Replication Fidelity tials for various dNTPs and template bases, dependent differences a demand for equivalent base-pair geometry (for review, see Echols For the three viral polymerases mentioned the amount binding discrimination that predicted from the free-energy ferences between correct and incorrect base pairs DNA polymerase active site, hydrogen-bonded water molecules are displaced from the transition for complementary base pairs (Fersht 1985), and from the active amplify base-pair free-energy differences, thus (Petruska et al.1986; Abbotts 1991; Johnson 1993).A water from the active site might partly explain polymerase-dependent differences Klenow polymerase utilizes discrimination to other three polymerases examined.has led to the suggestion that the "ground the inferred tional change initial binding Klenow polymer- not involve base-pairing (Johnson has also been gested (Capson et al.that any reduction matic efficiency resulting from the three incorrect might be tolerated because the pri- mary role for this polymerase DNA repair RNA primers during replication.contrast, reduced efficiency might less acceptable for polymerases that replicate entire genomes.
All three viral polymerases mentioned above, role is discriminate more strongly against incorrect binding than Klenow polymerase.next selectivity the polymerization inferred from several lines evidence (Johnson 1993; Zinnen et al.conformational change the ternary position the subsequent phosphodiester bond formation (Fig.polymerase, this change to "closed" structure which the is thought to lock is much more rapid correct base pairs that can adopt Watson- Crick geometry for incorrect base pairs that cannot.
This leads 2000- to 4000-fold the rate conformation with correctly incorrectly bound has been suggested to enhance the selectivity to 17,000-fold Zinnen et al.1994), depending on the mispair increasing the concentration the next correct incorporated after misinsertion.
Alternatively, adding to the reaction (dNMP), the end product action.Proofreading can also be changing amino acid residues essential for exonuclease residues are found three conserved sequence motifs common to the coding sequences DNA polymerases containing exonucleases (Ito and Braithwaite 1990; Blanco et al.1991; Chung et al.
1991; Morrison et al.1991; Simon et al.et al.
1991).The results obtained from these three approaches suggest that proofreading contributes on average to fidelity.
consistent with estimates from vivo studies proofreading activity (Schaaper and with cal- culations suggesting that the energetic cost improving fidelity this amount using exonucleolytic activity could be unacceptably due to too much excision correctly paired exonucleolytic proofreading to base fidelity can vary over a wide range, from only few-fold (Bebenek to almost 1000-fold Frey et al.
1993).This results partly from the different rate constants for polymerization from the also reflects are expected on the basis the idea originally proposed and subsequently supported extensive data Bloom et al.Carver et al.
containing a terminal mispair has higher probability being single-stranded does a correctly paired terminus.frayed end will preferentially bind to the exonuclease active site, which prefers single-stranded DNA.Similarly, a matched and, therefore, double-stranded terminus will preferentially bind to the polymerase ac- tive site, which prefers double-stranded (for review, see Joyce Because the stability the duplex region the template- primer will depend on its DNA sequence, proofreading efficiency is sequence contexts Moreover, the degree needed to allow single-stranded bind to the exonuclease active site vary, depending on the between the polymerase and active sites.This distance, estimated to be for the Klenow polymerase, could be greater for some enzymes compare data al. to Capson et al.; for review, see Joyce Steitz 1994), ing to enzyme-mediated proofreading efficiency.Replication Fidelity with reduced blue-color intensity.polymerase error by sequencing the identified average values per detectable nucleotide polymerized, possible single-base sequence contexts (Bebenek and Kunkel wide range of substitution fidelity the composition local template- examined at specific template positions kinetic approaches both the misinsertion and estimated sepa- the eukaryotic studied eukaryotic polymerase.from several sources average base substitution error rate of errors sequence contexts have been obtained when the the yeast lytic subunit alone compared to complex (Kunkel et al.Copeland et Similar values for misinsertion fidelity, representing have also observed by kinetic analyses of Drosophila melanogaster polymerase-a:DNA primase (Men- et al.highly purified DNA pol-a is not particularly accurate relative fidelity required below).However, error rate of a small number of Mistakes made here could also estimates are from exogenously recent observa- tions suggest synthesis coupled to primase may differ the fidelity present data consistent with the fact many preparations lack the three conserved sequence motifs character- of about Replication Fidelity for mispair extension Likewise, two the motif mutants, having single-amino-acid residues suggested to be important for the incom- for interacting with the primer, have improved insertion delity (Dong et al.
1993a), another has reduced discrimination against mispair extension et al.1993b).A this ap- proach with should increase the fidelity polymerase.
Polymerases with reduced enhanced fidelity should also be useful "biomarkers" defining their roles replication and the eukaryotic is also the least ac- The single subunit polymerases purified from chicken embryos have substitution error rates (Kunkel and Alexander For individual mispairs, range from Similar, and a few instances even higher, error rates have been obtained direct misinsertion enzyme, using steady-state kinetic analyses (Boosalis et al.1989).rates are consistent with the fact that purified pol-p lacks exonuclease activity the three motifs conserved proofreading polymerases).However, although pol- shares this property with is even that, independent proofreading, selectivity against substitution depends on the DNA polymerase.with a modest catalytic role filling gaps one or a few nucleotides during base excision repair.
Alternatively, pol-p may have higher accuracy than current estimates suggest.
Thus far, pol-p fidelity has been measured using template primers containing long single-stranded template regions, where synthesis distributive rather than processive.Recently, shown to template adjacent to -phosphoryl end (Singhal and Wilson 1993).As this type more closely resemble that occurring during base excision repair, it will be interesting to determine pol-p fidelity using substrates containing one or a few possible that pol-p fidelity accessory proteins.For malian DNase a 12-kD protein having both nuclease activity (Mosbaugh and Meyer associates with Foury and Vanderstraeten Gray and Wong 1992), and the the yeast has the three conserved exonuclease motifs (Foury and Vanderstraeten 1992).catalytic properties the exonuclease and the that fidelity is containing a (Kunkel and Soni Kunkel and Mosbaugh are consistent proofreading role ERRORS INVOLVING TEMPLATE-PRIMER MISALIGNMENT addition to direct misincorporation noncomplementary nucleotides, base-addition, -deletion, and even -substitution errors can processes involving template-primer misalignments Frameshifts* Initiated by Template-primer Slippage Strand slippage during replication misaligned intermediates stabilized correct base pairs (Fig.2A).sequent polymerization from the misaligned intermediate to dele- the unpaired nucleotide(s) is the template strand the unpaired nucleotide(s) is the primer strand (not shown).mechanism predicts (Streisinger et al.that the should increase the length of the increases, because the potential correct base pairs that could stabilize the misaligned interme- diates increases, as does the number potential misaligned intermedi- ates that can form (Fig.
2).Furthermore, the longer the the distance between the extra nucleotide and the terminus, potentially reducing interference the extra base formation within the active site.logic, frameshift rates, expressed per nucleotide polymerized correct for differences the number different lengths, indeed increase the length increases, for DNA DNA polymerase (Kunkel addition, error rates base deletions homopolymeric runs pol-@ (Kunkel frameshift mutation refers to the number that are three, for refers here resulting from any the number and 3 that are nucleotide considered.
An has at least repeated; reiterated Replication Fidelity Base Substitutions Initiated Strand Slippage Following slippage, correct incorporation another nucleotide realignment before continued incorporation generates a mispair (Kunkel 1985a).This can yield base substitution, but case, initiated rather than process has been termed dislocation mutagenesis (Kunkel and Alexander analogy with shoulder joint alignment but ultimately resumes normal position.Strong support for the model from fidelity with pol-p (Kunkel and Soni 1988b; (Bebenek et al.
vitro.With both base substitution hot are observed at the several different homopolymeric runs, and the substitution specificity depends on the immediate template neighbor.the situation shown but can involve the primer strand (Fig.
Bebenek et al.
paired template nucleotides (Fig.Bebenek et al.1993), or nucleotides (Fig.Kunkel and Soni 1988a; for review, see also Ripley 1990).
Frameshifts Initiated distinctly different way to generate a is misincor- poration followed template-primer rearrangement terminal base pair for continued polymerization (Fig
.misalignment ultimately frameshift error, but this case misinsertion rather than strand slippage.was sug- (for review, that "difficult-to-extend" mispairs realign such that extra nucleotide the template strand but correct base pair at the terminus.principle, this mechanism is possible any template position and is not limited the production minus-one-base errors.frameshift errors at template runs, minus errors varying numbers model has with yeast (Kunkel et al.1989), Klenow DNA polymerase (Bebenek et al.1990), and RT (Bebenek et al.
termini led to the suggestion (Kunkel and Soni 1988b) that incorporation opposite damaged templates might also involving replication several different lesions (Wang and Taylor 1992; Shibutani and Grollman support this suggestion.
Replication Fidelity rates were and these often processive synthesis within et al.a consistent wherein low processivity with low frameshift fidelity, con- sistent with the idea that the formation utilization of misaligned template primers phase of polymerization reaction.Accessory Proteins Polymerase Fidelity support for a relationship between processivity and study of the with and without processivity protein, thioredoxin et al.DNA polymerase alone has low processivity, adding only 1-50 nucleotides before dissociating.However, with its accessory subunit thioredoxin, proceeds for thou- nucleotides without dissociation.Fidelity with an that the rate for one-base 46-fold higher of thioredoxin than may have nificance, given that accessory proteins that are a general feature multiprotein replication complexes (see Stillman, volume).Frameshift fidelity is particularly interesting inasmuch replication infidelity is one sible explanation the instability reported for several diseases (Loeb thioredoxin, the found to be more accurate during vitro for and 2-nucleotide deletions (Kunkel et al.One possible explanation that the premutational intermediates not successfully with thioredoxin.that, under some circumstances, accessory protein-mediated alteration the extension rate from an unusual primer, e.g., opposite a damaged base a damaged site, could serve a seal the error before transfer to the exonuclease active site for accessory protein that logically could influence fidelity substitution fidelity several polymerases is increased reactions con- (Kunkel et al.1979, 1983).Similarly, the rate of Replication Fidelity have mismatch repair activity et al.1991a), these rates represent the sum both replication fidelity heteroduplex repair the extract.below) suggest extract only affects error that replication itself is high.errors are detected with undamaged centrations, further understanding how high replication achieved and how can be perturbed requires manipulation errors.Several approaches have been used to address specific questions.Proofreading and the Fidelity Leading- and One obvious question is whether proofreading responsible for high replication fidelity.question, reactions were formed with unequal force specific misinser- tions.
For example, misincorporation to revert codon can be (Fig.3).
to replication can then be examined either polymerization at the by inhibit- adding deoxynucleoside monophosphate cation reaction.
Results from approaches suggest lagging-strand synthesis across text for lagging-strand replication lagging 1agging:leading from extract reconstituted reaction compared to discontinuous synthesis Okazaki fragments which involves polymerase and/or possible influence replication fidelity cell extract fractions, neither which has replication activity.When combined, these fractions reconstitute replication activity that devoid of mismatch repair activity (Roberts 1994).When the fidelity examined using those observed extracts having mismatch repair activity, replication is indeed highly accurate.Moreover, the error extracts (Table et al.
1995), including unequal leading- and lagging-strand rates Thus, at these error replication activity but mismatch repair mismatch repair will facilitate future studies the human replication machinery replication reconstituted ponents, reactions performed with pol-a as polymerase (Carty found to intermediate be- tems fail, lesions may persist DNA or precursor pools.A lesions have been described have different thus potentially affect replication For example, an alkylated hydrogen-bonding potential lead to direct misinsertion errors, abasic site has lost base hydrogen-bonding potential altogether.
Bulky stacking interactions leading may be inconsistent with template-primer rearrangement frameshift errors.could also polymerase and exonuclease active sites Lesion-induced replication infidelity has been a number with purified polymerases (for see Echols Discussing all this information is beyond the this chapter; briefly review only a few of the replication using damaged substrates.generate mutagenic compounds within cells, of which is 8-0x0-deoxyguanosine.
several lines defense against mutations resulting from this base analog and Miller and Moriya is biologically important.A variety of extracts, misincorporate the triphosphate form of this base analog, 8-O-dGTP7 opposite template adenines, yielding transversions (Cheng et al.Minnick et al.data suggest mutagenic during genomic case for other modified dNTPs as Feig et in human the human (Sakumi et al.inactivating mutations (or other enzymes pools) might a mutator phenotype Mutagenic Translesion Replication DNA Containing Cyclobutane Pyrimidine Among the insults that generate lesions ultraviolet radiation perhaps the greatest attention, to its (Brash et al.mutagenic potential The values listed depend on simplifying assumptions often involve caveats and in references cited and 200-fold with the balance (factors 2,000,000) representing the tivity of the replication machinery (Table replication fidelity complicated by the influence of spontaneous damage, multiple damage-repair ways.
Moreover, are based genes, providing replication fidelity for large eukaryotic genomes.Despite these qualifications, mutation rate similar picture Mutation rates eukaryotic cells are generally mutations per base pair replicated per and references mutation rate repair-defective yeast Strand et references therein) and human tumor cells Eshleman et several hundred-fold.Moreover, yeast mutants of pol-&, and for conserved exonuclease also have spontaneous mutation rates that by up to several hundred-fold (Morrison 1991, 1993; and Vander- 1992), emphasizing ing both nuclear and mitochondria1 many ways, replication fidelity is Eukaryotic genomes huge compared scanned by current genes.For example, the human genome contains sequence elements error-prone replication human immunodeficiency J.S.
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