Recent studies of the fidelity of DNA synthesis

Biochimica et Biophysica Acta, 951 (1988) 1-15 Elsevier

1

BBA 91867

Review Recent studies of the fidelity of D N A synthesis T h o m a s A. K u n k e l a n d K a t a r z y n a B e b e n e k Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC (U.S.A.) (Received 2 August 1988)

Key words: DNA polymerase; Fidelity; Mutagenesis; DNA synthesis

Con~n~ I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

II.

Fidelity of purified DNA polymerases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Base substitution fidelity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Nucleotide selectivity of polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Exonucleolytic proofreading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Base deletion/addition fidelity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

i11. Fidelity of complex systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

12

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13

References

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I. Introduction The biological consequences of mutations in genetic material can be either positive, neutral or negative. Some mutations must occur to permit species evolution and some may be actively promoted when diversity is desirable. Conversely, errors that lead to alteration or loss of regulatory or structural information can be lethal. Mutation rates must therefore be carefully controlled.

Correspondence: D.A. Kunkel, Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, U.S.A.

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14

In organisms spanning the range from bacteria to higher eukaryotes, the mutation rate is remarkably low, between 10 -9 and 10-~2 errors per basepair per generation [1-4]. These mutation rates are achieved by multiple steps to discriminate against errors [5]. In this article, we focus on the two discrimination steps that operate during DNA synthesis. The first is the ability of a DNA polymerase to incorporate the correct rather than the incorrect nucleotide onto a properly aligned template-primer. The second is the ability of a DNA polymerase-associated 3' ---, 5' exonuclease activity to remove (proofread) an error prior to continued synthesis, thus giving the polymerase a second chance to insert the correct nucleotide. We begin with studies of how these two steps influence base

0167-4781/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

substitution fidelity, with emphasis on efforts to define mechanisms and understand how D N A and protein structures determine error rates. This is followed by a description of how the structurally dynamic properties of D N A can lead to errors resulting from template-primer misalignments. Finally, while most fidelity studies have involved highly purified D N A polymerases, we review several efforts to describe the fidelity of D N A synthesis catalyzed by more complex systems. We focus here on recent studies: for descriptions of earlier work and, more importantly, for perspectives that are in some instances different from ours, the reader can consult any of several reviews

I5-81. I!. Fidelity of purified DNA polymerams II-A. Bare substitution fidelity Base substitution fidelity during D N A synthesis in vitro has been monitored in assays that use single-stranded circular D N A templates. The first templates to be used contained nonsense codons in essential genes of bacteriophage q~X174 [9] (for a review see Ref. 7). A similar approach has more recently been developed [10] using the lacZa-complementation gene of bacteriophage M13mp2. Base substitution errors produced at the nonsense codon during synthesis with these viral D N A template are detected by transfecting the appropriate competent E. coli host cells with the products of the reaction, to determine the frequency of phage that have revertant phenotypes. These reversion assays are highly sensitive (i.e., they have a low background spontaneous revertant frequency) and are most useful for high fidelity synthesis reactions. The second assay [11] monitors D N A synthesis errors in the non-essential lacZa-complementation gene of M l 3 m p 2 as phage plaques that are light blue or colorless. All 12 possible base-mispairs can be detected, thus providing a detailed description of base substitution fidelity. Since these can be scored at a variety of template positions, the influences of neighboring base sequences can be evaluated.

I I- A. 1. Nucleotide selectivity during polymerization Nucleotide selectivity during polymerization has been described by measuring the fidelity of D N A

synthesis catalyzed by D N A polymerases lacking associated proofreading exonuclease activity. A striking conclusion of these studies is that error rates span at least a 6000-fold range, from 1 error in 130 bases incorporated at a mutational hot spot [12] to less than 1 error in 830000 bases polymerizcd [13]. Attempts to understand this wide variation in fidelity at the polymerization step require an appreciation for the parameters that affect single base substitution error rates. First, an influence of the D N A polymerase on nucleotide selectivity has been observed in a number of studies. One example is shown in Table I, A. At position 87 in the lacZa gene in Ml3mp2, the incorrect nucleotide d G T P is misinserted opposite a template T by chick embryo D N A polymerase/3 (Pol /3) once for every 490 insertions of the correct nucleotide, dATP. The same error is committed 12-fold less frequently by human KB cell D N A polymerase a (Pol a), clearly demonstrating that nucleotide selectivity is influenced by

TABLE I VARIABLES THAT EFFECT NUCLEOTIDE SELECTIVITY DURING POLYMERIZATION This is not intended to be a comprehensive compilation of published fidelity results that describe enzyme, site and mispair differences. These effects have also been described using a gel electrophoresis assay for misincorporation (e.g., see Ref. 16). Error rates were calculated per detectable nucleotide polymerized, as described in Refs. 12 and 14. Mispair

DNA polymerase

Errorrate

Rela- Reference tive value

(A) Same site. same mispair, different polymerase T.G chick Pol 13 1/490 1 14 KB cell Pol a 1/6000 12 14 (B) Same mispair, same polymerase, different site Gis,~. A chick Pol a 1/1 200 1 14 Gother. A 1/11 000 9 14 (C) Same site, same polymerase, different mispair T.G calf Pol ~x 1/14000 1 15 T.T

< 1/500000

~ 36

15

(D) Same mispair, same polymerase, different s y m m e t ~ G.A calf Pol a 1/36000 1 15 A'G < 1/250000 > 7 15 C.A chick Pol a 1/2000 1 14 A.C 1/67000 33 14

the interaction of the protein with the templateprimer. While a polymerases select the correct base more often than fl polymerases at most sites, Pol fl can actually be more accurate than Pol a at particular sites [14]. Nucleotide selectivity for the same polymerase and mispair can vary, depending on the surrounding template-primer sequence. Thus, in a single reaction, chick embryo DNA polymerase a produced G. A mispairs (our convention for mispairs is to list the template base first then the incoming nucleotide) at least 9-fold more often at position 159 in the lacZa sequence than at 14 other positions where this error can be detected (Table I, B). Selectivity likewise depends on the composition of the mispair, because at the same template site, the same polymerase [15] misincorporates different nucleotides with very different efficiencies (Table 1, C). Finally, the error rate depends on the symmetry of the mispair; large differences are observed for the same enzyme and mispair depending on which nucleotide was in the template and which was the incoming triphosphate (Table 1, D). These results demonstrate that during DNA synthesis there are indeed 12 possible mispairs by composition and symmetry. Since the surrounding DNA sequence and the protein can affect the structure of each of these, the number of possible structures that must be discriminated against to assure high base substitution fidelity is potentially enormous. Given this complexity, it is not surprising that wide variations in nucleotide selectivity are observed. Diverse approaches are being taken to understand the molecular basis of nucleotide discrimination during polymerization. In the simplest model for discrimination at the insertion step, the probability of nucleotide insertion is determined only by free energy differences between mispaired and correctly paired nucleotides [5]. However, the observed accuracy of polymerases for certain single base substitution errors is substantially better than is predicted from physical measurements of free energy differences for various base-pairs and mispairs in aqueous solution [5,17]. Petruska et al. [18] have suggested a possible explaination for this. Free energy differences in aqueous solution may be an underestimate of the actual free energy differences that are possible, because hydrogen

bonding can occur between bases and surrounding water molecules. However, water molecules may be partially excluded within the active site of DNA polymerase, magnifying free energy differences to the degree necessary to account for the observed base substitution fidelity of exonucleasedeficient polymerases. One approach for describing the enzymatic basis for nucleotide selectivity is to determine the enzyme kinetic constants for insertion of incorrect and correct nucleotides. Using a simple gel electrophoresis assay to measure polymerization at a single defined template position [19], nucleotide selectivity during catalysis by highly purified Drosophila DNA polymerase a has been shown to rely primarily on differences in the K m for insertion of the incorrect versus the correct dNTP substrate. In contrast, observations with E. coli DNA polymerase I suggest that the nucleotide selectivity of this polymerase depends primarily on a large difference in the rate of phosphodiester bond formation for incorrect versus correct substrates [20]. This effect may reflect a conformational change in the polymerase to reject incorrectly paired incoming nucleotides. Evidence that Pol I changes conformation during polymerization has been obtained by rapid-quench kinetic techniques [21,22], which have revealed a slow, ratelimiting step during polymerization that precedes chemical bond formation, and from nuclear magnetic resonance studies [23]. The data suggest that a first-order isomerization of the enzyme-polynucleotide-dNTP complex may occur to place the dNTP substrate in the appropriate position for chemical bond formation. These fidelity and kinetic assays are complemented by new information on the structure of DNA polymerases. For example, the gene fragment for the 'large (Klenow) fragment' of E. coil DNA polymerase I has been cloned and the protein has been overproduced in quantities sufficient to permit a determination of its structure by X-ray crystallography [24]. The enzyme has two distinct domains, one for the polymerase and one for the proofreading exonuclease (Fig. 1). The polymerase active site resides in the C-terminal domain [25], which contains 408 amino acids and a deep cleft with dimensions that can accommodate doublestranded B-DNA. Attempts are in progress to

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determine the exact location of the active site [26,27] and to define critical residues for substrate binding and catalysis. In addition to polymerase structural studies, the structures of base-mispairs are being defined in solution by nuclear magnetic resonance techniques (for a review, see Ref. 28) and in the solid state by X-ray crystallography (for a review, see Ref. 29). While results with these approaches must be interpreted with caution, because they define the structure of mispairs in double-stranded D N A

rather than at a template-primer tertninus within the active site of a polymerase, they nevertheless provide an excellent framework for considering the effects described in Table I. Information on the structure of G . I - , A • (" and G . A mispairs is now available. All three mispairs are easily accomodated in double-stranded D N A without dramatic effects on the local structure of the helix. Therefore, differences in nucleotide selectivity are unlikely to depend on the structure of the sugar-phosphate backbone itself. Discrimination is more likely' to depend on differences in the orientation of the bases relative to the backbone, and on the nature and pattern of functional groups. For example, the crystal structure of the G . A mispair in the B-type helix suggests that the sugar-base orientation is a n t i for G but ,~yn for A. creating two hydrogen bonds. The A - G mispair symmetry difference in Table ID could refect the fact that a template A cannot assume the svn conformation required to mispair with an incoming G because stacking interactions with neighboring bases prevent this rotation, while an incoming A opposite G can easily rotate from a n t i to s v n .

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NMR results with the G. A mispair in a different sequence context [28] demonstrate that both bases are in the anti conformation, suggesting that the surrounding sequence may influence the orientation of the bases within mispairs. This may reflect base stacking effects; the presence of mispairs in double-stranded DNA can actually improve certain stacking interactions with neighboring bases [29]. The orientation of each basepair or mispair can also be defined by the angle, lambda, between the C - 1 ' - . . C-1' vector and the glycosyl bond, C1'-N-1 (pyrimidine) or C-1'-N-9 (purine). The degree of symmetry between the lambda values for any two bases in a pair is characteristic and very different for correct versus incorrect basepairs (Fig. 2), providing one possible basis for discrimination. Enzyme- and mispair-dependent differences in fidelity may also reflect interactions between polymerase active sites and functional groups of mispairs that project into the major and minor grooves. The distribution of these groups is displayed in Fig. 3 (taken from Ref. 29) by stick diagrams, where those groups that point into the major groove are above the base line and those that point into the minor groove are below the line. Comparison of the distribution patterns between correct and incorrect basepairs shows dis-

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tinctive features (discussed in greater detail in Ref. 29) that may be important for nucleotide discrimination during polymerization. Existing X-ray [29] and NMR data [30] suggest that the A. C mispair contains two hydrogen bonds, one of which involves protonation of adenine at the N-1 position. This suggests the possibility that polymerases could influence nucleotide selection by changing the pK a values of the bases.

II-A.2. Exonucleolytic proofreading Proofreading is the 3'--* 5' exonucleolytic reversal of polymerization to remove nucleotides that have been incorrectly inserted during a polymerization reaction. Since proofreading is the subject of a recent review [31], in this section, we briefly discuss only certain key points. These include information on the structure of proofreading enzymes and on the balance of enzyme rate constants that determine the contribution of proofreading to fidelity, and evidence that proofreading operates during DNA synthesis by eukaryotic DNA polymerases. One of the most exciting advances in the study of proofreading is the availability of information on the structure, spatial relationship and binding properties of the polymerase and exonuclease active sites of the Klenow fragment of E. coli DNA polymerase I. In addition to the large C-terminal polymerase domain, the Klenow fragment has an N-terminal domain (approx. 200 amino acids) containing the exonuclease active site [32]. In the absence of DNA, the polymerase and exonuclease active sites are remote from each other, about 30 ,~ apart (Fig. 1). The physical separation of the two active sites and the differences in their binding characteristics may be essential for the editing decision [27]. Misinsertion of a base creates an unstable 'frayed' primer terminus that is less likely to remain bound to the polymerase active site, which prefers to bind duplex DNA. In order to proofread the error, the primer terminus would be required to slide on the protein over a distance equivalent to 8 base-pairs (Fig. 1). This sliding would occur in a spiral fashion in order to maintain optimal DNA-protein contacts in the binding cleft of the polymerase domain. The terminal 3 or 4 basepairs melt out, permitting efficient bind-

ing of the single-stranded DNA to the exonuclease active site for excision of the terminal base. While detailed structural information is not yet available for other proofreading enzymes, several studies suggest common features among different enzymes [33-35]. Like Pol I, for example, "I'4 DNA polymerase contains both polymerase and proofreading exonuclease activities within the same polypeptide chain. By comparing the primary DNA sequence of the gene for the T4 DNA polymerase with the DNA sequences of the genes for other DNA polymerases, Spicer et al, [35] suggest that the T4 DNA polymerase activity resides in the C-terminal portion of the coding sequence, whereas the exonuclease is encoded by the N-terminal sequence. The same conclusion has been derived from the DNA sequence analysis of the polymerase gene from 17 different T4 DNA polymerase mutator mutants [36]. This latter approach identifies amino acids that are critical for polymerization and proofreading fidelity. One of several criteria suggesting that an exonuclease may function to proofread errors during polymerization is its physical association with the DNA polymerase. This association is apparent, even with a multimeric protein like E. coli DNA polymerase 1II. However, unlike the Pol I or T4 DNA polymerases, here, the polymerase and exonuclease activities reside in separable polypeptide chains. Polymerase activity resides in the ot subunit, the product of the dnaE gene [37,38], and the 3' ---, 5' exonuclease activity resides in the c subunit, the product of the dnaQ ( = m u t D ) gene [39]. The deduced sequence of the c subunit shares limited homology with the exonuclease domain of the Pol I Klenow fragment. The presence of polymerase and exonuclease activities on separate subunits has led to speculation that fidelity may be regulated by the relative levels of a and c in the cell [40]. Indeed, the exonuclease activity of the c subunit alone is 10- to 80-fold lower than when it associates with the polymerase subunit [38]. The difference is due to a greatly increased affinity of the c subunit for the 3'-OH primer terminus, resulting from DNA binding by the polymerase subunit. Information on the extent to which proofreading enhances the fidelity of DNA synthesis in vitro comes from studies of excision of mis-

matched bases [41 4 3 ] and from fidelity studies [44-47]. Reaction conditions that either permit or diminish exonuclease activity are usually employed (for a review, see Ref. 31). These studies suggest that, just as for discrimination at the polymerization step, the efficiency of editing base substitution errors can vary over a wide range, depending on the enzyme, the mispair and the templateprimer position. This variation in proofreading efficiency reflects a balance between the competing processes of polymerization and excision. A complete understanding of proofreading therefore requires a description of the reaction rates that influence editing. The polymerase inserts a base at a rate characteristic of the basepair or mispair formed. There are then two choices. The polymerase can incorporate the next correct base onto the resulting primer terminus: extension from a mispair is more difficult than from a correctly paired terminus [17]. Alternatively, the exonuclease may excise the terminal base before further polymerization. The final fidelity of the reaction depends on the relative rates of each of these three steps. Each rate is expected to vary not only for correct versus incorrect bases, but also for each different mispair and each template-primer position, since each position provides a new set of protein-DNA contacts and a new set of base stacking interactions known to influence fidelity [48]. These considerations imply that even a polymerase with a relatively weak exonuclease could gain enormous benefit from proofreading if the polymerase has difficulty extending from a particular mispair. Furthermore, a proofreading exonuclcase need not exhibit a stronger preference for excision of a mismatched rather than a matched base as detected in a simple exonuclease assay. All that is required is discrimination against extension from a mispair within the polymerase active site. It remains to be established whether, during normal polymerization, the exonuclease corrects an error during a processive polymerization-excision cycle without dissociating from the DNA, or whether it corrects the error after dissociating and then re-associating. Polymerase dissociation at a mispair may be important for explaining reactiondependent differences in the extent and specificity of proofreading [47], and the observation that the

exonuclease activity of Pol I increases (warms up) as the enzyme progresses along the template [49]. Also, in those systems for which no polymeraseassociated exonuclease has yet been detected (e.g., with certain eukaryotic DNA polymerases), were the polymerase to routinely dissociate from a mismatched template-primer, the opportunity would exist for any exonuclease with the appropriate specificity to compete with the polymerase for binding. Thus, in some situations, proofreading might result from exonucleases not necessarily tightly associated with the DNA polymerase, which are separated from the polymerase during purification. Higher eukaryotes contain four DNA polymerases, designated Pol a, /3, 8 and ~, (for an extensive review, see Ref. 6). Evidence that higher eukaryotic DNA polymerase proofread comes primarily from recent measurements of mismatch excision and the fidelity of DNA synthesis [10,43,50,51]. A distinctive characteristic of DNA polymerase & an enzyme that functions in both nuclear DNA replication and DNA repair, is the presence of an associated 3' ~ 5' exonuclease [52]. Proofreading does indeed operate during DNA synthesis by calf thymus DNA polymerase & enhancing fidelity for some base substitution errors 100-fold [10]. In most reports on polymerase & the exonuclease activity copurifies with polymerase activity, demonstrating their physical association. However, Crute et al. [53] described one form of DNA polymerase 8 containing a dissociable exonuclease, suggesting either that the activities reside on separate subunits or that a single polypeptide was proteolyzed. The replicative DNA polymerase for the mitochondrial genome is DNA polymerase ~,. Highly purified preparations of chick embryo polymerase "t contain a 3' ~ 5' exonuclease activity that efficiently excises a mismatched base from a 3'-OH primer terminus [51]. The exonuclease remains associated with the polymerase during sedimentation in a high-salt glycerol gradient. Chick DNA polymerase ~, exhibits very high fidelity for some single base substitution errors, and this high accuracy results at least in part from exonucleolytic proofreading. DNA polymerase a, the putative replicative

polymerase for the nuclear genome and an enzyme that functions in DNA repair as well, has generally been purified devoid of exonuclease. However, there are several exceptions: Pol a containing associated 3' --* 5' exonuclease activity has been purified from mouse myeloma cells [54], calf thymus [55], HeLa cells [56] and Drosophila melanogaster embryos [50]. In addition to 3' ~ 5' exonuclease activity, the mouse and HeLa cell enzyme preparations both contain a second exonuclease activity that digests DNA in the 5' ---, 3' direction. In the case of the mouse myeloma activities, both exonucleases copurify with the polymerase during polyacrylamide gradient gel electrophoresis and cosediment with the polymerase through a high-salt glycerol gradient. For HeLa cells, both exonucleases can be separated from the polymerase and both activities reside in a single polypeptide of M r 69000 [57]. Interestingly, the 3'--* 5' exonuclease activity in the Drosophila a polymerase preparation is relatively inactive when purified with the polymerase as a four-polypeptide complex. However, the exonuclease becomes highly active and cosediments with the 182000 Da polymerase when the complex is subjected to sedimentation in a glycerol gradient containing 50% ethylene glycol. Although proofreading during a polymerization reaction has not yet been demonstrated by nextnucleotide or monophosphate effects on fidelity for any of these preparations of exonuclease-containing DNA polymerase a, both the HeLa cell and the Drosophila exonuclease efficiently excise mismatched bases from primer termini [43,50,57]. Furthermore, the most purified form of the Drosophila enzyme (182 kDa) is highly accurate for certain base substitution errors, consistent with a role for the exonuclease in proofreading. DNA polymerase fl contains no detectable exonuclease activity when extensively purified. This polymerase can associate in vitro with mammalian DNAase V, a 12000 Da protein containing both 3' -, 5' and 5' --, 3' exonuclease activities [58,59]. A role for DNAase V in proofreading has not been established. Just as for prokaryotic enzymes, proofreading in eukaryotes is not equally active against all mispairs in all DNA contexts [43,50,51]. This presumably reflects some of the same structural and

kinetic principles discussed above. However, the mechanisms of proofreading by' these cnzymcs have not been investigated and could be different.

II-B. Base deletion/addition fidelity D N A is a structurally dynamic molecule, capable of assuming a variety of conformations. This flexibility provides a significant opportunity to commit errors that result in the gain or loss of one or more bases due to misalignment of strands. Such mutations are produced in vivo at a significant frequency and represent a substantial proportion of the spontaneous mutant collections described at the D N A sequence level in both prokaryotes (Refs. 60-62 and references therein) and eukaryotes [63-67]. Nevertheless, while base substitution fidelity has been extensively studied in vitro for more than 20 years, base a d d i t i o n / deletion fidelity has been examined only recently. Errors due to template-primer misalignments have been described using the M l 3 m p 2 forward mutation assay, with purified D N A polymcrases a, /3 and "/ [68]. More recently, a reversion assay has been used to describe errors by the Klenow fragment of E. coli Pol I (69). Table lI lists error produced b~ D N A polyTABI,E II VARIABLES T H A T A F F E C T F R A M E S H I F T FIDEI,ITY Data are taken from from Ref. 68. In all cases, the error rates arc expressed per detectable nucleotide incorporated. Sincc these are calculated using defined target sizes, the differences are real and are not an artifact of different target sizes. Error catego~'

Error rate H u m a n Pol a Rat Pol/3

Base substitutions (average)

1 / 3 100

Frameshifts: Overall average 1 / 7 800 Plus-one-base 1/110(R)O Minus-one-base 1/ 8 500 template pyrimidine runs 1 / 5 600 template purine runs 1/9900 T T T T (i'u)sition 70) l / 2 200 AAAA (position 91) < 1/13000 CCCC(" (position 132) 1/1 400 CCC (position 106) < 1/13000 CCC (position 166) < 1/1300~) template non-runs (all bases) 1/12000 template non-run G positions 1/5000

1 / I 300

1/ 1 100 1/90000 1/ 1 100 l/400 1/4700 1/ 57 1 / I 900 < 1/7000 1/260 1 / I 400 1/17000 1/8400

merases a and ft, both of which lack associated proofreading exonuclease activity. Both DNA polymerases produce one-base frameshift errors at a frequency' similar to or even greater than base substitution errors. As with base substitutions, the frameshifts error rate varies widely and depends on several variables. Both D N A polymerases a and /3 produce minus-one-base errors at least 10-fold more often than plus-one-base errors. There are at least two possible explanations for this bias. It may reflect the fact that formation of the misaligned intermediate necessary to produce a plus-one frameshift (an extrahelical nucleotide in the primer strand) requires the disruption of at least one more basepair than is required to create a minus-one intermediate (Fig. 4). Alternatively, the preference for the loss of a base could reflect less stringent polymerase constraints on an extrahelical template base. Both D N A polymerase a and fl produce minus-one-base errors more frequently at reiterated base sequences than at non-reiterated sites. As first suggested by Streisinger and co-workers [70,71] and subsequently directly demonstrated during D N A synthesis in vitro catalyzed by D N A polymerase ,8 [68], frameshifts within runs arise as if they result from movement of the template relative to the primer, to create a misaligned premutational intermediate containing an extrahelical template nucleotide. Presumably, the error frequency is higher at such positions because additional correct basepairs are possible on the 3 ' O H - p r i m e r side of the misalignment (Fig. 4). These can stabilize the intermediate as well as place the extrahelical base some distance away from the polymerase active site involved in the next phosphodiester bond formation. Several parameters affect the frequency of minus-on-base errors created by a slippage mechanism during D N A synthesis in vitro (Table II). First, such errors occur more frequently in runs of template pyrimidines than in runs of template purines. Whether this is so with other polymerases and other mutational target sequences remains to be established. One possible explanation for this bias is that template pyrimidines may unstack to create the necessary misalignment more easily than template purines.

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( , ) C G T T T T A C

C G T T T T A C id)

l

A

A.A.A

T G

(4)

(j) C G T T T T A C

C.A-A.A-A.TG C G T T T T A C

Fig. 4. The possible intermediates leading to plus-one or minus-one base errors in a run of T residues are shown. The numbers of parentheses are the number of base pairs that must be disrupted to produce the intermediate shown• (Reprinted from Ref. 68.) Second, the sequence surrounding the template run influences the frameshift error rate. Thus, the frequency of m i n u s - o n e base errors by Pol fl in a

run of three template Cs varies 5-fold d e p e n d i n g on the neighboring base sequence (compare positions 106 and 166 in Table II). Even more dramatic

10

is the comparison between position 106, which has an error rate 1/260 for a run of three Cs, and position 132, having an error rate of under 1 / 7 000 for a run of live Cs. Not unexpectedly, a third parameter that influences frameshift error rates within runs is the D N A polymerase. D N A polymerase ,8 is usually less accurate than D N A polymerase a. For example, at positions 106 in the M l 3 m p 2 lacZe~ sequence, Pol /3 is 50-fold less accurate than Pol a. However, at a second site (position 132), Pol a is at least 5-fold less accurate than Pol '8. Such effects presumably reflect different polymeraseD N A interactions, for which there is considerable evidence (see Ref. 6 for a review). For example, Pol /3, a single subunit enzyme, is the smallest known D N A polymerase (M~ = 40000), binds tightly to nicked double-stranded DNA, completely fills gaps to the last nucleotide, polymerizes bases in a distributive manner and efficiently adds bases to a mismatched primer. These observations imply that Pol /3 can incorporate nucleotides even without extensive or perfect contacts with the single-stranded template or the primer. In contrast, Pol a is a multisubunit D N A polymerase whose catalytic subunit is much larger ( M~ = 180000), does not bind at nicks or fill gaps to completion, is more processive, absolutely requires single-stranded D N A for binding and needs at least 3 5 correctly paired bases for efficient catalysis. The more extensive D N A - p r o t e i n interactions implied by these data may result in greater a n d / o r different constraints on either the frequency of formation or the subsequent stability of misaligned template-primers. Such interactions may explain the substantial difference between a and ,8 polymerases in the ratio of run to non-run frameshifts (Table II), and the fact that most of the Pol a frameshifts within runs are in runs of three or more bases, while Pol ,8 prtx:tuces a high frequency of minus-one events, even in two-base runs. They may also be related to the observation that the average fidelity of o~ and ,8 polymerases for frameshifts (and for base substitutions as well) correlates with the processivity of these enzymes [72]. The more accurate a polymerases are also more processive, i.e., they add more nucleotides to the template-primer per association event. This suggests that some of the same p o l y m e r a s e - t e m -

p l a t e - p r i m e r interactions that determine whether a polymerase remains bound during cycles of d N T P binding, phosphodiester bond formation and translocation also determine the base substitution a n d / o r frameshift fidelity of the reaction. This correlation between processivity and fidelity has only been examined once. Using reaction conditions that allow more processive D N A synthesis by D N A polymerase '8, frameshift fidelity improved [72]. Further experiments are required to examine the influence of processivity on fidelity. Under some conditions, such as with damaged D N A templates, increased processivity could actually lead to lower fidelity. For example, highly processive E. coli D N A polymerase Ill has been shown to more efficiently bypass certain D N A adducts [73] than does a simpler, less processive form of this same polymerase [74]. Surprisingly, during I)NA synthesis by Pol ft, misalignments at positions 103 and 104 of the lacZet sequence in M l 3 m p 2 can produce not only frameshifts but also base substitutions at position 103 at a very high frequency. r h c base substitution errors result from a "dislocation" mechanism (Fig. 5 and Ref. 12). Here, the initial event is slippage, designated pathway F in Fig. 5, for 'frameshift'. This is followed by incorporation of one additional correct nucleotide onto the misaligned template-primer. However, rather than follow the usual pathway of further incorporation to fix the frameshift intermediate (pathway FF), the extrahelical template base realigns to create a primer-terminal mispair (pathway FB). Note that this mispair is not produced by classical miscoding, but by a transient misalignment. Since Pol /3 lacks a proofreading exonuclease to remove the mispaired base and can efficiently polymerize from mismatched ends, a base substitution error results. This model was tested by replacing the template G at position 102 (underlined) with an A, and then re-examining the error specificity at position 103. As predicted by the dislocation model, T - - , G errors disappeared and T - - , A errors were produced at a high frequency [12]. These data demonstrate that, at least during Pol /3-dependent D N A synthesis in vitro, a base at one position can act as a template for a mutation at another position. The logic of the dislocation model may explain a much larger class of errors created by D N A

11 .A-T-~

,L,C-§-T-T-A-C3, (F)

,~ ~

c-~-tA-T-G ~-~ I,T.~,o,.,,.. .c-A.-T-.G

(FF) ~

c-~-r A-C 'T

G-C-A-T-G

,~-_~-~ i-,~

(8)

C-.A-T-.G c-a-r-r-A-c

(aa)~ .G-C.-C-.A-!-.G

C'A. -T- ?

c-~-~-~-,-c

(BF) C-A.-I-.G

C-~-T-T-A-C

C-f-T A-C T

c-_~-o-,-,-c

~.~_-~ i-~

(FB)

T

l

,,.

G-C-A-T-G

(T~G103)

I inc°rp°r4ti°n

T

1

.G-c-C-A.-T-G. C-G-T-A-C

(~.T103)

C-§-T-T-A-C

1

C-6-T-A'C

(,~TI031

C-G-6-T- A-C

(T~GI03)

Fig. 5. Mutational pathways and intermediated for a 'dislocation' hotspot for DNA polymerase ft. The underlined base is position 102 of the lacZa gene, where position 1 is the first transcribed base of the lacZa gene. The open triangle indicates a minus-one base frameshift. (Reprinted from Ref. 12.)

polymerases in vitro, frameshifts at non-run template positions (e.g., see Table II and Ref. 69). The frequent production of non-run frameshifts is not predicted in an obvious way by a Streisinger slippage mechanism, since at non-reiterated base sites, simple slippage would not create a misaligned intermediate stabilized by correct basepairs. However, we have proposed that the principles of the slippage mechanism can nonetheless be fulfilled, even at non-run sites. Such errors could arise by misincorporation of a single base (Fig. 5, pathway B) followed by rearrangement of the templateprimer before the next base is added (pathway BF), such that the misinserted base in the primer strand forms a single correct basepair, with a template base at some other position (in Fig. 5, pairing with the next base is shown). Alternatively, non-run frameshift could result from other mechanisms. For example, a non-run template base might assume an unusual and stable conformation in the enzyme-template-primer complex that would prevent the polymerase from recognizing its presence, allowing the base to be skipped. This possibility might be examined by combining information on the structure of the

active site of a polymerase with information on the structure of DNA containing extrahelical bases, currently being examined by N M R [75,76]. During D N A synthesis in vitro, D N A polymerases also commit errors that lead to the loss of more than one base [11,69,72]. Some of these are readily explained by a Streisinger misalignment mechanism involving directly repeated sequences rather than monotonous runs to establish the intermediate that provides the required stability and a functional template-primer. As for other classes of polymerase errors, the frequency and specificity of deletion errors are influenced by the DNA sequence. However, while both the length and the sequence of the repeated sequence are important, there are insufficient data to establish rules concerning sequence effects. DNA polymerases also create multibase deletion errors that are not explained by a simple direct repeat. Some of these result from unknown mechanisms. However, some can be explained using the principles of the dislocation mechanism described above: misalignment, limited incorporation, realignment and continued incorporation. This model has been used to explain a very com-

12 plex mutation created by Pol ,8 [12] and both two-base deletions and complex frameshifts produced by the Klenow fragment of E. coli I)NA polymerase I [69]. In these instances, aberrant DNA synthesis moves a "block" of genetic information from one site in a genome to an entirely new position. The idea that distant sequences can template mutations during DNA replication, repair or recombination could explain a variety of mutations generated in vivo [59-65]. Transient misalignment during DNA synthesis provides one mechanism to explain the origin of some of these mutations. This mechanism could have biological significance for the evolution of mammalian muhigene families, such as the human interferon genes [77] or for systems requiring rapid generation of diversity, such as the immunoglobulin genes [78]. We have, in fact, already reported that synthesis by terminal transferase, either alone or in combination with Pol ,8, produces, through imperfect, misaligned intermediates, complex errors that are similar to sequences in rearranged immunoglobulin genes

[791. II!. Fidelity of complex systems DNA replication in vivo is catalyzed by a DNA polymerase acting in concert with additional proteins as part of a replication complex. Several studies have examined the fidelity of DNA synthesis using multicomponent DNA synthesis systems. The first studies, performed with prokaryotic enzymes, focused on single base substitution errors using OX174 amber codon reversion assays. The replicative polymerase in E. coil, DNA polymerase IIl holoenzyme, contains at least five subunits (and possibly more) in addition to the polymerase and proofreading exonuclease catalytic subunits. This highly processive enzyme complex, in the presence of rep and single-stranded DNA-binding proteins of E. coli and the q~X174encoded gene A protein, synthesizes multiple copies of q~X174 single-stranded DNA from double-stranded DNA by a rolling circle mechanism. This synthesis is highly accurate, producing single base substitution errors at rates approaching in vivo mutation rates [80,81]. Using q)X174

single-stranded DNA. complementary-strand synthesis can be performed even in the absence of rep, SSB and the gene A protein. The holoenzyme complex remains highly accurate [82], demonstrating that high fidelity results from the components of the holoenzyme itself. These results, and the observation that mutant alleles of both &laE and & u l Q ( m u t D ) have ahered mutation rates m vivo [83 85]. clearly demonstrate that high base substitution accuracy results from the combination of proofreading by' the exonuclease catalvtic subunit (~) and nucleotide selectivity by the polymerasc catalytic subunit (~). Whether these discrimination steps are affected bv the additional subunits of the holoenzyme remains to be established. However. the recent demonstration that the E. c.oli m u t T mutator specifically affects replication fidelity' for A. d G M P mispairs [86] suggests that gent products other than the a and c subunits can influence one or boil1 of these discrimination mechanisms. The base substitution fidelity' of the T4-encoded I)NA replication complex reconstituted from seven purified proteins has been determined at several amber codons during synthesis with nicked double-stranded q~X174 DNA [87,88]. This muhicomponent system commits errors very infrequently. While the contribution of proofreading has not yet been quantitated in vitro, both genetic studies (Refs. 89. 90 and see Refs. within Ref. 36) of mutant alleles of the polymerase gene (gene 43) and in vitro measurements of terminal mismatch excision [42] suggest that exonucleolytic proofreading contributes significantly to the fidelity of Ta DNA replication. The observation that the base substitution fidelity of mutant forms of purified T4 DNA polymerase is only 2- to 4-fold different than the fidelity of the wild-type polymerase [91], yet 100-fold effects are observed in vivo, suggests that additional components of the replication apparatus may modulate fidelity. This is supported by genetic studies of mutant alleles of genes encoding other components of the complex [92,93]. However, an effect of these accessory proteins on the fidelity of I)NA synthesis has not been established in vitro and, surprisingly, the T4 polymerase alone can be as accurate as the entire complex, at least for base substitution errors at one template position [91].

13 In higher eukaryotes, the fidelity of DNA synthesis catalyzed by multisubunit forms of DNA polymerase a has been examined. The intact, four-subunit DNA polymerase a-primase complexes isolated from Drosophila melanogaster embryos, calf thymus, human lymphoblasts and hamster lung cells are all highly accurate for base substitution errors involving the misincorporation of dCMP a n d / o r dAMP opposite a template A at position 587 in ~X174 DNA [13,43,50]. Fidelity is several-fold higher than that previously reported for other a polymerases, even though no proofreading exonuclease is detectable in these intact polymerase-primase complexes (except for the Drosophila enzyme, see section II-B above). The results imply greater nucleotide selectivity at the polymerization step, which could result either from a more intact (i.e., less proteolyzed) form of the catalytic subunit or from contributions by the additional polypeptides. Given the variety of all possible DNA synthesis errors and the effects described in Tables I and II, results from studies of single base-mispairs at only one position should obviously be interpreted with caution. The fidelity of two different intact DNA polymerase a-primase complexes has been examined in the M13mp2 forward mutation assay, which detects a variety of errors at numerous positions. The human KB cell DNA polymerase a-primase was highly accurate for some base substitution errors, with error rates lower than 1/100000. However, for the sum of all possible base substitution and deletion errors, this more complex enzyme was neither highly accurate nor significantly more accurate than other forms of DNA polymerase a [72]. More recently, similar forward mutant frequency results were obtained with intact DNA polymerase a-primase complex freshly purified from human HeLa cells [94]. These studies suggest that, while fidelity can indeed be high for certain errors at particular template sites, the final error rate is limited by the least accurate of all the reactions that occur. Thus, the overall error rate of the high-molecular-weight polymerase catalytic subunit in the presence of the additional polypeptides of the polymerase-primase complex is only 1/5 000. One explanation for such inaccuracy may be the loss, during purification, of subunits that func-

tion during replication to improve fidelity. To determine whether eukaryotic replication complexes contain additional fidelity components, the M13mp2-based forward and reversion assays were adapted to measure the error rate of the human (HeLa cell) replication complex during DNA replication in vitro [94]. In this system [95], synthesis initiates at a unique site (the SV40 origin) and is bidirectional and semiconservative. Only a single viral protein is required, the SV40 large tumor antigen ( T a g ) . All other proteins are of human cell origin and include (at least) the four-subunit Pol a-primase complex, topoisomerases, DNA ligase, single-strand DNA binding protein(s), proliferating cell nuclear antigen and, possibly, Pol 8. In the forward mutation assay, the error rate for replicative synthesis was maximally 1/150 000, much more accurate than gap-filling DNA synthesis catalyzed by the HeLa cell DNA polymerase a-primase complex purified by immunoaffinity chromatography. The data suggest that additional fidelity factors are operating during replication in vitro. Increased fidelity could result from additional protein subunits that alter processivity by increasing the affinity of the polymerase for template-primers, or from exonucleolytic proofreading, either by some form of Pol a or by Pol 8, or from novel mechanisms. In the more sensitive Ml3mp2 opal codon reversion assay, single base substitution errors were readily produced in replication reactions containing an excess of incorrect over correct dNTP substrates. Error rates ranged from 1/110000 to less than 1/1500000, depending on the mispair. These data suggest that, just as with purified DNA polymerases and just as in chromosomal replication in vivo, the mutation frequency in this eukaryotic replication system varies widely, depending on the mispair examined. The observation that the replication complex commits errors even with only slightly biased substrate pools suggests that certain fidelity factors may be missing or inoperative, and that the fidelity of this replication complex alone cannot account for the low rate of spontaneous mutations in vivo.

Acknowledgements We are grateful to our colleagues in the Mutagenesis Section of the Laboratory Molecular

14

Genetics here at the N I E H S for many stimulating discussions. We thank John D. Roberts and John W. Drake for critical evaluation of the manuscript.

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