Evolving views of DNA replication (in)fidelity.

"It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material" (Watson and Crick 1953). In the years since this remarkable understatement, we have come to realize the enormous complexity of the cellular machinery devoted to replicating DNA with the accuracy needed to maintain genetic information over many generations, balanced by the emergence of mutations on which selection can act. This complexity is partly based on the need to remove or tolerate cytotoxic and mutagenic lesions in DNA generated by environmental stress. Considered here is the fidelity with which undamaged and damaged DNA is replicated by the many DNA polymerases now known to exist. Some of these seriously violate Watson-Crick base-pairing rules such that, depending on the polymerase, the composition and location of the error, and the ability to correct errors (or not), DNA synthesis error rates can vary by more than a millionfold. This offers the potential to modulate rates of point mutations over a wide range, with consequences that can be either deleterious or beneficial.

Evidence that errors made by DNA polymerase alpha are corrected by DNA polymerase delta.

Eukaryotic replication begins at origins and on the lagging strand with RNA-primed DNA synthesis of a few nucleotides by polymerase alpha, which lacks proofreading activity. A polymerase switch then allows chain elongation by proofreading-proficient pol delta and pol epsilon. Pol delta and pol epsilon are essential, but their roles in replication are not yet completely defined . Here, we investigate their roles by using yeast pol alpha with a Leu868Met substitution . L868M pol alpha copies DNA in vitro with normal activity and processivity but with reduced fidelity. In vivo, the pol1-L868M allele confers a mutator phenotype. This mutator phenotype is strongly increased upon inactivation of the 3' exonuclease of pol delta but not that of pol epsilon. Several nonexclusive explanations are considered, including the hypothesis that the 3' exonuclease of pol delta proofreads errors generated by pol alpha during initiation of Okazaki fragments. Given that eukaryotes encode specialized, proofreading-deficient polymerases with even lower fidelity than pol alpha, such intermolecular proofreading could be relevant to several DNA transactions that control genome stability.

Indirect readout of DNA sequence at the primary-kink site in the CAP-DNA complex: alteration of DNA binding specificity through alteration of DNA kinking.

The catabolite activator protein (CAP) sharply bends DNA in the CAP-DNA complex, introducing a DNA kink, with a roll angle of approximately 40 degrees and a twist angle of approximately 20 degrees, between positions 6 and 7 of the DNA half-site, 5'-A(1)A(2)A(3)T(4)G(5)T(6)G(7)A(8)T(9)C(10)T(11)-3' ("primary kink"). CAP recognizes the base-pair immediately 5' to the primary-kink site, T:A(6), through an "indirect-readout" mechanism involving sequence effects on the energetics of primary-kink formation. CAP recognizes the base-pair immediately 3' to the primary-kink site, G:C(7), through a "direct-readout" mechanism involving formation of a hydrogen bond between Glu181 of CAP and G:C(7). Here, we report that substitution of the carboxylate side-chain of Glu181 of CAP by the one-methylene-group-shorter carboxylate side-chain of Asp changes DNA binding specificity at position 6 of the DNA half site, changing specificity for T:A(6) to specificity for C:G(6), and we report a crystallographic analysis defining the structural basis of the change in specificity. The Glu181-->Asp substitution eliminates the primary kink and thus eliminates indirect-readout-based specificity for T:A(6). The Glu181-->Asp substitution does not eliminate hydrogen-bond formation with G:C(7), and thus does not eliminate direct-readout-based specificity for G:C(7).

Asymmetric recognition of DNA local distortion. Structure-based functional studies of eukaryotic Msh2-Msh6.

Crystal structures of bacterial MutS homodimers bound to mismatched DNA reveal asymmetric interactions of the two subunits with DNA. A phenylalanine and glutamate of one subunit make mismatched base-specific interactions, and residues of both subunits contact the DNA backbone surrounding the mismatched base, but asymmetrically. A number of amino acids in MutS that contact the DNA are conserved in the eukaryotic Msh2-Msh6 heterodimer. We report here that yeast strains with amino acids substituted for residues inferred to interact with the DNA backbone or mismatched base have elevated spontaneous mutation rates consistent with defective mismatch repair. Purified Msh2-Msh6 with substitutions in the conserved Phe(337) and Glu(339) in Msh6 thought to stack or hydrogen bond, respectively, with the mismatched base do have reduced DNA binding affinity but normal ATPase activity. Moreover, wild-type Msh2-Msh6 binds with lower affinity to mismatches with thymine replaced by difluorotoluene, which lacks the ability to hydrogen bond. The results suggest that yeast Msh2-Msh6 interacts asymmetrically with the DNA through base-specific stacking and hydrogen bonding interactions and backbone contacts. The importance of these contacts decreases with increasing distance from the mismatch, implying that interactions at and near the mismatch are important for binding in a kinked DNA conformation.

High affinity cooperative DNA binding by the yeast Mlh1-Pms1 heterodimer.

We demonstrate here that the Saccharomyces cerevisiae Mlh1-Pms1 heterodimer required for DNA mismatch repair and other cellular processes is a DNA binding protein. Binding was evaluated using a variety of single and double-stranded DNA molecules. Mlh1-Pms1 bound short substrates with low affinity and showed a slight preference for single-stranded DNA. In contrast, Mlh1-Pms1 exhibited a much higher affinity for long DNA molecules, suggesting that binding is cooperative. High affinity binding required a duplex DNA length greater than 241 base-pairs. The rate of association with DNA was rapid and dissociation of protein-DNA complexes following extensive dilution was very slow. However, in competition experiments, we observed a rapid active transfer of Mlh1-Pms1 from labeled to unlabeled DNA. Binding was non-sequence specific and highly sensitive to salt type and concentration, suggesting that Mlh1-Pms1 primarily interacts with the DNA backbone via ionic contacts. Cooperative binding was observed visually by atomic force microscopy as long, continuous tracts of Mlh1-Pms1 protein bound to duplex DNA. These images also showed that Mlh1-Pms1 simultaneously interacts with two different regions of duplex DNA. Taken together, the atomic force microscope images and DNA binding assays provide strong evidence that Mlh1-Pms1 binds duplex DNA with positive cooperativity and that there is more than one DNA binding site on the heterodimer. These DNA binding properties of Mlh1-Pms1 may be relevant to its participation in DNA mismatch repair, recombination and cellular responses to DNA damage.

Error rate and specificity of human and murine DNA polymerase eta.

We describe here the error specificity of mammalian DNA polymerase eta (pol eta), an enzyme that performs translesion DNA synthesis and may participate in somatic hypermutation of immunoglobulin genes. Both mouse and human pol eta lack intrinsic proofreading exonuclease activity and both copy undamaged DNA inaccurately. Analysis of more than 1500 single-base substitutions by human pol eta indicates that error rates for all 12 mismatches are high and variable depending on the composition and symmetry of the mismatch and its location. pol eta also generates tandem base substitutions at an unprecedented rate, and kinetic analysis indicates that it extends a tandem double mismatch about as efficiently as other replicative enzymes extend single-base mismatches. This ability to use an aberrant primer terminus and the high rate of single and double-base substitutions support the idea that pol eta may forego strict shape complementarity in order to facilitate highly efficient lesion bypass. Relaxed discrimination is further indicated by pol eta infidelity for a wide variety of nucleotide deletion and addition errors. The nature and location of these errors suggest that some may be initiated by strand slippage, while others result from additional mechanisms.

In vivo consequences of putative active site mutations in yeast DNA polymerases alpha, epsilon, delta, and zeta.

Several amino acids in the active site of family A DNA polymerases contribute to accurate DNA synthesis. For two of these residues, family B DNA polymerases have conserved tyrosine residues in regions II and III that are suggested to have similar functions. Here we replaced each tyrosine with alanine in the catalytic subunits of yeast DNA polymerases alpha, delta, epsilon, and zeta and examined the consequences in vivo. Strains with the tyrosine substitution in the conserved SL/MYPS/N motif in region II in Pol delta or Pol epsilon are inviable. Strains with same substitution in Rev3, the catalytic subunit of Pol zeta, are nearly UV immutable, suggesting severe loss of function. A strain with this substitution in Pol alpha (pol1-Y869A) is viable, but it exhibits slow growth, sensitivity to hydroxyurea, and a spontaneous mutator phenotype for frameshifts and base substitutions. The pol1-Y869A/pol1-Y869A diploid exhibits aberrant growth. Thus, this tyrosine is critical for the function of all four eukaryotic family B DNA polymerases. Strains with a tyrosine substitution in the conserved NS/VxYG motif in region III in Pol alpha, -delta, or -epsilon are viable and a strain with the homologous substitution in Rev3 is UV mutable. The Pol alpha mutant has no obvious phenotype. The Pol epsilon (pol2-Y831A) mutant is slightly sensitive to hydroxyurea and is a semidominant mutator for spontaneous base substitutions and frameshifts. The Pol delta mutant (pol3-Y708A) grows slowly, is sensitive to hydroxyurea and methyl methanesulfonate, and is a strong base substitution and frameshift mutator. The pol3-Y708A/pol3-Y708A diploid grows slowly and aberrantly. Mutation rates in the Pol alpha, -delta, and -epsilon mutant strains are increased in a locus-specific manner by inactivation of PMS1-dependent DNA mismatch repair, suggesting that the mutator effects are due to reduced fidelity of chromosomal DNA replication. This could result directly from relaxed base selectivity of the mutant polymerases due to the amino acid changes in the polymerase active site. In addition, the alanine substitutions may impair catalytic function to allow a different polymerase to compete at the replication fork. This is supported by the observation that the pol3-Y708A mutation is recessive and its mutator effect is partially suppressed by disruption of the REV3 gene.

The fidelity of human DNA polymerase gamma with and without exonucleolytic proofreading and the p55 accessory subunit.

Mutations in human mitochondrial DNA influence aging, induce severe neuromuscular pathologies, cause maternally inherited metabolic diseases, and suppress apoptosis. Since the genetic stability of mitochondrial DNA depends on the accuracy of DNA polymerase gamma (pol gamma), we investigated the fidelity of DNA synthesis by human pol gamma. Comparison of the wild-type 140-kDa catalytic subunit to its exonuclease-deficient derivative indicates pol gamma has high base substitution fidelity that results from high nucleotide selectivity and exonucleolytic proofreading. pol gamma is also relatively accurate for single-base additions and deletions in non-iterated and short repetitive sequences. However, when copying homopolymeric sequences longer than four nucleotides, pol gamma has low frameshift fidelity and also generates base substitutions inferred to result from a primer dislocation mechanism. The ability of pol gamma both to make and to proofread dislocation intermediates is the first such evidence for a family A polymerase. Including the p55 accessory subunit, which confers processivity to the pol gamma catalytic subunit, decreases frameshift and base substitution fidelity. Kinetic analyses indicate that p55 promotes extension of mismatched termini to lower the fidelity. These data suggest that homopolymeric runs in mitochondrial DNA may be particularly prone to frameshift mutation in vivo due to replication errors by pol gamma.

Functional analysis of the mismatch repair system in bladder cancer.

In bladder cancer the observed microsatellite instability indicates that mismatch repair deficiency could be a frequently involved factor in bladder cancer progression. To investigate this hypothesis we analysed extracts of seven bladder cancer cell lines and, as a novel approach, five clinical cancer samples for mismatch repair activity. We found that one cell line (T24) and three of the clinical samples had a reduced repair capacity, measured to approximately 20% or less. The T24 cell extract was unable to repair a G-G mismatch and showed reduced repair of a 2-base loop, consistent with diminished function of the MSH2-MSH6 heterodimer. The functional assay was combined with measurement for mutation frequency, microsatellite analysis, sequencing, MTT assay, immunohistochemical analysis and RT-PCR analysis of the mismatch repair genes MSH2, MSH3, MSH6, PMS1, PMS2 and MLH1. A >7-fold relative increase in mutation frequency was observed for T24 compared to a bladder cancer cell line with a fully functional mismatch repair system. Neither microsatellite instability, loss of repair nor mismatch repair gene mutations were detected. However, RT-PCR analysis of mRNA levels did detect changes in the ratio of expression of the Mut S and Mut L homologues. The T24 cell line had the lowest MSH6 expression level of the cell lines tested. Identical RT-PCR analysis of seventeen clinical samples (normal urothelium, 7; pTa low stage, 5; and pT1-4 high stage, 5) indicated a significant change in the expression ratio between MSH3/MSH6 (P< 0.004), MSH2/MSH3 (P< 0.012) and PMS2/MLH1 P< 0.005, in high stage bladder tumours compared to normal urothelium and low stage tumours. Collectively, the data suggest that imbalanced expression of mismatch repair genes could lead to partial loss of mismatch repair activity that is associated with invasive bladder cancer.

Identification of an intrinsic 5'-deoxyribose-5-phosphate lyase activity in human DNA polymerase lambda: a possible role in base excision repair.

Base excision repair (BER) is a major repair pathway in eukaryotic cells responsible for repair of lesions that give rise to abasic (AP) sites in DNA. Pivotal to this process is the 5'-deoxyribose-5-phosphate lyase (dRP lyase) activity of DNA polymerase beta (Pol beta). DNA polymerase lambda (Pol lambda) is a recently identified eukaryotic DNA polymerase that is homologous to Pol beta. We show here that human Pol lambda exhibits dRP lyase, but not AP lyase, activity in vitro and that this activity is consistent with a beta-elimination mechanism. Accordingly, a single amino acid substitution (K310A) eliminated more than 90% of the wild-type dRP lyase activity, thus suggesting that Lys(310) of Pol lambda is the main nucleophile involved in the reaction. The dRP lyase activity of Pol lambda, in coordination with its polymerization activity, efficiently repaired uracil-containing DNA in an in vitro reconstituted BER reaction. These results suggest that Pol lambda may participate in "single-nucleotide" base excision repair in mammalian cells.

Mutator effects of overproducing DNA polymerase eta (Rad30) and its catalytically inactive variant in yeast.

DNA polymerase eta synthesizes DNA in vitro with low fidelity. Based on this, here we report the effects of deletion or increased expression of yeast RAD30 gene, encoding for polymerase eta (Pol eta), on spontaneous mutagenesis in vivo. Deletion of RAD30 did not affect spontaneous mutagenesis. Overproduction of Rad30p was slightly mutagenic in a wild-type yeast strain and moderately mutagenic in strains with inactive 3'-->5'-exonuclease of DNA polymerase epsilon or DNA mismatch repair. These data suggest that excess Rad30p reduces replication fidelity in vivo and that the induced errors may be corrected by exonucleolytic proofreading and DNA mismatch repair. However, the magnitude of mutator effect (only up to 10-fold) suggests that the replication fork is protected from inaccurate synthesis by Pol eta in the absence of DNA damage. Overproduction of catalytically inactive Rad30p was also mutagenic, suggesting that much of the mutator effect results from indirect perturbation of replication rather than from direct misincorporation by Pol eta. Moreover, while excess wild-type Pol eta primarily induced base substitutions in the msh6 and pms1 strains, excess inactive Rad30p induced both base substitutions and frameshifts. This suggests that more than one mutagenic mechanism is operating when RAD30 is overexpressed.

Somatic mutation hotspots correlate with DNA polymerase eta error spectrum.

Mutational spectra analysis of 15 immunoglobulin genes suggested that consensus motifs RGYW and WA were universal descriptors of somatic hypermutation. Highly mutable sites, "hotspots", that matched WA were preferentially found in one DNA strand and RGYW hotspots were found in both strands. Analysis of base-substitution hotspots in DNA polymerase error spectra showed that 33 of 36 hotspots in the human polymerase eta spectrum conformed to the WA consensus. This and four other characteristics of polymerase eta substitution specificity suggest that errors introduced by this enzyme during synthesis of the nontranscribed DNA strand in variable regions may contribute to strand-specific somatic hypermutagenesis of immunoglobulin genes at A-T base pairs.

The 3'-->5' exonuclease of DNA polymerase delta can substitute for the 5' flap endonuclease Rad27/Fen1 in processing Okazaki fragments and preventing genome instability.

Many DNA polymerases (Pol) have an intrinsic 3'-->5' exonuclease (Exo) activity which corrects polymerase errors and prevents mutations. We describe a role of the 3'-->5' Exo of Pol delta as a supplement or backup for the Rad27/Fen1 5' flap endonuclease. A yeast rad27 null allele was lethal in combination with Pol delta mutations in Exo I, Exo II, and Exo III motifs that inactivate its exonuclease, but it was viable with mutations in other parts of Pol delta. The rad27-p allele, which has little phenotypic effect by itself, was also lethal in combination with mutations in the Pol delta Exo I and Exo II motifs. However, rad27-p Pol delta Exo III double mutants were viable. They exhibited strong synergistic increases in CAN1 duplication mutations, intrachromosomal and interchromosomal recombination, and required the wild-type double-strand break repair genes RAD50, RAD51, and RAD52 for viability. Observed effects were similar to those of the rad27-null mutant deficient in the removal of 5' flaps in the lagging strand. These results suggest that the 3'-->5' Exo activity of Pol delta is redundant with Rad27/Fen1 for creating ligatable nicks between adjacent Okazaki fragments, possibly by reducing the amount of strand-displacement in the lagging strand.

The Bloom's syndrome protein (BLM) interacts with MLH1 but is not required for DNA mismatch repair.

Bloom's syndrome (BS) is a rare autosomal recessive disorder characterized by pre- and postnatal growth deficiency, immunodeficiency, and a tremendous predisposition to a wide variety of cancers. Cells from BS individuals are characterized by a high incidence of chromosomal gaps and breaks, elevated sister chromatid exchange, quadriradial formations, and locus-specific mutations. BS is the consequence of mutations that lead to loss of function of BLM, a gene encoding a helicase with homology to the RecQ helicase family. To delineate the role of BLM in DNA replication, recombination, and repair we used a yeast two-hybrid screen to identify potential protein partners of the BLM helicase. The C terminus of BLM interacts directly with MLH1 in the yeast-two hybrid assay; far Western analysis and co-immunoprecipitations confirmed the interaction. Cell extracts deficient in BLM were competent for DNA mismatch repair. These data suggest that the BLM helicase and MLH1 function together in replication, recombination, or DNA repair events independent of single base mismatch repair.

5'-Deoxyribose phosphate lyase activity of human DNA polymerase iota in vitro.

DNA polymerase iota (pol iota) is one of several recently discovered DNA polymerases in mammalian cells whose function is unknown. We report here that human pol iota has an intrinsic 5'-deoxyribose phosphate (dRP) lyase activity. In reactions reconstituted with uracil-DNA glycosylase (UDG), apurinic/apyrimidinic (AP) endonuclease and DNA ligase I, pol iota can use its dRP lyase and polymerase activities to repair G*U and A*U pairs in DNA. These data and three distinct catalytic properties of pol iota implicate it in specialized forms of base excision repair (BER).

Purification of eukaryotic MutL homologs from Saccharomyces cerevisiae using self-affinity technology.

Self-cleaving affinity technology is an effective tool for rapid purification of native sequence recombinant proteins overproduced in Escherichia coli. In this report, we describe the adaptation of this technology to purify DNA mismatch repair proteins overproduced in the eukaryote Saccharomyces cerevisiae. Mlh1 and Pms1 are homologs of the E. coli MutL protein that participate in a variety of DNA transactions in cells, including correction of DNA replication errors, recombination, excision repair, and checkpoint control. Difficulties in preparing substantial quantities of highly purified MutL homologs have impeded descriptions of their biophysical and biochemical properties and mechanisms of action. To overcome this limitation, here we use self-cleaving affinity technology to purify to apparent homogeneity the yeast Mlh1--Pms1 heterodimer and the individual yeast and human Mlh1 subunit. The availability of these proteins should accelerate an understanding of their multiple functions in mismatch repair and other DNA transactions. The general approach is a valid alternative for simple, rapid purification of recombinant proteins in yeast when expression in bacteria is unsuitable.

Inactivation of DNA mismatch repair by increased expression of yeast MLH1.

Inactivation of DNA mismatch repair by mutation or by transcriptional silencing of the MLH1 gene results in genome instability and cancer predisposition. We recently found (P. V. Shcherbakova and T. A. Kunkel, Mol. Cell. Biol. 19:3177-3183, 1999) that an elevated spontaneous mutation rate can also result from increased expression of yeast MLH1. Here we investigate the mechanism of this mutator effect. Hybridization of poly(A)(+) mRNA to DNA microarrays containing 96.4% of yeast open reading frames revealed that MLH1 overexpression did not induce changes in expression of other genes involved in DNA replication or repair. MLH1 overexpression strongly enhanced spontaneous mutagenesis in yeast strains with defects in the 3'-->5' exonuclease activity of replicative DNA polymerases delta and epsilon but did not enhance the mutation rate in strains with deletions of MSH2, MLH1, or PMS1. This suggests that overexpression of MLH1 inactivates mismatch repair of replication errors. Overexpression of the PMS1 gene alone caused a moderate increase in the mutation rate and strongly suppressed the mutator effect caused by MLH1 overexpression. The mutator effect was also reduced by a missense mutation in the MLH1 gene that disrupted Mlh1p-Pms1p interaction. Analytical ultracentrifugation experiments showed that purified Mlh1p forms a homodimer in solution, albeit with a K(d) of 3.14 microM, 36-fold higher than that for Mlh1p-Pms1p heterodimerization. These observations suggest that the mismatch repair defect in cells overexpressing MLH1 results from an imbalance in the levels of Mlh1p and Pms1p and that this imbalance might lead to formation of nonfunctional mismatch repair complexes containing Mlh1p homodimers.

Proofreading of DNA polymerase eta-dependent replication errors.

Human DNA polymerase eta, the product of the skin cancer susceptibility gene XPV, bypasses UV photoproducts in template DNA that block synthesis by other DNA polymerases. Pol eta lacks an intrinsic proofreading exonuclease and copies DNA with low fidelity, such that pol eta errors could contribute to mutagenesis unless they are corrected. Here we provide evidence that pol eta can compete with other human polymerases during replication of duplex DNA, and in so doing it lowers replication fidelity. However, we show that pol eta has low processivity and extends mismatched primer termini less efficiently than matched termini. These properties could provide an opportunity for extrinsic exonuclease(s) to proofread pol eta-induced replication errors. When we tested this hypothesis during replication in human cell extracts, pol eta-induced replication infidelity was found to be modulated by changing the dNTP concentration and to be enhanced by adding dGMP to a replication reaction. Both effects are classical hallmarks of exonucleolytic proofreading. Thus, pol eta is ideally suited for its role in reducing UV-induced mutagenesis and skin cancer risk, in that its relaxed base selectivity may facilitate efficient bypass of UV photoproducts, while subsequent proofreading by extrinsic exonuclease(s) may reduce its mutagenic potential.