Bacterial DNA Polymerase I

Ying 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 lesion repair, as well as DNA replication. These enzymes have a multidomain structure containing a polymerase activity, a proofreading 3 5 exonuclease activity and/or a 5 3 exonuclease activity within one polypeptide chain.

Secondary article
Article Contents
. Introduction . Basic Features of DNA Polymerization: The Enzyme and Substrates . Escherichia coli DNA Polymerase I as the Archetype Polymerase . Assays . Mechanism . Multiple Activity (Polymerase, Nucleases, Reversal) . Structural Information

Introduction
Bacterial DNA polymerase I (pol I) enzymes play an important role in deoxyribonucleic acid (DNA) replication and the repair of DNA lesions in prokaryotic organisms. These enzymes are multidomain proteins with each domain corresponding to a DNA polymerase activity, a proofreading 3 5 exonuclease activity and/or a 5 3 exonuclease activity respectively. The basic reaction catalysed by the polymerase domain is the templatedirected addition of a deoxyribonucleotide onto the 3 OH group of a DNA primer strand. The 3 5 exonuclease domain catalyses the cleavage of a mismatched nucleotide from the 3 end of the DNA. The 5 3 exonuclease domain has not only the 5 exonuclease activity, which is the cleavage of a nucleotide from the 5 end of the DNA, but also a ap endonuclease activity, which recognizes specically 5 ap single-stranded DNA structures and cleaves the ap at the single-strandeddouble-stranded DNA (ssDNAdsDNA) junction. Based on sequence comparison and structural studies, all the known DNA polymerases can be grouped into ve families: the pol I (or A) family, the pol a (B) family, the pol b (X) family, the pol III (C) family, and the reverse transcriptases (RT) family. Bacterial DNA polymerase I, as well as DNA polymerase from bacteriophage T7, belong to the pol I (A) family.

. Biological Function . Applications: Polymerase Chain Reaction, etc. . Current Research Topics/Unanswered Questions

Basic Features of DNA Polymerization: The Enzyme and Substrates

DNA polymerases catalyse the template-directed DNA polymerization reaction, which is the addition of deoxyribonucleoside 5 triphosphate (dNTP) onto the 3 end of a DNA primer strand, as outlined below:
dNMPn dNTP $ dNMPn1 PPi DNA DNA

polymerization reaction are a new primer/template DNA with an elongated primer strand ((dNMP)n 1 1) and an inorganic pyrophosphate (PPi). The reaction consists of a nucleophilic attack by the 3 OH group on the template strand to the a-phosphate group of the incoming dNTP. The overall direction of the DNA elongation is from 5 to 3. The addition of each dNTP to the 3 end of the primer strand is directed by correct base pairing of the incoming dNTP with the bases on the template strand. DNA polymerases have a broad substrate range, which enables them to incorporate dierent kinds of dNTPs. However, during each cycle of the polymerization reaction, the polymerases have to select the right substrate against a pool of structurally similar dNTPs. This is accomplished by altering substrate specicity at each step of the catalytic cycle. During DNA synthesis by DNA polymerases, the DNA translocates along the polymerase with each cycle of polymerization; thus a new base on the template strand is presented to the polymerase active site. The incorporation of a nucleotide takes place when it forms a correct base pair with this template base. The rate of DNA polymerization by DNA polymerases as well as the processivity of these enzymes vary widely. DNA pol I from Escherichia coli (E. coli pol I) is a moderately processive enzyme which extends a primer chain by about 10100 nucleotides before dissociating from the DNA. Its rate of polymerization is about 50 nucleotides per second. In contrast, the DNA pol I enzyme for bacteriophage T7 extends DNA primer strands at a rate of 300 nucleotides per second and dissociates from the DNA only after incorporating  1500 bases (Johnson, 1993).

The enzyme has two substrates: one is the (dNMP)n, which is the primer/template DNA with n residues on the primer strand; the other is dNTP. The products of the DNA
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Bacterial DNA Polymerase I

Escherichia coli DNA Polymerase I as the Archetype Polymerase

E. coli pol I was the rst DNA polymerase identied Kornberg and Baker, 1956). As deduced from the sequence of its encoding polA gene, E. coli pol I is a single polypeptide chain containing 928 residues with calculated molecular weight of 103 kDa. E. coli pol I has at least three distinct functions: (1) a DNA polymerase activity, which is responsible for the 5 3 DNA synthesis by this enzyme; (2) a 3 5 exonuclease activity, which cleaves a mismatched base from the primer terminus, and thus supports the proofreading activity; (3) a 5 3 exonuclease activity, which degrades DNA from its 5 end and generates monoor oligonucleotides. Limited proteolytic cleavage of pol I generates two active components (Kornberg and Baker, 1992): a large Cterminal fragment (  600 residues), also known as the Klenow fragment, contains the polymerase activity and the 3 5 exonuclease activity; and a small N-terminal fragment (  300 residues) contains only the 5 3 exonuclease activity. The large fragment carries out DNA synthesis on the 3 OH side of a nick in a dsDNA. It also carries out 3 5 exonuclease activity on both ssDNA or unpaired regions in dsDNA. The small fragment degrades DNA from the 5 end and is capable of excising mismatched regions in duplex DNA. crystal structure of the Klenow fragment of E. The 3.3 A coli pol I complexed with a deoxythymidine monophosphate (dTMP) was the rst structure solved for a DNA polymerase (Ollis et al., 1985). This structure (Figure 1) shows that the enzyme is folded into two distinct domains. The smaller domain (approximately the rst 200 Nterminal residues) consists of a central parallel b sheet anked by a helices. The larger domain (about 400 residues at the C-terminus) is mainly a-helical. This domain has a shape reminiscent of a right hand, with a large cleft between the thumb, palm and ngers subdomains. Sitedirected mutagenesis studies have identied the separate functions of the large and small domains: the 3 5 exonuclease active site is located in the small domain and the polymerase active site is located in the cleft of the large ) between the domain. There is a large distance (  30 A polymerase active site and the 3 5 exonuclease active site in the Klenow structure.

tion of chemical quench ow techniques, has been instrumental in capturing the various steps in the pathway of nucleotide incorporation. These experiments can achieve high sensitivity by using radiolabelled substrates, while the amount of enzyme required is small. It enables measurements of single enzyme turnover reactions within millisecond time scales. Identication and quantication of individual steps along the reaction sequence is made possible by these methods. Most of the early work on the DNA polymerase mechanism was performed using E. coli pol I or the Klenow fragment of this enzyme. Bacteriophage T7 and T4 DNA polymerases have also been used as model systems for this kind of study. Although there are some functional and kinetic dierences between them, these enzymes show remarkably similar reaction pathways.

Mechanism
A minimal kinetic scheme (Johnson, 1993) of the DNA polymerization reaction by pol I enzymes is summarized below: 1. Binding of the primer/template DNA to the enzyme positions the 3 end of the primer strand near the polymerase active site in the cleft. 2. Binding of a dNTP to the enzymeDNA complex forms an enzymeDNAdNTP ternary complex. 3. A rate-limiting conformational change of the enzyme leads to the formation of a productive ternary complex, which is poised for chemical reaction of nucleotide addition. 4. The chemical reaction is a fast, kinetically nondetectable step. In the productive ternary complex, nucleophilic attack of the 3 OH to the a-phosphate of the incoming dNTP forms the phosphodiester bond and the products: the elongated DNA and a pyrophosphate. Divalent metal ions are required for this phosphoryl transfer reaction to happen. 5. A second rate-limiting conformational change returns the enzyme to its original conformation and allows release of the pyrophosphate and translocation of the DNA to present the next template nucleotide to the polymerase active site. The enzyme at this stage is ready to enter the next cycle of nucleotide incorporation (step 1). During each cycle of nucleotide incorporation, the enzymeDNA complex adopts two alternating conformations, referred to as open and closed (Figure 2). In the open conformation, the dNTP binds to the enzymeDNA complex at the dNTP-binding site. This binding event induces a rate-limiting conformational change in the enzyme from open to closed, which brings the dNTP within the enzymes active site. In the closed conformation,

Assays
The mechanism by which the pol I enzymes achieve their high delity DNA synthesis has been the subject of extensive studies. Chemical, kinetic and structural approaches have been used to investigate the mechanistic details of polymerization and proofreading activities. The use of transient kinetic methods, especially the introduc2

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Bacterial DNA Polymerase I

Figure 1 Structure of the Klenow fragment of Escherichia coli DNA polymerase I showing the large polymerase domain and the small 3 5 exonuclease domain. Helices are labelled with letters from A to R, while strands are labelled with numbers from 1 to 14. The division between the two domains is the loop 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 green, respectively, with the O-helix in the fingers domain shown in red; the primer strand of the DNA is shown in silver and the template strand is 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 open ternary complex has not been captured in a crystal structure. Modified from Li et al. (1998).

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Bacterial DNA Polymerase I

the enzyme and the substrates form a tight ternary complex in which they are positioned properly for the chemical reaction to occur. Once the nucleotide is incorporated, the enzyme switches to the open conformation: DNA translocation and product release then occur. Comparison of studies on DNA pol I with those on HIV (Human immunodeciency virus) I reverse transcriptase and mammalian DNA polymerase b suggests that this open-toclosed conformational switch may be a common mechan et al., 1999). ism for all polymerases (Doublie The delity of DNA pol I enzymes is about one mismatch per 108 to 1010 bases (Johnson, 1993). This is much higher than that predicted by the free energy dierence between the correct and incorrect base pairs, which is about 13 kcal mol 2 1 and gives an error frequency of about one mismatch per 5150 bases. The two-conformational state mechanism described above explains how the enzyme achieves high delity (Johnson, 1993). It has indeed been shown that the rate-limiting conformational change before the chemical reaction is very sensitive to proper base pairing geometry. For T7 DNA polymerase, the conformational change is 20004000-fold slower when incorporating a mismatched nucleotide, contributing a factor of 20004000 to the delity of the enzyme. Furthermore, as suggested by kinetic and structural studies, the rate-limiting conformational change is not only sensitive to the base pairing between the incoming nucleotide and the single-stranded base on the template, but also sensitive to the proper pairing of the base incorporated in the previous cycle. When a mismatch is incorporated, the incorporation of the next nucleotide is stalled, allowing sucient time for the 3 5 exonuclease to remove the mismatched nucleotide. The Klenow structure shows that the 3 5 exonuclease away from the polymerase active active site is about 30 A site. This suggests that at least 89 base pairs have to be melted for the mismatched 3 end of the primer strand to move from the polymerase site into the exonuclease site. The kinetic mechanism for the exonuclease function has been established based on studies on T7 DNA pol I (Johnson, 1993). Kinetic partitioning between the polymerase site and the exonuclease site determines whether the polymerase undergoes the polymerization reaction or the exonuclease reaction. Under normal conditions, the polymerase favours primarily the polymerization reaction. However, when a mismatch is incorporated, the rate of incorporation of the next correct nucleotide is greatly reduced, while the rate of exonuclease activity is increased. These eects cumulate to increase overall delity by a factor of 200. A two-metal ion mechanism for the catalysis at the polymerase active site was rst proposed by T. A. Steitz (Figure 3), based on comparison with the well-studied 3 5 exonuclease site (Beese and Steitz, 1991; Steitz, 1999). This mechanism is supported by mutagenesis and structural studies. According to this mechanism, two divalent metal
4

ions are required for the catalysis of the phosphoryl transfer reaction at the polymerase active site. One metal ion, metal A, promotes the deprotonation of the 3 OH and facilitates the 3 O 2 attack on to the a-phosphate; the other metal ion, metal B, facilitates the leaving of the pyrophosphate group. Both metal ions help stabilize the pentacovalent transition state formed at the a-phosphate by facilitating the formation of a 908 O-P-O angle. The two metal ions are coordinated by three acidic residues lying at the bottom of the polymerase cleft, which are highly conserved among all polymerases. In the case of the exonuclease active site, the mechanism is similar except that the attacking group comes from a water molecule and the leaving group is the 3 OH of a primer strand. There are also three highly conserved acidic residues coordinating the two metal ions at the exonuclease site.

Multiple Activity (Polymerase, Nucleases, Reversal)

As described above, E. coli pol I has three distinct enzymatic activities in one polypeptide chain. The large fragment (Klenow fragment) has a polymerase activity that is capable of synthesizing DNA on the 3 OH side of a nick in a dsDNA. It also has 3 5 exonuclease activity on both ssDNA and unpaired regions in dsDNA. The small fragment contains the 5 3 exonuclease/5 ap endonuclease activity. It degrades DNA into mono- or oligonucleotides from the 5 end. It also cleaves 5 ribonucleic acid (RNA) primers at an RNADNA junction. Biochemical and structural studies suggest that this fragment has very similar structure and functions as eukaryotic ap endonuclease (FEN-1) enzymes and nucleotide excision repair enzymes such as XP-G and RAD2 (Lieber, 1997). These enzymes recognize 5 end branched DNA structures and cleave the DNA at the branch junction. The product of the cleavage can be monoor polynucleotides. Under some circumstances, the 5 3 exonuclease domain can make endonucleolytic incision on a mismatched or distorted DNA region, in the absence of the 5 terminus. The structure and mechanism of the 5 3 exonuclease is less well understood compared with that of the large fragment and will not be discussed in detail in this review. The pol I enzymes also catalyse pyrophosphorolysis, which is the reversal of the polymerization reaction. Because of the high concentration of pyrophosphate required for this reaction and the relatively low concentration of inorganic pyrophosphate in the cells, the reversal of polymerization reaction may happen with very low frequencies in vivo and the possible biological signicance for this reaction has not been investigated.

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Bacterial DNA Polymerase I

Figure 3 The two metal ion mechanism for the catalysis at the polymerase active site. Modified from Steitz (1999).

Structural Information
Structural studies on the Klenow fragment of E. coli and Bacillus stearothermophilus pol I enzymes, T7 DNA polymerase, as well as on the full-length and Klenow fragment of DNA pol I from Thermus aquaticus (Taq) show that DNA binding to these polymerases shares et al., 1999). signicant similarities (reviewed in Doublie The primer/template DNA binds to the palm domain of the polymerase with its 3 end close to the polymerase active site, which is formed by a cluster of three highly conserved acidic side-chains (D705, D882 and E883 in Klenow) at the bottom of the palm domain. The thumb domain grips the DNA and holds it in position. The wrapping of the tip of the thumb domain around the duplex DNA may contribute partially to the processivity of pol I enzymes. The interactions between the DNA and the protein are primarily sequence-independent interactions with the phosphodiester backbone. The DNA is predominantly B form with a kink in the single-stranded 5 end of the template strand. In none of these structures does the DNA go through the cleft formed by the palm, ngers and thumb domains (Figure 2). About two base pairs near the active site are partially unwound with decreased helical twist and widened minor groove, which are characteristics of A form

DNA. The N3 position of purine bases and the O2 position of pyrimidine bases on the minor groove side of the rst base pair at the 3 terminus form hydrogen bonds with two highly conserved side-chains (corresponding to R668 and Q849 in E. coli pol I). Since only the N3 and O2 groups provide the same, twofold symmetric hydrogen-acceptor patterns in both G.C and A.T base pairs, the minor groove recognition of these universal hydrogen-bonding acceptors by conserved protein side-chains provides sequence-independent selectivity for correct base pairs. A large conformational change in the ngers domain was revealed by the structure of a quaternary complex of T7 polymerase with primer/template DNA, incoming dideoxyribonucleoside 5 triphosphate (ddNTP) and a processivity factor, thioredoxin, as well as by the ternary complexes of the Klenow fragment of Taq polymerase (Klentaq) with primer/template DNA and incoming et al., 1999). The conformaddNTP (Figure 2) (Doublie tional change involves an  468 inward rotation of the tip of the ngers domain, resulting in a partial closing of the cleft. This closed conformation positions the 3 OH and incoming dNTP at the critical positions, allowing transient immobilization of DNA and formation of a tight ternary complex ready for catalysis. In the closed form of the enzyme, a narrow pocket is formed around the incoming nucleotide base pair, allowing only the correct base pair to t in. This open-to-closed conformational change probably corresponds to the rate-limiting conformational change identied by kinetic studies. In the closed complex structures, two divalent metal ions are observed at the polymerase active site. Their positions relative to the various elements of the active site and the substrates are consistent with the proposed two-metal ion mechanism for polymerase catalysis (Steitz, 1999; Figure 3). A cocrystal structure of the Klenow fragment of E. coli pol I with duplex DNA containing a 3 overhang (Beese et al., 1993) shows that the duplex part of the DNA binds in a similar way to that seen in other polymerase complexes. However, the 3 end overhang binds to the 3 5 exonuclease site, in contrast to that seen in other polymerase complexes, in which the 3 primer end is part of the duplex DNA and binds to the polymerase site. A shuttle mechanism whereby the exonuclease domain exerts its function was proposed (see Mechanism above).

Biological Function
DNA pol I enzymes play an important role in DNA damage repair in bacteria. Some physical or chemical agents (ultraviolet light (UV), X-rays, alkylating agents, etc.) produce distorting lesions in DNA. In E. coli, excision of UV-induced pyrimidine dimers and bulky lesions is initiated by a protein complex, the UvrABC nuclease, which makes endonucleolytic cleavages near the lesions
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Bacterial DNA Polymerase I

and generates free 5 phosphate groups. The pyrimidine dimer is then cleaved by the 5 3 exonuclease of pol I and the gap is lled out by the polymerase, leaving only a nick on the DNA. The coupling of the 5 3 exonuclease activity to the polymerase activity in a single enzyme enables lesion excision and gap lling at the same time, thus preventing the 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 end and displacing it with a newly synthesized chain from the 3 end, resulting in a translated nick (nick translation). The repair is completed by dissociation of pol I and ligation of the nick. The 5 3 exonuclease domain of DNA pol I also recognizes branched RNA structures and is able to cleave an RNA branch from an RNADNA junction. This function is essential in Okazaki fragment processing during bacterial DNA replication. Okazaki fragments are oligonucleotide fragments of 10002000 residues. They are generated as intermediates of lagging strand synthesis during DNA replication. Each Okazaki fragment is initiated from a short RNA primer which is subsequently excised by the 5 3 exonuclease activity of DNA pol I and RNAase H: the resulting gaps are lled in by DNA pol I and DNA fragments are ligated together to become one strand.

incubation at 958C, Taq DNA polymerase remains active during the heat denaturation step. Thus, it is no longer necessary to add fresh DNA polymerases after this step. Although lacking the 3 5 exonuclease proofreading activity, the Taq DNA polymerase catalyses DNA synthesis in vitro with high accuracy. Furthermore, due to the high temperature optimum of the Taq polymerase, the primer annealing and extension steps can be performed at elevated temperatures (558C instead of 378C), which signicantly improves the specicity, the homogeneity of product size and the yield of product. DNA sequencing is also an important application of DNA polymerases in molecular biology. Current DNA sequencing protocols rely on the incorporation of ddNTPs in order to terminate chain extension and generate sequence ladders (Sanger et al., 1977). Because of its ability to incorporate ddNTPs with high eciency and to generate sequencing patterns with even band intensity and peak height, T7 DNA pol I has been extensively used in DNA sequencing. However, because of the advantages of thermocycling mentioned above, thermostable DNA polymerases are also commonly used in DNA sequencing. In particular, due to its high turnover number, lack of a proofreading activity and ability to incorporate dyelabelled nucleotide analogue with high eciency, Taq DNA polymerase and its variants are the most used enzymes in automated sequencing methods.

Applications: Polymerase Chain Reaction, etc.

The most important applications of DNA pol I enzymes are in the polymerase chain reaction (PCR) and in DNA sequencing. PCR is a method of producing large quantities of a DNA fragment (Saiki et al., 1988). It requires two oligonucleotides that ank the target DNA sequence to serve as primers. After annealing to the target DNA sequence, these primers are extended by DNA polymerases. After denaturing and reannealing to primers, these newly synthesized DNA strands can serve as templates for the next cycle of primer extension. Thus, in theory, n cycles of heat denaturing, annealing and primer extension result in exponential accumulation (2n) of the target DNA. Since its invention, the PCR method has proved a powerful technique in molecular biology for DNA cloning, mutagenesis and DNA sequence analysis. The method is not limited to DNA amplication: using reverse transcriptases, PCR is also used to amplify RNA sequences. Amplication of target DNA sequences by PCR can be accomplished by DNA polymerases from many sources. These polymerases dier from each other in eciency and delity. The introduction of the thermostable Taq DNA polymerase has greatly simplied the PCR method (Saiki et al., 1988). With the ability to survive extended
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Current Research Topics/Unanswered Questions

Although considerable progress has been made toward dening the molecular principles of template-directed DNA polymerization, much remains unclear. A fundamental question is that of the determinant of delity. Several reports have emphasized the observation of snug and close t of the polymerase structure around the nascent base pair as the basis for delity. However, this argument is only valid if one accepts the conventional view that a mismatched base pair (wobble A.C or G.T base pairs) has a conguration widely dierent from that of a matched base pair (A.T or G.C). However, several reports have challenged this view and shown that classical Watson Crick hydrogen-bonding interactions between bases in the base pair may not contribute much specicity during nucleotide incorporation. Hence, the conguration of a mismatch may not be as dierent as believed previously, and therefore it remains unclear how the protein can discriminate between similarly congured mismatched and proper base pairing. Another property of DNA pol I enzyme has not been claried: how does the DNA translocate to present the next single-stranded template base to the active site of the polymerase. A possible mechanism for DNA translocation suggests that the DNA

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Bacterial DNA Polymerase I

is prevented from moving in the wrong direction by the presence of a tyrosine residue (Y671 in Taq polymerase or Y766 in E. coli pol I) in the nger domains onto which the DNA would calibrate itself properly against the active site of the protein (Li et al., 1998). In this mechanism, the DNA would 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 the DNA: the interior of that cylinder is lined mostly with positively-charged residues, which interact in a sequencenonspecic manner with the ribose-phosphate backbone of the duplex DNA. Hence, it is hypothesized that this cylinder forms an electrostatic tunnel where the DNA is free to move. Only when the nucleotide is brought to the active 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 a working hypothesis that remains to be tested.

Kornberg A, Lehman IR, Bessman MJ and Simms ES (1956) Enzymatic synthesis of deoxyribonucleic acid. Biochimica et Biophysica Acta 21: 197198. Li Y, Korolev S and Waksman G (1998) Crystal structures of open and closed forms of binary and ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I: structural basis for nucleotide incorporation. EMBO Journal 17: 75147525. Lieber MR (1997) The FEN-1 family of structure-specic nucleases in eukaryotic DNA replication, recombination and repair. Bioessays 19: 233240. Ollis DL, Brick P, Hamlin R, Xuong NG and Steitz TA (1985) Structure of large fragment of Escherichia coli DNA polymerase I complexed with dTMP. Nature 313: 762766. Saiki RK, Gelfand DH, Stoel S et al. (1988) Primer-directed enzymatic amplication of DNA with a thermostable DNA polymerase. Science 239: 487491. Sanger F, Nicklen S and Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences of the USA 74: 54635467. Steitz TA (1999) DNA polymerases: structure diversity and common mechanisms. Journal of Biological Chemistry 274: 1739517398.

References
Beese LS, Derbyshire V and Steitz TA (1993) Structure of DNA polymerase I Klenow fragment bound to duplex DNA. Science 260: 352355. 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: 2533. S, Sawaya MR and Ellenberger T (1999) An open and closed Doublie case for all polymerases. Structure 7: R31R35. Johnson KA (1993) Conformational coupling in DNA polymerase delity. Annual Review of Biochemistry 62: 685713. Kornberg A and Baker TA (1992) DNA Replication, 2nd edn. New York: Freeman.

Further Reading
Goodman MF (1997) Hydrogen bonding revisited: geometric selection as a principal determinant of DNA replication delity. Proceedings of the National Academy of Sciences of the USA 94: 1049310495. Guckian KM, Krugh TR and Kool ET (1998) Solution structure of a DNA duplex containing a replicable diuorotoluene-adenine pair. Nature Structural Biology 5: 954959. Matray TJ and Kool ET (1999) A specic partner for abasic damage in DNA. Nature 399: 704708. Moran S, Ren R X-F and Kool ET (1997) A thymidine triphosphate shape analog lacking WatsonCrick pairing ability is replicated with high sequence selectivity. Proceedings of the National Academy of Sciences of the USA 94: 1050610511.

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