonCrick hydrogen-bonding interactions between bases in th

base pair may not contribute much specificity durinnucleotide incorporation. Hence, the configuration of mismatch may not be as different as believed previouslyand therefore it remains unclear how the protein cadiscriminatebetweensimilarly configured mismatched anproper base pairing. Another property of DNA pol enzyme has not been clarified: how does the DNAtranslocate to present the next single-stranded templatbase to the active site of the polymerase. A possibmechanism for DNA translocation suggests that the DNA





is prevented from moving in the wrong direction by thepresence of a tyrosine residue (Y671 in Taq polymerase orY766 in E. coli pol I) in the finger domains onto which theDNA would calibrate itself properly against the active siteof the protein (Li etal ., 1998). In this mechanism, the DNAwould then be allowed to move only in the direction of polymerization. The crystal structures show that the

protein bound to DNA forms a quasicylinder around theDNA: the interior of that cylinder is lined mostly withpositively-charged residues, which interact in a sequence-nonspecific manner with the ribose-phosphate backboneof the duplex DNA. Hence, it is hypothesized that thiscylinder forms an electrostatic ‘tunnel’ where the DNA isfree to move. Only when the nucleotide is brought to theactive site, and forms the complex shown in Figure 2b,would the DNA be immobilized and the reaction of nucleotide addition occur (Johnson, 1993; Li et al ., 1998).However, this mechanism for DNA translocation is only aworking hypothesis that remains to be tested.

References

Beese LS, Derbyshire V and Steitz TA (1993) Structure of DNA

polymerase I Klenow fragment bound to duplex DNA. Science 260:

352–355.

Beese LS and Steitz TA (1991) Structural basis for the 3 ’ –5’ exonuclease

activity of Escherichia coli DNA polymerase I: a two metal ion

mechanism. EMBO Journal 10: 25–33.

Doublie S, Sawaya MR and Ellenberger T (1999) An open and closed

case for all polymerases. Structure 7: R31–R35.

Johnson KA (1993) Conformational coupling in DNA polymerase

fidelity. Annual Review of Biochemistry 62: 685–713.

Kornberg A andBakerTA (1992)DNA Replication, 2ndedn.New York:

Freeman.

Kornberg A, Lehman IR,Bessman MJ andSimmsES (1956)Enzymat

synthesis of deoxyribonucleic acid. Biochimica et Biophysica Acta 2

197–198.

Li Y, Korolev S and Waksman G (1998) Crystal structures of open an

closed forms of binary and ternary complexes of the large fragment o

Thermus aquaticus DNA polymerase I: structural basis for nucleotid

incorporation. EMBO Journal 17: 7514–7525.

Lieber MR (1997) The FEN-1 family of structure-specific nucleases i

eukaryotic DNA replication, recombination and repair. Bioessays 1233–240.

Ollis DL,Brick P, HamlinR, Xuong NG andSteitz TA (1985)Structu

of large fragment of Escherichia coli DNA polymerase I complexe

with dTMP. Nature 313: 762–766.

Saiki RK, Gelfand DH, Stoffel S etal . (1988) Primer-directed enzymat

amplification of DNA with a thermostable DNA polymerase. Scienc

239: 487–491.

Sanger F, Nicklen S and Coulson AR (1977) DNA sequencing wit

chain-terminating inhibitors. Proceedings of the National Academy o

Sciences of the USA 74: 5463–5467.

Steitz TA (1999) DNA polymerases: structure diversity and commo

mechanisms. Journal of Biological Chemistry 274: 17395–17398.

Further Reading

Goodman MF (1997) Hydrogen bonding revisited: geometric selectio

as a principal determinant of DNA replication fidelity. Proceedings

the National Academy of Sciences of the USA 94: 10493–10495.

Guckian KM, Krugh TR and Kool ET (1998) Solution structure of

DNA duplex containing a replicable difluorotoluene-adenine pai

Nature Structural Biology 5: 954–959.

Matray TJ and Kool ET (1999) A specific partner for abasic damage i

DNA. Nature 399: 704–708.

Moran S, Ren R X-F and Kool ET (1997) A thymidine triphosphat

shape analog lacking Watson–Crick pairing ability is replicated wit

high sequence selectivity. Proceedings of the National Academy

Sciences of the USA 94: 10506–10511.



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