Expansions of CAG repeat tracts are frequent in a yeast mutant defective in Okazaki fragment maturation
Expansions of CAG repeat tracts are frequent in a yeast mutant defective in Okazaki fragment maturationJill Kuglin Schweitzer and Dennis M. Livingston*
Department of Biochemistry, 4-225 Millard Hall, 435 Delaware Street SE, University of Minnesota, Minneapolis, MN 55455-0347, USA
Received August 18, 1997;Revised and Accepted October 21, 1997
To understand the causes of CAG repeat tract changes that occur in the passage of human disease alleles, we are studying the effect of replication and repair mutations on CAG repeat tracts embedded in a yeast chromosome. In this report, we examine the effect of a mutation in the RTH1/RAD27 gene encoding a deoxyribonuclease needed for removal of excess nucleotides at the 5'-end of Okazaki fragments. Deletion of the RTH1/RAD27 gene has two effects on CAG tracts. First, the rth1/rad27 mutation destabilizes CAG tracts. Second, although most tract length changes in wild-type yeast cells are tract contractions, approximately half of the changes that occur as a result of the rth1/rad27 mutation are expansions of one or more repeat units. These results support the hypothesis that tract expansions that occur during passage of human disease alleles bearing expanded CAG tracts result from excess DNA synthesis on the lagging strand of replication.
DNA tracts containing repetitions of the trinucleotide CAG are the mutational cause of a number of inherited human neurodegenerative and neuromuscular diseases. Among many of the disease genes, CAG tracts are found to encode a string of glutamine residues in the respective protein products (1 -8 ). Disease alleles are caused by tracts that are abnormally long. In the diseases where the repeat tracts encode glutamine residues, tracts that become longer than ~40 repeat units cause the disease.
A prominent feature of these disease alleles is that while the repeat tracts remain relatively stable during somatic division (9 -12 ), they often change in length when passed from parent to child. Tract length changes most likely occur during gametogenesis, and are sometimes biased towards expansion of the repeat tract. Among the diseases where repeat tracts encode polyglutamine, the bias towards expansion is almost always more prevalent when the disease allele is passed from the father (3 ,6 ,13 -18 ). The bias towards tract expansion provides a molecular explanation for the anticipation, i.e. the earlier onset and more severe clinical presentation, as the disease allele is passed through the generations of a pedigree.
To understand why CAG repeat tracts are unstable, we have been studying the stability of a repeat tract embedded in a yeast chromosome (19 ,20 ). Tract length changes in mitotically dividing, wild-type yeast cells are overwhelmingly contractions. Repeat tract stability in yeast is greatly affected by the orientation of the tract with respect to the leading and the lagging strand of replication (19 ,21 ). An advantage of yeast is that mutations affecting many aspects of DNA replication, repair and recombination are available to apply to the study of CAG tract stability in this simple eukaryote. We have already shown that the mismatch repair system prevents small changes in repeat tract length from occurring but does not prevent longer tract length changes (20 ).
In this report, we examine the effect of a deletion of the RTH1/RAD27 gene (22 ,23 ) encoding a deoxyribonuclease needed to process the ends of Okazaki fragments (24 -27 ). Deletion of this gene has been shown to cause small expansions of dinucleotide repeat tracts (28 ) and to favor sequence expansions in tests of forward and reverse mutations of particular yeast genes (29 ). In addition, rth1/rad27 mutant strains have a number of phenotypes that indicate a role in DNA replication and repair. These phenotypes include sensitivity to the radiomimetic agent methylmethane sulfonate, partial temperature sensitivity for growth, and cell cycle arrest (23 ,30 ). Mutants are not particularly sensitive to ionizing radiation and are also resistant to UV irradiation in some strain backgrounds (23 ,30 ). Kunkel, Resnick and Gordenin (31 ,32 ) have commented on the role of the RTH1/RAD27 gene product to produce expansions of repeated sequences including trinucleotide repeats. This study shows that deletion of RTH1/RAD27 not only has the effect of destabilizing CAG tracts in a yeast chromosome, but also alters the pattern of tract changes from one where tract contractions are the overwhelming majority of events to a new state where tract expansions represent approximately half of the changes.
CAG repeat tracts copied from a disease allele of the human ataxin1 gene (4 ) have been appended to the 3'-end of the yeast ADE2 gene (Fig. 1 ) (19 ). In turn, the appended ADE2 gene was placed in the ARO2 gene and the resulting aro2 disruption was then used to replace the ARO2 locus on yeast chromosome VII. The repeat has been placed with either CAG or its complement CTG in the ADE2 coding strand, and the ADE2 gene has been oriented in both directions with respect to the ARO2 coding sequence (Fig. 1 ). The ADE2 clone contains a replication origin, termed ARS, 5' to the ADE2 coding sequence that greatly affects the stability of the repeat tracts. Tracts are relatively stable when CAG is the lagging strand template, and relatively unstable, yielding many tract contractions, when CTG is the lagging strand template (19 ). In one case, tract H-, the ARS element has been removed from the ADE2 fragment so that replication into the repeats proceeds from a replication origin that is upstream of ARO2 (Fig. 1 ). A rth1/rad27 deletion-disruption was introduced into these strains by mating. Because tracts are very unstable in yeast, most tracts used in this study are different from the tract length found in the original ataxin1 disease allele.
Tract C is oriented with CAG as the lagging strand template and is relatively stable in wild-type yeast cells (19 ). Combining results from this study with our two previous studies on tract C in wild-type yeast cells (19 ,20 ), we have recorded only four changes in 291 sibling colonies in tracts ranging in size from 28 to 82 repeat units. Of the four changes, three were tract contractions and one was a tract expansion. When a tract containing 33 repeat units was introduced into a rth1/rad27 mutant, 10 of the 62 sibling colonies contained tract length changes, and nine of these changes were expansions (Fig. 2 ). The expansions ranged from five to 35 repeat units in length. Thus, not only does a rth1/rad27 deletion destabilize the repeat tract but it also biases the changes to favor expansions over contractions. A longer tract containing 78 repeat units was even more unstable in this mutant. Analysis of 61 sibling colonies showed that 33 had tract length changes. Of these, approximately half were expansions and half were contractions (Table 1 ). As with the shorter tract, the expansions ranged from a single repeat unit to one as large as 40 repeat units. As a control, this same tract was examined in a wild-type strain that was derived from a sister spore of the tetrad giving rise to the rth1/rad27 mutant. No tract changes were found among 64 sibling colonies (Table 1 ).
The same general result was observed using another tract, H-, oriented with CAG as the lagging strand template (Fig. 1 ). Like tract C, tract H- is relatively stable in a wild-type strain, yielding only one change, a contraction, among 63 sibling colonies from a tract that began with 52 repeat units (Table 1 ). In a rth1/rad27 mutant, a short tract of 38 repeat units underwent 11 changes in 63 sibling colonies. Of those changes, slightly more than half were expansions (Table 1 ). A longer tract of 52 repeat units yielded 16 changes among 63 sibling colonies, of which six of the changes were expansions (Table 1 ).
Tracts D and H are oriented with CTG as the lagging strand template and differ from each other only in the chromosomal orientation of the ADE2 gene (Fig. 1 ). These tracts are relatively unstable in wild-type yeast cells and mostly undergo tract contractions (19 ). The results from the wild-type strain used in this study showed that a tract of 71 repeat units of orientation D yielded 11 contractions and no expansions among 64 sibling colonies (Table 1 ), while a tract of 87 repeat units of orientation H yielded 14 contractions and one expansion among 63 sibling colonies (Table 1 ). In a rth1/rad27 mutant, tract D underwent 44 changes among 62 sibling colonies, of which eight were expansions and the rest contractions (Table 1 ). Similarly, in the rth1/rad27 mutant, tract H underwent 43 changes among 63 sibling colonies, of which 15 were expansions and the remainder contractions (Table 1 ). Thus, the rth1/rad27 mutation further destabilizes these tracts. If the number of contraction events that occur in wild-type cells are subtracted from the distribution in the mutant cells, it appears that approximately half of the events that are created by the rth1/rad27 mutation are expansions and half are contractions.
Shorter D and H tracts were also examined in a rth1/rad27 mutant. These tracts underwent few changes: three changes among 63 sibling colonies for a D tract of 30 repeat units and three changes among 63 sibling colonies for an H tract of 33 repeat units (Table 1 ). Two of the H tract changes were expansions, but the three D tract changes were contractions. Thus, the behavior of these shorter tracts appear to differ from the behavior of tracts C and H- in that they are only slightly destabilized by the rth1/rad27 mutation.
To ensure that the tract expansions in the rth1/rad27 mutant resulted from direct duplication of CAG repeat units, three of the expansion alleles were sequenced (data not shown). An unusual expansion allele of tract C that was recorded as an increase from 33 to 68 repeat units and two expansion alleles from tract H that were recorded as changes from 33 repeat units to 55 and 56, respectively, were sequenced. In all three cases, the expansions resulted solely from increases in repeat units. We did not observe a change in the flanking sequences surrounding the repeat tracts, an inversion of repeat units within the tracts or the introduction of interruptions into the tracts. Thus, most expansions are likely to be direct duplications within the repeat tracts. The unusual nature of the tract that increased from 33 to 68 units is most likely accounted for by the culmination of more than one expansion event during colony growth.
[chi]2 analysis of comparisons between the distributions of tract expansions and contractions in mutant and wild-type strains reveals that in all cases the distributions are significantly different (P <0.001). Furthermore, the mutant distributions are significantly different from each other (P <0.001). Although this might be expected for comparisons between the tracts that are normally stable and unstable in wild-type cells, the differences are also significant between the two tracts of the stable (C and H-) orientation and between the two tracts of the unstable (D and H) orientation as well. This may reveal an underlying role of the context of surrounding sequences. One trend that the results in Table 1 reveal is that tracts become more unstable in mutant cells as they become longer. This trend was substantiated by additional results showing that tracts of intermediate length yield an intermediate number of tract changes (data not shown). What is also evident from the results of Table 1 is that in all cases, except in the case of tract D, the ratio of expansions to contractions is higher for the shorter tract than for the longer tracts. Again, this trend was followed by the tracts of intermediate length (data not shown). Finally, the mean sizes of the expansions ranged from 8.9 (tract D:71) to 16.6 (tract C:78), while the mean sizes of the contractions ranged from -9.2 (tract H-:52) to -41.4 (tract H:87), showing that in general the contractions were larger than the expansions. As discussed below, this difference probably results from the different mechanisms involved in recovering from the damage created by the absence of RTH1/RAD27.
Although the normal inclination of yeast cells is to contract the length of CAG repeat tracts, numerous examples of tract expansions are found in a rth1/rad27 mutant. Tract expansions occur when either CAG or CTG is the lagging strand template. In the case of tracts with CAG providing the lagging strand template, the tracts are relatively stable in wild-type cells and all the events are attributable to the rth1/rad27 mutation. When CTG is the lagging strand template, tracts undergo frequent contractions in wild-type cells. The effect of the rth1/rad27 mutation is to destabilize these tracts further and to add a new class of events containing expansions.
We interpret the results in terms of events occurring on the lagging strand because the RTH1/RAD27 gene product encodes a deoxyribonuclease that is used in the maturation of Okazaki fragments (23 ,26 -30 ). In order to join Okazaki fragments together, RNA primers must be removed, DNA must be synthesized in its place and ligase must seal the interruption. Enzymological studies have shown that eukaryotic RNase H removes all but one ribonucleotide of the primer (24 ,25 ). This ribonucleotide prevents ligation of the adjoining Okazaki fragments. The RTH1/RAD27 gene product, referred to as the FLAP endonuclease or FEN1, is responsible for removing the last ribonucleotide. Nuclease action may require the displacement of a portion of the Okazaki fragment before it can act. DNA polymerization from the 3'-end of the newest Okazaki fragment into the displaced flap creates an excess of single strand DNA (Fig. 3 ). When the displaced strand contains a repeated sequence, it can potentially reanneal to the template, creating a loop structure. Without its removal, the loop of DNA may be fixed during a subsequent round of replication, creating an expansion of the sequence (Fig. 3 ).
Isogenic strains SSL204a and SSL204[alpha] containing CAG repeat tracts inserted into yeast chromosome VII in different orientations have been described previously (19 ). A deletion of RTH1/RAD27 disrupted by HIS3 was created in the parental strains SSL204a and SSL204[alpha] lacking repeat tracts using the PCR primers described by Tishkoff et al. (29 ) based upon the method of Manivasakam et al. (37 ). These strains were mated to the strains containing the CAG repeats and the resulting diploids were sporulated. Sister spores containing the repeat tract and either the rth1/rad27 deletion or its wild-type allele provide isogenic strains with repeat tracts of the same length.
The methods to propagate yeast strains for the purpose of determining the frequency with which a CAG repeat tract changes in length and the PCR assay performed on chromosomal DNA from sibling yeast colonies have been described (19 ,20 ). A yeast colony harboring a CAG repeat tract is designated the parental colony and a portion of the colony is used to prepare DNA in order to establish the parental tract length. Another portion of the colony is dispersed into single cells that grow into sibling colonies. Analysis of the tract lengths in the DNA from the sibling colonies reveals that some of the sibling colonies no longer contain a tract of parental length but instead contain a repeat tract of a different length. This signifies that the parental colony contained cells that had undergone tract length changes. The frequency of such colonies yields a metric of the instability. Because of the instability of repeat tracts in yeast, colonies that contain both the parental repeat tract length and tracts of larger or smaller length are often observed (Fig. 2 ). These are not counted in the analysis because they arise from changes that occur after dispersal of the parental colony. Approximately 30 sibling colonies are sampled for each parental colony, and more than one parental colony is used for each tract and strain.
We thank Harry Orr for his encouragement and for his comments on the manuscript. This work was supported by grant P01NS33718 from the National Institutes of Health.
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*To whom correspondence should be addressed. Tel: +1 612 625 1484; Fax: +1 612 625 2163; Email: livin001@maroon.tc.umn.edu
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R. Pelletier, B. T. Farrell, J. J. Miret, and R. S. Lahue Mechanistic features of CAG*CTG repeat contractions in cultured cells revealed by a novel genetic assay
Nucleic Acids Res.,
September 30, 2005;
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S. Bhattacharyya and R. S. Lahue Srs2 Helicase of Saccharomyces cerevisiae Selectively Unwinds Triplet Repeat DNA
J. Biol. Chem.,
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R. D. Wells, R. Dere, M. L. Hebert, M. Napierala, and L. S. Son Advances in mechanisms of genetic instability related to hereditary neurological diseases
Nucleic Acids Res.,
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W. Wang and R. A. Bambara Human Bloom Protein Stimulates Flap Endonuclease 1 Activity by Resolving DNA Secondary Structure
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C. D. Bayliss, W. A. Sweetman, and E. R. Moxon Destabilization of tetranucleotide repeats in Haemophilus influenzae mutants lacking RnaseHI or the Klenow domain of PolI
Nucleic Acids Res.,
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V. I. Hashem, M. J. Pytlos, E. A. Klysik, K. Tsuji, M. Khajav, T. Ashizawa, and R. R. Sinden Chemotherapeutic deletion of CTG repeats in lymphoblast cells from DM1 patients
Nucleic Acids Res.,
December 1, 2004;
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R. Dere, M. Napierala, L. P. W. Ranum, and R. D. Wells Hairpin Structure-forming Propensity of the (CCTG{middle dot}CAGG) Tetranucleotide Repeats Contributes to the Genetic Instability Associated with Myotonic Dystrophy Type 2
J. Biol. Chem.,
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S. Bhattacharyya and R. S. Lahue Saccharomyces cerevisiae Srs2 DNA Helicase Selectively Blocks Expansions of Trinucleotide Repeats
Mol. Cell. Biol.,
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B. L. Ruggiero and M. D. Topal Triplet Repeat Expansion Generated by DNA Slippage Is Suppressed by Human Flap Endonuclease 1
J. Biol. Chem.,
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Y. Liu, H. Zhang, J. Veeraraghavan, R. A. Bambara, and C. H. Freudenreich Saccharomyces cerevisiae Flap Endonuclease 1 Uses Flap Equilibration To Maintain Triplet Repeat Stability
Mol. Cell. Biol.,
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S. Sharma, M. Otterlei, J. A. Sommers, H. C. Driscoll, G. L. Dianov, H.-I Kao, R. A. Bambara, and R. M. Brosh Jr. WRN Helicase and FEN-1 Form a Complex upon Replication Arrest and Together Process Branchmigrating DNA Structures Associated with the Replication Fork
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B. L. Heidenfelder and M. D. Topal Effects of sequence on repeat expansion during DNA replication
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J. L. Callahan, K. J. Andrews, V. A. Zakian, and C. H. Freudenreich Mutations in Yeast Replication Proteins That Increase CAG/CTG Expansions Also Increase Repeat Fragility
Mol. Cell. Biol.,
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J. Veeraraghavan, M. L. Rossi, and R. A. Bambara Analysis of DNA Replication Intermediates Suggests Mechanisms of Repeat Sequence Expansion
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C. Spiro and C. T. McMurray Nuclease-Deficient FEN-1 Blocks Rad51/BRCA1-Mediated Repair and Causes Trinucleotide Repeat Instability
Mol. Cell. Biol.,
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P. J. A. Gutierrez and T. S.-F. Wang Genomic Instability Induced by Mutations in Saccharomyces cerevisiae POL1
Genetics,
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S.-R. Yoon, L. Dubeau, M. de Young, N. S. Wexler, and N. Arnheim Huntington disease expansion mutations in humans can occur before meiosis is completed
PNAS,
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J. Parenteau and R. J. Wellinger Differential Processing of Leading- and Lagging-Strand Ends at Saccharomyces cerevisiae Telomeres Revealed by the Absence of Rad27p Nuclease
Genetics,
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M. J. Hartenstine, M. F. Goodman, and J. Petruska Weak Strand Displacement Activity Enables Human DNA Polymerase beta to Expand CAG/CTG Triplet Repeats at Strand Breaks
J. Biol. Chem.,
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S. Bhattacharyya, M. L. Rolfsmeier, M. J. Dixon, K. Wagoner, and R. S. Lahue Identification of RTG2 as a Modifier Gene for CTG{middle dot}CAG Repeat Instability in Saccharomyces cerevisiae
Genetics,
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L. A. Henricksen, J. Veeraraghavan, D. R. Chafin, and R. A. Bambara DNA Ligase I Competes with FEN1 to Expand Repetitive DNA Sequences in Vitro
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G. B. Panigrahi, J. D. Cleary, and C. E. Pearson In Vitro (CTG){middle dot}(CAG) Expansions and Deletions by Human Cell Extracts
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E. A. Sia, M. Dominska, L. Stefanovic, and T. D. Petes Isolation and Characterization of Point Mutations in Mismatch Repair Genes That Destabilize Microsatellites in Yeast
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J. K. Schweitzer, S. S. Reinke, and D. M. Livingston Meiotic Alterations in CAG Repeat Tracts
Genetics,
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C. J. M. Bontekoe, C. E. Bakker, I. M. Nieuwenhuizen, H. van der Linde, H. Lans, D. de Lange, M. C. Hirst, and B. A. Oostra Instability of a (CGG)98 repeat in the Fmr1 promoter
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PNAS,
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M. L. Rolfsmeier, M. J. Dixon, L. Pessoa-Brandão, R. Pelletier, J. J. Miret, and R. S. Lahue Cis-Elements Governing Trinucleotide Repeat Instability in Saccharomyces cerevisiae
Genetics,
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M. L. Moseley, L. J. Schut, T. D. Bird, M. D. Koob, J. W. Day, and L. P.W. Ranum SCA8 CTG repeat: en masse contractions in sperm and intergenerational sequence changes may play a role in reduced penetrance
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M. W. Walberg Applicability of Yeast Genetics to Neurologic Disease
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M. J. Ireland, S. S. Reinke, and D. M. Livingston The Impact of Lagging Strand Replication Mutations on the Stability of CAG Repeat Tracts in Yeast
Genetics,
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H. Seznec, A.-S. Lia-Baldini, C. Duros, C. Fouquet, C. Lacroix, H. Hofmann-Radvanyi, C. Junien, and G. Gourdon Transgenic mice carrying large human genomic sequences with expanded CTG repeat mimic closely the DM CTG repeat intergenerational and somatic instability
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Q. Liu, W.-c. Choe, and J. L. Campbell Identification of the Xenopus laevis Homolog of Saccharomyces cerevisiae DNA2 and Its Role in DNA Replication
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R. R. Iyer, A. Pluciennik, W. A. Rosche, R. R. Sinden, and R. D. Wells DNA Polymerase III Proofreading Mutants Enhance the Expansion and Deletion of Triplet Repeat Sequences in Escherichia coli
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S. Simeoni, M. A. Mancini, D. L. Stenoien, M. Marcelli, N. L. Weigel, M. Zanisi, L. Martini, and A. Poletti Motoneuronal cell death is not correlated with aggregate formation of androgen receptors containing an elongated polyglutamine tract
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R. Gary, M. S. Park, J. P. Nolan, H. L. Cornelius, O. G. Kozyreva, H. T. Tran, K. S. Lobachev, M. A. Resnick, and D. A. Gordenin A Novel Role in DNA Metabolism for the Binding of Fen1/Rad27 to PCNA and Implications for Genetic Risk
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P. J. White, R. H. Borts, and M. C. Hirst Stability of the Human Fragile X (CGG)n Triplet Repeat Array in Saccharomyces cerevisiae Deficient in Aspects of DNA Metabolism
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J. K. Schweitzer and D. M. Livingston The Effect of DNA Replication Mutations on CAG Tract Stability in Yeast
Genetics,
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J. Parenteau and R. J. Wellinger Accumulation of Single-Stranded DNA and Destabilization of Telomeric Repeats in Yeast Mutant Strains Carrying a Deletion of RAD27
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H. Cohen, D. D. Sears, D. Zenvirth, P. Hieter, and G. Simchen Increased Instability of Human CTG Repeat Tracts on Yeast Artificial Chromosomes during Gametogenesis
Mol. Cell. Biol.,
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F. Paques and J. E. Haber Multiple Pathways of Recombination Induced by Double-Strand Breaks in Saccharomyces cerevisiae
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PNAS,
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R. R. Iyer and R. D. Wells Expansion and Deletion of Triplet Repeat Sequences in Escherichia coli Occur on the Leading Strand of DNA Replication
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R. J. Kokoska, L. Stefanovic, A. B. Buermeyer, R. M. Liskay, and T. D. Petes A Mutation of the Yeast Gene Encoding PCNA Destabilizes Both Microsatellite and Minisatellite DNA Sequences
Genetics,
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C.-Y. Kim, B. Shen, M. S. Park, and G. A. Olah Structural Changes Measured by X-ray Scattering from Human Flap Endonuclease-1 Complexed with Mg2+ and Flap DNA Substrate
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G. Frank, J. Qiu, M. Somsouk, Y. Weng, L. Somsouk, J. P. Nolan, and B. Shen Partial Functional Deficiency of E160D Flap Endonuclease-1 Mutant in Vitro and in Vivo Is Due to Defective Cleavage of DNA Substrates
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D. J. Hosfield, G. Frank, Y. Weng, J. A. Tainer, and B. Shen Newly Discovered Archaebacterial Flap Endonucleases Show a Structure-Specific Mechanism for DNA Substrate Binding and Catalysis Resembling Human Flap Endonuclease-1
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J. J. Miret, L. Pessoa-Brandao, and R. S. Lahue Orientation-dependent and sequence-specific expansions of CTG/CAG trinucleotide repeats in Saccharomyces cerevisiae
PNAS,
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D. J. Maurer, B. L. O'Callaghan, and D. M. Livingston Mapping the Polarity of Changes That Occur in Interrupted CAG Repeat Tracts in Yeast
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R. D. Wells, P. Parniewski, A. Pluciennik, A. Bacolla, R. Gellibolian, and A. Jaworski Small Slipped Register Genetic Instabilities in Escherichia coli in Triplet Repeat Sequences Associated with Hereditary Neurological Diseases
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R. J. Kokoska, L. Stefanovic, H. T. Tran, M. A. Resnick, D. A. Gordenin, and T. D. Petes Destabilization of Yeast Micro- and Minisatellite DNA Sequences by Mutations Affecting a Nuclease Involved in Okazaki Fragment Processing (rad27) and DNA Polymerase delta (pol3-t)
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A. Pluciennik, R. R. Iyer, P. Parniewski, and R. D. Wells Tandem Duplication. A NOVEL TYPE OF TRIPLET REPEAT INSTABILITY
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M. E. Budd, W.-c. Choe, and J. L. Campbell The Nuclease Activity of the Yeast Dna2 Protein, Which Is Related to the RecB-like Nucleases, Is Essential in Vivo
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PNAS,
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