Multi-stage proofreading in DNA replication

Beckman, Robert A.; Loeb, Lawrence A.

doi:10.1017/s0033583500002869

Cited by 55 publications

(39 citation statements)

References 169 publications

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“…However, in reality, the fraction of mutator loci that result in ␣ above a certain cutoff will be smaller the higher the cutoff is. Mutations of proofreading, base selection, DNA repair, and chromosomal instability can produce values of ␣ within the ranges described in Table 1 (9,30,31,33). Mutations in Escherichia coli DNA polymerase I exhibit up to a 50,000-fold increase in mutation rate (31).…”

Section: Discussionmentioning

confidence: 99%

Efficiency of carcinogenesis with and without a mutator mutation

Beckman¹,

Loeb

2006

Proc. Natl. Acad. Sci. U.S.A.

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104

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Carcinogenesis involves the acquisition of multiple genetic changes altering various cellular phenotypes. These changes occur within the fixed time period of a human lifespan, and mechanisms that accelerate this process are more likely to result in clinical cancers. Mutator mutations decrease genome stability and, hence, accelerate the accumulation of random mutations, including those in oncogenes and tumor suppressor genes. However, if the mutator mutation is not in itself oncogenic, acquiring that mutation would add an extra, potentially time-consuming step in carcinogenesis. We present a deterministic mathematical model that allows quantitative prediction of the efficiency of carcinogenesis with and without a mutator mutation occurring at any time point in the process. By focusing on the ratio of probabilities of pathways with and without mutator mutations within cell lineages, we can define the frequency or importance of mutator mutations in populations independently of absolute rates and circumvent the question of whether mutator mutations are ''necessary'' for cancers to evolve within a human lifetime. We analyze key parameters that predict the relative contribution of mutator mutants in carcinogenesis. Mechanisms of carcinogenesis involving mutator mutations are more likely if they occur early. Involvement of mutator mutations in carcinogenesis is favored by an increased initial mutation rate, by greater fold-increase in mutation rate due to the mutator mutation, by increased required steps in carcinogenesis, and by increased number of cell generations to the development of cancer.cancer ͉ mathematical model ͉ mutator hypothesis T he development of a cancer requires multiple steps as suggested by epidemiologic data and animal models (1-5). At least some of these steps are believed to involve oncogenic mutations or other genetic changes.The mutator hypothesis originally stated that mutations in DNA polymerases and DNA repair enzymes would play a critical role in carcinogenesis by accelerating the acquisition of oncogenic mutations (6). This concept has been broadened to include a variety of mutator mutations that create genetic instability by mechanisms including microsatellite instability, chromosomal instability, and aberrations of checkpoint control (7-10).An alternative hypothesis is that enhanced rates of genetic instability are not necessary for carcinogenesis and that cancer arises from mutations occurring at the normal rate followed by multiple rounds of lineage selection and expansion (11)(12)(13)(14).These two hypotheses are not mutually exclusive. Indeed it is likely that carcinogenic pathways corresponding to both hypotheses contribute concurrently. For example, carcinogenesis could involve a mutator mutation and subsequent mutations augmented by lineage selection and expansion.The common outcome of a malignant lineage is likely approachable by numerous pathways, and we postulate that these pathways will be seen in clinical cancers in proportion to their relative efficiencies, irrespective of w...

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Section: Discussionmentioning

confidence: 99%

Efficiency of carcinogenesis with and without a mutator mutation

Beckman¹,

Loeb

2006

Proc. Natl. Acad. Sci. U.S.A.

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104

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“…Like other small-size DNA alterations and point mutations, small palindromic additions create variability. Intriguingly, they appear to be produced exclusively by V(D)J recombination or excision of transposons of the hAT family, as they are not otherwise within the range of DNA alterations produced by repair of chromosomal doublestrand breaks (15,25,30), nor are they produced by replication errors (5). Since V(D)J recombination is a somatic process, however, the mutations that it produces do not serve as raw material for evolution.…”

Section: Discussionmentioning

confidence: 99%

Extensive, Nonrandom Diversity of Excision Footprints Generated by Ds-Like Transposon Ascot-1Suggests New Parallels with V(D)J Recombination

Colot

Haedens

Rossignol

1998

Molecular and Cellular Biology

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Upon insertion, transposable elements can disrupt or alter gene function in various ways. Transposons moving through a cut-and-paste mechanism are in addition often mutagenic when excising because repair of the empty site seldom restores the original sequence. The characterization of numerous excision events in many eukaryotes indicates that transposon excision from a given site can generate a high degree of DNA sequence and phenotypic variation. Whether such variation is generated randomly remains largely to be determined. To this end, we have exploited a well-characterized system of genetic instability in the fungus Ascobolus immersus to perform an extensive study of excision events. We show that this system, which produces many phenotypically and genetically distinct derivatives, results from the excision of a novel Ds-like transposon, Ascot-1, from the spore color gene b2. A unique set of 48 molecularly distinct excision products were readily identified from a representative sample of excision derivatives. Products varied in their frequency of occurrence over 4 orders of magnitude, yet most showed small palindromic nucleotide additions. Based on these and other observations, compelling evidence was obtained for intermediate hairpin formation during the excision reaction and for strong biases in the subsequent processing steps at the empty site. Factors likely to be involved in these biases suggest new parallels between the excision reaction performed by transposons of the hAT family and V(D)J recombination. An evaluation of the contribution of small palindromic nucleotide additions produced by transposon excision to the spectrum of spontaneous mutations is also presented.Transposons are ubiquitous components of both prokaryotic and eukaryotic genomes, contributing to structural organization, gene activity, and evolution. Two classes of transposons have been distinguished according to their modes of transposition (12). Class I transposons, or retroelements, move by reverse transcription of their RNA, whereas class II transposons are mobilized by either excision/reinsertion or cointegration. Although class I and class II transposons generate variability primarily by their insertion into host DNA, class II elements that move by a cut-and-paste mechanism often produce additional variability at a given site as a result of the excision footprint left at that site. Understanding how these footprints are formed is therefore essential for the proper evaluation of the full mutational impact of the class II transposons.The excision reaction can be separated into two steps, double-strand break formation at both ends of the transposon and repair of the gapped molecule. In vivo and in vitro studies performed with bacterial and animal DNA transposons have shown that all transposases examined cleave precisely at the 3ЈOH ends of the element, which serve for the subsequent strand transfer reaction. On the other hand, the position of the 5Ј cleavage required for the excision of the element need not occur precisely at the tran...

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“…A second explanation for the reduced fidelity of our mutants involves a pyrophosphate error prevention mechanism (44,45). It might have been predicted that amino acid substitutions adjacent to the Phe and Tyr (Fig.…”

Section: Low Fidelity Mutants Of T Aquaticus Dna Polymerase Imentioning

confidence: 98%

“…The assignment of individual amino acid residues that interact with specific atoms in the incoming dNTPs is tentative, at best (46). It is based primarily on inferences from kinetic studies (45), binding studies with binary complexes (44), and cross-linking studies with wild-type and mutant DNA polymerases (11). Nevertheless, evidence suggests that both Lys-663 and Arg-659, and the homologous residues in other DNA polymerases, interact with the ␤-and ␥-phosphates in the incoming dNTPs and could thus facilitate removal of pyrophosphate during catalysis (44).…”

Section: Low Fidelity Mutants Of T Aquaticus Dna Polymerase Imentioning

confidence: 99%

“…This error preventing step follows binding of the substrate and precedes primer elongation. Enhancement of misincorporation by the addition of pyrophosphate has been demonstrated for DNA polymerases (49) and has been attributed to a kinetic proofreading step in a multi-step proofreading mechanism (45).…”

Section: Low Fidelity Mutants Of T Aquaticus Dna Polymerase Imentioning

confidence: 99%

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Low Fidelity Mutants in the O-Helix of Thermus aquaticus DNA Polymerase I

Suzuki¹,

Avicola²,

Hood

et al. 1997

Journal of Biological Chemistry

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We screened 67 mutants in the O-helix of Thermus aquaticus (Taq) DNA polymerase I (pol I) for altered fidelity of DNA synthesis. These mutants were obtained (Suzuki, M., Baskin, D., Hood, L., and Loeb, L. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9670 -9675) by substituting an oligonucleotide containing random sequences for codons 659 -671, and selecting for complementation of a growth defect in Escherichia coli caused by temperature-sensitive host pol I. Thirteen mutants decreased fidelity in a screen that employed primer extension reactions lacking one of four complementary deoxynucleoside triphosphates (dNTPs). Three mutants were purified and exhibited 29 -68% of wild-type specific activity. Homogeneous polymerases A661E, A661P, and T664R extended primers further than the wild-type, synthesizing past template nucleotides for which the complementary dNTP was absent. The data indicate that both misinsertion of incorrect nucleotides and extension of mispaired primer termini were increased. In a lacZ␣ forward mutation assay, A661E and T664R yielded mutation frequencies at least 7-and 25-fold greater, respectively, than that of the wild-type polymerase. These findings emphasize the importance of the O-helix in substrate recognition and are compatible with a role for pyrophosphate release in enhancing fidelity of DNA synthesis. Thermus aquaticus (Taq) DNA polymerase I (pol I)1 is used extensively in the amplification of DNA by the polymerase chain reaction. Based on its primary sequence, crystal structure, and catalytic mechanism, Taq pol I is classified in the same family as Escherichia coli pol I (1-3). The amino acid sequences of Taq pol I and E. coli pol I are 38% homologous, both enzymes have 5Ј-3Ј exonuclease domains (4, 5), and the crystal structures of the polymerase domains are nearly identical (5, 6). The structure of the vestigial 3Ј-5Ј exonuclease domain of Taq pol I is very different, however; unlike E. coli pol I, purified Taq pol I exhibits no proofreading activity (7).The O-helix in E. coli pol I is constituted of 15 amino acids which form part of the DNA binding cleft (8). Crystallographic, biochemical, and mutagenesis studies indicate that four conserved residues in the O-helix which face toward the cleft interact with either the incoming dNTP or the templateprimer. Considerable evidence indicates that in E. coli pol I, Arg-754 and Lys-758 interact with the phosphate groups in the incoming dNTP, that Phe-762 interacts with the deoxyribose moiety in the dNTP, and that Tyr-766 binds to the templateprimer (9 -11). The nearly identical structures of the polymerase domains of Taq pol I and E. coli pol I (6, 7) suggest that the corresponding amino acids in Taq pol I have analogous functions. By using random sequence mutagenesis together with genetic complementation, we recently observed that the Ohelix of Taq pol I contains four amino acids which are immutable (or nearly immutable) and are apparently essential for catalysis in vivo (12). These residues, Arg-659, Lys-663, Phe-667, and Tyr-671, corres...

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Multi-stage proofreading in DNA replication

Cited by 55 publications

References 169 publications

Efficiency of carcinogenesis with and without a mutator mutation

Efficiency of carcinogenesis with and without a mutator mutation

Extensive, Nonrandom Diversity of Excision Footprints Generated by Ds-Like Transposon Ascot-1Suggests New Parallels with V(D)J Recombination

Low Fidelity Mutants in the O-Helix of Thermus aquaticus DNA Polymerase I

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