FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2001231205> ?p ?o ?g. }

• W2001231205 endingPage "42866" @default.
• W2001231205 startingPage "42854" @default.
• W2001231205 abstract "We previously developed a system to investigate the mechanism of repeat sequence expansion during eukaryotic Okazaki fragment processing. Upstream and downstream primers were annealed to a complementary template to overlap across a CAG repeat region. Annealing by the competing primers lead to structural intermediates that ligated to expand the repeat segment. When an equal number of repeats overlapped on the upstream and downstream primers, a 2-fold expansion was expected, but no expansion occurred. We show here that such substrates do not expand irrespective of their repeat length. To reveal mechanism, we tested different hairpin loop intermediates expected to form and facilitate ligation. Substrates configured to form large loops in either the upstream or downstream primer alone allowed expansion. Large or small fixed position single loops allowed expansion when located at least six nucleotides up- or downstream of the nick. Fixed loops in both primers, simulating a double loop intermediate, allowed expansion as long as each loop was nine nucleotides from the nick. Thus, neither the double loop configuration required to form with equal length overlaps nor the large single loop configuration are fundamental structural impediments to expansion. We propose a model for the expansion mechanism based on the relative stabilities of single loop, double loop, hairpin, and flap intermediates that is consistent with the observed expansion efficiency of equal and unequal overlap substrates. The model suggests that the equilibrium concentration of double loop intermediates is so vanishingly small that they are not likely contributors to sequence expansion. We previously developed a system to investigate the mechanism of repeat sequence expansion during eukaryotic Okazaki fragment processing. Upstream and downstream primers were annealed to a complementary template to overlap across a CAG repeat region. Annealing by the competing primers lead to structural intermediates that ligated to expand the repeat segment. When an equal number of repeats overlapped on the upstream and downstream primers, a 2-fold expansion was expected, but no expansion occurred. We show here that such substrates do not expand irrespective of their repeat length. To reveal mechanism, we tested different hairpin loop intermediates expected to form and facilitate ligation. Substrates configured to form large loops in either the upstream or downstream primer alone allowed expansion. Large or small fixed position single loops allowed expansion when located at least six nucleotides up- or downstream of the nick. Fixed loops in both primers, simulating a double loop intermediate, allowed expansion as long as each loop was nine nucleotides from the nick. Thus, neither the double loop configuration required to form with equal length overlaps nor the large single loop configuration are fundamental structural impediments to expansion. We propose a model for the expansion mechanism based on the relative stabilities of single loop, double loop, hairpin, and flap intermediates that is consistent with the observed expansion efficiency of equal and unequal overlap substrates. The model suggests that the equilibrium concentration of double loop intermediates is so vanishingly small that they are not likely contributors to sequence expansion. Repeat sequences are distributed widely in all organisms, forming the micro- and mini-satellite regions of their chromosomal DNAs (1Bennett P. Mol. Pathol. 2000; 53: 177-183Crossref PubMed Scopus (68) Google Scholar). Triplet repeat sequences have attracted particular attention, because they are involved in pathogenesis of at least 14 neurological disorders (2Usdin K. Grabczyk E. Cell. Mol. Life Sci. 2000; 57: 914-931Crossref PubMed Scopus (86) Google Scholar). Of interest are the CAG, CGG, and GAA repeat tracts that are present in the normal population in lengths of 10–25 triplets and show significant length polymorphisms. In a subset of the population repeat lengths reach the relatively stable pre-mutational length of 30–50 repeats, which then undergo large intergenerational expansion by mechanisms that are poorly understood. Repeat sequences present in coding regions expand to a smaller extent than repeats that are located in 3′-untranslated region or regulatory regions that show expansion into thousands of repeats (2Usdin K. Grabczyk E. Cell. Mol. Life Sci. 2000; 57: 914-931Crossref PubMed Scopus (86) Google Scholar). Locus-specific expansion of CAG/CTG and CGG/CCG sequences suggests that the instability is an inherent property of repeat DNA (3Goellner G.M. Tester D. Thibodeau S. Almqvist E. Goldberg Y.P. Hayden M.R. McMurray C.T. Am. J. Hum. Genet. 1997; 60: 879-890PubMed Google Scholar). One characteristic of such DNA is the ability of repeat sequences to slip and mispair because of partial self-complementarity in the region. This gives rise to secondary structures in DNA (4Chastain P.D. Sinden R.R. J. Mol. Biol. 1998; 275: 405-411Crossref PubMed Scopus (71) Google Scholar, 5Pearson C.E. Wang Y.H. Griffith J.D. Sinden R.R. Nucleic Acids Res. 1998; 26: 816-823Crossref PubMed Scopus (135) Google Scholar). Although slip mispairing can occur at all repeat sequences, only GAA, CGG, and CTG repeats exhibit expansion in vivo (6Sutherland G.R. Richards R.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3636-3641Crossref PubMed Scopus (301) Google Scholar). These sequences have the additional quality that they can form secondary structures with high stability. Single strands of CTG and CGG repeats have higher melting temperatures than repeats of the other triplets (7Gacy A.M. Goellner G. Juranic N. Macura S. McMurray C.T. Cell. 1995; 81: 533-540Abstract Full Text PDF PubMed Scopus (519) Google Scholar, 8Mitas M. Yu A. Dill J. Kamp T.J. Chambers E.J. Haworth I.S. Nucleic Acids Res. 1995; 23: 1050-1059Crossref PubMed Scopus (134) Google Scholar, 9Mitas M. Yu A. Dill J. Haworth I.S. Biochemistry. 1995; 34: 12803-12811Crossref PubMed Scopus (112) Google Scholar). Furthermore, the single strands of these sequences form more stable hydrogen-bonded fold-back structures than those of the other triplets, as determined by NMR (7Gacy A.M. Goellner G. Juranic N. Macura S. McMurray C.T. Cell. 1995; 81: 533-540Abstract Full Text PDF PubMed Scopus (519) Google Scholar, 10Gacy A.M. McMurray C.T. Biochemistry. 1998; 37: 9426-9434Crossref PubMed Scopus (71) Google Scholar). In Escherichia coli and yeast, CTG and CGG repeats show significantly higher propensity for expansions and deletions than repeat tracts of non-structure-forming sequences, indicating that DNA structure plays a role in instability in vivo (11Ohshima K. Kang S. Wells R.D. J. Biol. Chem. 1996; 271: 1853-1856Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 12Miret J.J. Pessoa-Brandao L. Lahue R.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12438-12443Crossref PubMed Scopus (160) Google Scholar). Slip mispairing can occur when DNA is in a single stranded form during replication, repair, and recombination (13Ohshima K. Wells R.D. J. Biol. Chem. 1997; 272: 16798-16806Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 14Hartenstine M.J. Goodman M.F. Petruska J. J. Biol. Chem. 2000; 275: 18382-18390Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Examination of imperfect repeat sequences in patients showed that repeats were most frequently added on the 3′ end of the segment (15Eichler E.E. Holden J.J. Popovich B.W. Reiss A.L. Snow K. Thibodeau S.N. Richards C.S. Ward P.A. Nelson D.L. Nat. Genet. 1994; 8: 88-94Crossref PubMed Scopus (416) Google Scholar). This polarity in expansion suggested that the mechanism is associated with synthesis of DNA. Moreover, synthesis by DNA polymerase in vitro was found to pause frequently across repeat sequences indicating the presence of secondary structures (13Ohshima K. Wells R.D. J. Biol. Chem. 1997; 272: 16798-16806Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 16Kang S. Ohshima K. Shimizu M. Amirhaeri S. Wells R.D. J. Biol. Chem. 1995; 270: 27014-27021Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). This decreased rate of synthesis was expected to further facilitate structure formation. Additional orientation dependence was observed for repeat instability first in yeast and E. coli and more recently in mammalian cell extracts and cell lines (17Cleary J.D. Nichol K. Wang Y.H. Pearson C.E. Nat. Genet. 2002; 31: 37-46Crossref PubMed Scopus (169) Google Scholar, 18Freudenreich C.H. Stavenhagen J.B. Zakian V.A. Mol. Cell. Biol. 1997; 17: 2090-2098Crossref PubMed Scopus (200) Google Scholar, 19Panigrahi G.B. Cleary J.D. Pearson C.E. J. Biol. Chem. 2002; 277: 13926-13934Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Expansion and deletion of CTG repeats occurred mostly on the lagging strand, implicating lagging strand synthesis as a source of instability (12Miret J.J. Pessoa-Brandao L. Lahue R.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12438-12443Crossref PubMed Scopus (160) Google Scholar, 18Freudenreich C.H. Stavenhagen J.B. Zakian V.A. Mol. Cell. Biol. 1997; 17: 2090-2098Crossref PubMed Scopus (200) Google Scholar, 20Freudenreich C.H. Kantrow S.M. Zakian V.A. Science. 1998; 279: 853-856Crossref PubMed Scopus (363) Google Scholar, 21Kang S. Jaworski A. Ohshima K. Wells R.D. Nat. Genet. 1995; 10: 213-218Crossref PubMed Scopus (317) Google Scholar). CTG sequences tended to expand when present on the Okazaki fragment strand, whereas they underwent deletions when present on the lagging strand template (12Miret J.J. Pessoa-Brandao L. Lahue R.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12438-12443Crossref PubMed Scopus (160) Google Scholar, 18Freudenreich C.H. Stavenhagen J.B. Zakian V.A. Mol. Cell. Biol. 1997; 17: 2090-2098Crossref PubMed Scopus (200) Google Scholar, 22Maurer D.J. O'Callaghan B.L. Livingston D.M. Mol. Cell. Biol. 1996; 16: 6617-6622Crossref PubMed Scopus (108) Google Scholar). This correlates with the particular stability of secondary structure in CTG hairpins, which were found to be more stable than CAG hairpins. Based on the ability of repeat sequences to form stable secondary structure and their propensity to expand when present on the lagging strand, Gordenin et al. (23Gordenin D.A. Kunkel T.A. Resnick M.A. Nat. Genet. 1997; 16: 116-118Crossref PubMed Scopus (189) Google Scholar) proposed a model for repeat sequence expansion. They hypothesized that during lagging strand synthesis, strand displacement within the repeat sequence will result in generation of a flap that could form stable secondary structure. Such secondary structures, they proposed, would be resistant to processing by the structure-specific 5′ flap endonuclease (FEN1) that is involved in resolution of Okazaki fragments (23Gordenin D.A. Kunkel T.A. Resnick M.A. Nat. Genet. 1997; 16: 116-118Crossref PubMed Scopus (189) Google Scholar, 24Bambara R.A. Murante R.S. Henricksen L.A. J. Biol. Chem. 1997; 272: 4647-4650Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Re-annealing of the flap without the removal of repeats within the flap sequence would then result in generation of sequence expansion in the daughter strand. Indeed yeast FEN1 null mutants show an increased propensity to expand repeat sequences (20Freudenreich C.H. Kantrow S.M. Zakian V.A. Science. 1998; 279: 853-856Crossref PubMed Scopus (363) Google Scholar, 25Ireland M.J. Reinke S.S. Livingston D.M. Genetics. 2000; 155: 1657-1665Crossref PubMed Google Scholar). Further, CTG and CGG containing flaps were found to be resistant to FEN1 cleavage in vitro (26Spiro C. Pelletier R. Rolfsmeier M.L. Dixon M.J. Lahue R.S. Gupta G. Park M.S. Chen X. Mariappan S.V. McMurray C.T. Mol. Cell. 1999; 4: 1079-1085Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). We have previously described a model system designed to recapitulate strand displacement within a CAG/CTG repeat region in vitro, to study the cis and trans factors that influence sequence expansion (27Henricksen L. Veeraraghavan J. Chafin D.R. Bambara R.A. J. Biol. Chem. 2002; 277: 22361-22369Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The oligonucleotide model consists of a template strand containing 10 CAG repeats that are flanked by random sequences. To its 3′ end we annealed a complementary downstream primer with CTG repeats at its 5′ end. An upstream primer with a varying number of CTG repeats at its 3′ end was also annealed, such that it strand-displaced varying lengths of downstream primer into a 5′ flap or caused slip mispairing that could result in formation of expansion intermediates (27Henricksen L. Veeraraghavan J. Chafin D.R. Bambara R.A. J. Biol. Chem. 2002; 277: 22361-22369Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). We found that the repeat sequence substrate and DNA ligase I alone were sufficient to allow expansion by ligation of the overlapping strands (27Henricksen L. Veeraraghavan J. Chafin D.R. Bambara R.A. J. Biol. Chem. 2002; 277: 22361-22369Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Addition of FEN1 to this reaction resulted in a decrease in expanded product and increase in correctly sized DNA segment. However, as the number of repeats on the upstream primer was increased such that more repeats were displaced into a 5′ flap on the downstream primer, FEN1 cleavage of these flaps and the subsequent formation of correct sized product were greatly decreased (27Henricksen L. Veeraraghavan J. Chafin D.R. Bambara R.A. J. Biol. Chem. 2002; 277: 22361-22369Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). These results were consistent with the proposed model for sequence expansion resulting from decreased processing of repeat containing flaps. Surprisingly, whereas flap cleavage decreased with increase in primer overlap, ligation of the overlapped segments did not concomitantly increase. On the contrary, as the repeat number on the upstream primer approached that on the downstream primer, ligation efficiency dropped precipitously. This suggested that repeat sequence expansion to lengths of 2-fold or greater may not occur by simple joining of unprocessed flaps that re-anneal to ligatable intermediates. In the current study, we examine why ligation efficiency of triplet repeat sequences decreases with increase in the length of overlap. To establish mechanism, we determined the properties of slipped DNA intermediates that can mediate ligation-based expansion during lagging strand synthesis and processing. We find that very few slipped DNA intermediates are refractory to ligation-based expansion. The factor limiting expansion of certain length overlaps during Okazaki fragment processing is a feature of the DNA itself and not of the transacting factors. We propose a model that is based on the propensity of repeat DNA to form certain types of intermediates and the stability of these intermediates as the basis for sequence expansion. Materials—Oligonucleotides were synthesized either by Integrated DNA Technologies (Coralville, IA) or by Genosys Biotechnologies (Woodlands, TX). Radionucleotides [α-32P]dCTP and [γ-32P]ATP (3000–6000 Ci/mmol) were obtained from PerkinElmer Life Sciences. T4 polynucleotide kinase and the Klenow fragment of E. coli DNA polymerase I (labeling grade) were from Roche Diagnostics. Recombinant N-terminal His-tagged human DNA ligase I was cloned into the pHIS expression vector and was expressed and purified from E. coli as reported previously (27Henricksen L. Veeraraghavan J. Chafin D.R. Bambara R.A. J. Biol. Chem. 2002; 277: 22361-22369Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Yeast FEN1 expression and purification was performed as described previously (28Xie Y. Liu Y. Argueso J.L. Henricksen L.A. Kao H.I. Bambara R.A. Alani E. Mol. Cell. Biol. 2001; 21: 4889-4899Crossref PubMed Scopus (56) Google Scholar). All other reagents were the best available commercial grade. Oligonucleotide Substrates—Repeat sequence model substrates were generated as described previously (27Henricksen L. Veeraraghavan J. Chafin D.R. Bambara R.A. J. Biol. Chem. 2002; 277: 22361-22369Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Briefly, oligonucleotides were designed to mimic the last steps of Okazaki fragment processing. Each substrate consisted of a template strand annealed with a downstream primer at its 5′ end and an upstream primer at its 3′ end. The sequences of oligonucleotides used are listed in Table I. The template and downstream primers contained CAG/CTG repeats, having complete complementarity. Upstream primers containing varying number of repeats, which overlap with the CTG repeats of the downstream primer, are added to complete substrate formation. The (CTG) n substrates each contained n number of CTG repeats at the 3′ ends of the upstream primer. In the final substrate, the upstream and downstream primers contain sequences complimentary to the template and overlap across the repeat region. These overlapping regions should result in a dynamic equilibrium between the primers. This would produce intermediates involving strand displacement of one primer to form flap and loop structures involving one or both primers. A nick substrate that lacked overlapping CTG repeats was used as a control for ligation efficiency.Table IOligonucleotide sequence Fixed CTG bubbles on downstream or upstream primer were configured by annealing a primer with six internal CTG repeats, flanked by non-repeat sequences, to a template complementary to the non-repeat sequences. Such primers were aligned to create a nick with an adjacent primer. This annealing configuration would result in the formation of a six-CTG repeat hairpin at varying distances from the nick. Prior to annealing the downstream primer was radiolabeled at its 3′ end. The downstream primer (10 pmol) was annealed to labeling template T1 (25 pmol) generating a recessed 3′ end and was then extended with [α-32P]dCTP using Klenow polymerase at 37 °C for 3 h. Unincorporated radionucleotides were removed using Micro Bio-spin 30 chromatography columns (Bio-Rad). Upstream primers were labeled at the 5′ end with [γ-32P]ATP in an end labeling reaction with T4 polynucleotide kinase. The labeled primers were isolated and purified on a 10% 7 m urea denaturing polyacrylamide gel. Substrates were generated by annealing the downstream primer, template, and upstream primer at a molar ratio of 1:2:4, respectively. Downstream, template, and upstream primers were diluted into 50 μl of TE (10 mm Tris-Cl, pH 8, and 1 mm EDTA) and heated to 100 °C for 5 min. The reaction was placed at 70 °C and allowed to slowly cool to room temperature. Enzyme Assays—Assays contained indicated amounts of substrate, DNA ligase I, or yeast FEN1 in reaction buffer in a final volume of 20 μl. Reaction buffer contained 30 mm Hepes, pH 8.0 (diluted from a 1 m stock), 40 mm KCl, 4 mm MgCl2, 0.5 mm ATP, 0.01% Nonidet P-40, 0.5% inositol, 0.1 mg/ml bovine serum albumin, and 1 mm dithiothreitol. Assays were assembled on ice and then incubated at 37 °C for 15 min. The reactions were stopped by the addition of 10 μl of termination dye (95% formamide (v/v), 1 mm EDTA with 0.5% bromphenol blue and xylene cyanol and heated at 95 °C for 5 min. Products were separated on an 8% polyacrylamide, 7 m urea denaturing gel and detected by PhosphorImager (Amersham Biosciences). Quantitation was done using ImageQuant v1.2 software from Amersham Biosciences. All assays were performed at least in triplicate. Analysis for Stability of Structural Intermediates—Free energies for various structural intermediates formed by overlapping CTG repeats was calculated using M-fold, a software used to determine structures formed by single stranded DNA. The software calculates the free energy of formation for various structures formed by user specified sequences because of intra-strand base pairing (29Zuker M. Curr. Opin. Struct. Biol. 2000; 10: 303-310Crossref PubMed Scopus (203) Google Scholar). The free energy estimates predict the stability of various intermediates and the abundance in solution of these structures (30SantaLucia Jr., J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1460-1465Crossref PubMed Scopus (2285) Google Scholar). The algorithm allows users to force the formation of specific base pairs and therefore various structures formed by a given sequence. This makes possible the estimation of free energy of various intermediates expected to form by overlapping repeats. The three primers used to form the overlapping repeat substrate were entered into M-fold as one single stranded DNA molecule. The 5′ end of the downstream primer was used as the 5′ end of the single stranded DNA. To its 3′ end, the 5′ end of the template followed by the 5′ end of the upstream primer was linked using a six-nucleotide random sequence designated the N string. The N string allows for the formation of hairpin loops at the junction of upstream and downstream primer to the template sequence. The structures formed by overlap of 10 CTG repeats on the downstream and upstream primers competing to base pair to the complimentary CAG repeat on the template was obtained at 150 mm NaCl and 10 mm MgCl2. 1J. SantaLucia, Jr., M. Zuker, A. Bommarito, and R. J. Irani, unpublished results. Double loop and flap-loop intermediates were formed by forcing specific bases in the sequence to base pair as permitted by the software. The free energy of flap formation was obtained by subtracting the free energy of structure formed by a string of 10 CTG repeats from the free energy of the fold-back flap structure. We set out to test the hypothesis that equal length overlapping repeat segments lead to the formation of intermediates unfavorable for ligation. The intention was to use the results of that analysis to discern the intermediates and mechanisms that favor expansions. To assess expansion efficiency, complementary oligonucleotides were annealed to simulate the template, downstream, and upstream fragments in the configuration formed during Okazaki fragment synthesis and processing (27Henricksen L. Veeraraghavan J. Chafin D.R. Bambara R.A. J. Biol. Chem. 2002; 277: 22361-22369Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The template oligonucleotide included an internal repeat segment of 10 CAG repeats, to which a complementary 3′ downstream primer containing a 10-CTG repeat and 5′ upstream primers with varying numbers of repeats were annealed such that they overlapped within the repeat segment (Fig. 1a). The overlapping configuration of upstream and downstream primer results in transient strand displacement of the downstream primer into a 5′ flap. However, as the substrates contain sequence repeats, they can slip and mispair to generate a nick that can be joined by DNA ligase to expand the daughter strand. Our experimental design does not address the potential effects of template folding. Such folding might be expected to promote repeat sequence contraction. Decreased Expansion Efficiency with Increase in Overlap Length—In the presence of increasing amounts of human DNA ligase I, overlapping repeat segments on the upstream and downstream primers were joined, resulting in expansion of the daughter strand (Fig. 1b). The ligation efficiency increased as the repeat number on the upstream primer was increased to six CTGs (Fig. 1b, lanes 1–24). However, as the repeat length on the upstream primer was increased to seven, there was a sharp decline in the formation of expanded product. Expansion by ligation was completely suppressed as the overlap length was increased from 8 to 10 repeats even at large enzyme excess (Fig. 1b, lanes 29–36). The same decrease in ligation efficiency with length of overlap also occurred when the number of repeats was fixed for the upstream primer and varied on the downstream primer (data not shown). This indicates some form of symmetry in the phenomenon. Both results would appear to be inconsistent with the acceleration of repeat expansion with increasing repeat length observed in vivo (2Usdin K. Grabczyk E. Cell. Mol. Life Sci. 2000; 57: 914-931Crossref PubMed Scopus (86) Google Scholar). Further, this result suggests a size limitation on expansions based on direct ligation of DNA replication intermediates. Overlapping repeat segments of equal length must form double loop intermediates to be joined by DNA ligase I for expansion. For example, in a 10-10 repeat overlap, a 9-1 intermediate will carry a 9-repeat loop on one primer and a 1-repeat loop on the other. Theoretically, many such loop intermediates may be formed. The lack of expansion products with 8-10 or 10-10 overlap (Fig. 1b, lanes 29–36) indicates that either the formation of a suitable double loop intermediate or the position of these loops might interfere with ligation and limit expansion. This issue lead us to examine the substrate structures that influence ligation. Suppressed Ligation Is Independent of Repeat Length—We first determined whether the decrease in ligation efficiency correlated with size of the repeat segment on the template. Templates were designed to contain either 5 or 20 CAG repeats, and each was annealed to a downstream primer containing 5 or 20 CTG repeats, respectively. These substrates were then annealed to upstream primers with 3′ repeats of varying lengths and tested for expansion by DNA ligase (Fig. 2). Surprisingly, the substrates with both 5 and 20 repeat-containing templates showed results similar to those with the 10-CAG substrate. Upstream primers producing small overlaps allowed formation of expanded products on both the 5- and 20-repeat substrate (Fig. 2, a and b, lanes 1–15). However, increasing the overlap length to 5 repeats on the (CAG)5 substrate resulted in complete prevention of expansion, as did increasing the repeat length to 18 in the case of (CAG)20 substrate (Fig. 2, a, lanes 16–20, and b, lanes 21–25). Again this phenomenon was independent of the enzyme concentration. These results verified that expansion does not occur when overlap regions contain about equal numbers of repeats, irrespective of the size of the repeat segment. This suggests that an equal length overlap across a repeat region, when produced during lagging strand DNA replication, would also inhibit ligation. A Mechanism for 2n Expansion—Lack of 2n expansion when repeat segments overlap suggested that the position or size of a loop on either upstream or downstream primer is inhibitory to the activity of DNA Ligase I. To manipulate the size and position of the loop in a systematic manner, we employed primers such that all of the additional repeats for expansion (up to 2n) were contributed by only one primer. In different experiments the extra repeats would be only on either the upstream or the downstream primer. For example, in the substrate used in Fig. 3a, we annealed downstream primers with 10, 15, 18, and 20 CTG repeats to a template with 10 CAG repeats and an upstream primer that has no repeats. The upstream primer is complementary to the random sequence on the 3′ end of the template. We tested repeat loops of various sizes on both up- and downstream primers that were annealed to (CAG)5 and (CAG)10 templates. We observed that on these substrates DNA ligase could join the two primers irrespective of the size and site of the loop relative to the nick (Fig. 3). Interestingly, a 2n expanded product was readily formed, resulting from joining of primers when (CTG)10 and (CTG)20 primers were annealed to a (CAG)5 and (CAG)10 template, respectively (Fig. 3, a, lanes 16–20, and b and c). Apparently, template size-independent 2n expansion can occur in our system when there is slip mispairing on only one of the fragments and may occur by a similar mechanism in vivo. A possible reason for the observed lack of 2-fold expansion in the previous experiments is the overlapping configuration in which the substrates were annealed across the repeat region. The overlapping segments may interact with the template and each other in a way that prevents the formation of stable double loop intermediates that could be substrates for expansion. Comparing the efficiency of ligation of the loops formed on upstream and downstream primers on (CAG)5 and (CAG)10 substrates, there were some subtle but noteworthy differences. We observed lower levels of ligation on the (CAG)5 template (Fig. 3b). Downstream and upstream (CTG)10 repeat-containing primers ligated, but at less than 30% efficiency, whereas the nick substrates were joined at 80% efficiency. Ligation efficiency of upstream and downstream (CTG)20 primers annealed to the (CAG)10 template was about 50%. Slip mispairing can result in formation of either a single large loop comprising the entire excess repeat, anywhere relative to the nick, or multiple smaller loops distributed across the repeat region. The fact that (CTG)20 primers ligated with comparatively high efficiency (about 50%) suggested that a single large loop is formed, as smaller loops would be equally as inhibitory as larger ones, in both smaller and larger repeat segments, if located at the same distance from the adjacent DNA fragment. Further, the lower ligation efficiency on the smaller repeat segment suggests that a relatively large loop, formed closer to the nick on smaller repeat segments, interferes with the ligase or is unstable so as not to form ligatable intermediates. Loop Position and Not Size Modulates DNA Ligase Activity— The substrates used thus far in the study are dynamic in that they allow the formation of multiple loop intermediates. The effect of any one intermediate is indistinguishable from that of the others. To study the effect of a specific intermediate on the activity of DNA ligase, we designed substrates that cont" @default.
• W2001231205 created "2016-06-24" @default.
• W2001231205 creator A5005315226 @default.
• W2001231205 creator A5027656802 @default.
• W2001231205 creator A5070794249 @default.
• W2001231205 date "2003-10-01" @default.
• W2001231205 modified "2023-10-14" @default.
• W2001231205 title "Analysis of DNA Replication Intermediates Suggests Mechanisms of Repeat Sequence Expansion" @default.
• W2001231205 cites W1492929429 @default.
• W2001231205 cites W1501673396 @default.
• W2001231205 cites W1597824427 @default.
• W2001231205 cites W1880539669 @default.
• W2001231205 cites W1901832225 @default.
• W2001231205 cites W1985242213 @default.
• W2001231205 cites W1987125440 @default.
• W2001231205 cites W1988744134 @default.
• W2001231205 cites W1991070590 @default.
• W2001231205 cites W2001087751 @default.
• W2001231205 cites W2001221504 @default.
• W2001231205 cites W2003329456 @default.
• W2001231205 cites W2007368142 @default.
• W2001231205 cites W2008668090 @default.
• W2001231205 cites W2014223000 @default.
• W2001231205 cites W2015324688 @default.
• W2001231205 cites W2017237069 @default.
• W2001231205 cites W2019043328 @default.
• W2001231205 cites W2033229710 @default.
• W2001231205 cites W2036456126 @default.
• W2001231205 cites W2042160009 @default.
• W2001231205 cites W2050346965 @default.
• W2001231205 cites W2052319774 @default.
• W2001231205 cites W2052893592 @default.
• W2001231205 cites W2055491585 @default.
• W2001231205 cites W2057787531 @default.
• W2001231205 cites W2059938757 @default.
• W2001231205 cites W2064642497 @default.
• W2001231205 cites W2069157381 @default.
• W2001231205 cites W2082159174 @default.
• W2001231205 cites W2085617522 @default.
• W2001231205 cites W2093986283 @default.
• W2001231205 cites W2104261681 @default.
• W2001231205 cites W2109234462 @default.
• W2001231205 cites W2113194569 @default.
• W2001231205 cites W2115237139 @default.
• W2001231205 cites W2117896563 @default.
• W2001231205 cites W2123218200 @default.
• W2001231205 cites W2125000841 @default.
• W2001231205 cites W2133167492 @default.
• W2001231205 cites W2135995702 @default.
• W2001231205 cites W2144325802 @default.
• W2001231205 cites W2169710836 @default.
• W2001231205 cites W4237207603 @default.
• W2001231205 doi "https://doi.org/10.1074/jbc.m305137200" @default.
• W2001231205 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12902352" @default.
• W2001231205 hasPublicationYear "2003" @default.
• W2001231205 type Work @default.
• W2001231205 sameAs 2001231205 @default.
• W2001231205 citedByCount "11" @default.
• W2001231205 countsByYear W20012312052012 @default.
• W2001231205 countsByYear W20012312052018 @default.
• W2001231205 crossrefType "journal-article" @default.
• W2001231205 hasAuthorship W2001231205A5005315226 @default.
• W2001231205 hasAuthorship W2001231205A5027656802 @default.
• W2001231205 hasAuthorship W2001231205A5070794249 @default.
• W2001231205 hasBestOaLocation W20012312051 @default.
• W2001231205 hasConcept C12590798 @default.
• W2001231205 hasConcept C159047783 @default.
• W2001231205 hasConcept C2778112365 @default.
• W2001231205 hasConcept C54355233 @default.
• W2001231205 hasConcept C552990157 @default.
• W2001231205 hasConcept C70721500 @default.
• W2001231205 hasConcept C73573662 @default.
• W2001231205 hasConcept C86803240 @default.
• W2001231205 hasConceptScore W2001231205C12590798 @default.
• W2001231205 hasConceptScore W2001231205C159047783 @default.
• W2001231205 hasConceptScore W2001231205C2778112365 @default.
• W2001231205 hasConceptScore W2001231205C54355233 @default.
• W2001231205 hasConceptScore W2001231205C552990157 @default.
• W2001231205 hasConceptScore W2001231205C70721500 @default.
• W2001231205 hasConceptScore W2001231205C73573662 @default.
• W2001231205 hasConceptScore W2001231205C86803240 @default.
• W2001231205 hasIssue "44" @default.
• W2001231205 hasLocation W20012312051 @default.
• W2001231205 hasOpenAccess W2001231205 @default.
• W2001231205 hasPrimaryLocation W20012312051 @default.
• W2001231205 hasRelatedWork W1828691184 @default.
• W2001231205 hasRelatedWork W1982771726 @default.
• W2001231205 hasRelatedWork W1991523530 @default.
• W2001231205 hasRelatedWork W2002128513 @default.
• W2001231205 hasRelatedWork W2005864317 @default.
• W2001231205 hasRelatedWork W2058462727 @default.
• W2001231205 hasRelatedWork W2080053800 @default.
• W2001231205 hasRelatedWork W2088301344 @default.
• W2001231205 hasRelatedWork W4238567963 @default.
• W2001231205 hasRelatedWork W2092874662 @default.
• W2001231205 hasVolume "278" @default.
• W2001231205 isParatext "false" @default.
• W2001231205 isRetracted "false" @default.

• next