Cruciform DNA Structure Underlies the Etiology for Palindrome-mediated Human Chromosomal Translocations

Abstract

There is accumulating evidence to suggest that palindromic AT-rich repeats (PATRRs) represent hot spots of double-strand breakage that lead to recurrent chromosomal translocations in humans. As a mechanism for such rearrangements, we proposed that the PATRR forms a cruciform structure that is the source of genomic instability. To test this hypothesis, we have investigated the tertiary structure of a cloned PATRR. We have observed that a plasmid containing this PATRR undergoes a conformational change, causing temperature-dependent mobility changes upon agarose gel electrophoresis. The mobility shift is observed in physiologic salt concentrations and is most prominent when the plasmid DNA is incubated at room temperature prior to electrophoresis. Analysis using two-dimensional gel electrophoresis indicates that the mobility shift results from the formation of a cruciform structure. S1 nuclease and T7 endonuclease both cut the plasmid into a linear form, also suggesting cruciform formation. Furthermore, anti-cruciform DNA antibody reduces the electrophoretic mobility of the PATRR-containing fragment. Finally, we have directly visualized cruciform extrusions from the plasmid DNA with the size expected of hairpin arms using atomic force microscopy. Our data imply that for human chromosomes, translocation susceptibility is mediated by PATRRs and likely results from their unstable conformation. There is accumulating evidence to suggest that palindromic AT-rich repeats (PATRRs) represent hot spots of double-strand breakage that lead to recurrent chromosomal translocations in humans. As a mechanism for such rearrangements, we proposed that the PATRR forms a cruciform structure that is the source of genomic instability. To test this hypothesis, we have investigated the tertiary structure of a cloned PATRR. We have observed that a plasmid containing this PATRR undergoes a conformational change, causing temperature-dependent mobility changes upon agarose gel electrophoresis. The mobility shift is observed in physiologic salt concentrations and is most prominent when the plasmid DNA is incubated at room temperature prior to electrophoresis. Analysis using two-dimensional gel electrophoresis indicates that the mobility shift results from the formation of a cruciform structure. S1 nuclease and T7 endonuclease both cut the plasmid into a linear form, also suggesting cruciform formation. Furthermore, anti-cruciform DNA antibody reduces the electrophoretic mobility of the PATRR-containing fragment. Finally, we have directly visualized cruciform extrusions from the plasmid DNA with the size expected of hairpin arms using atomic force microscopy. Our data imply that for human chromosomes, translocation susceptibility is mediated by PATRRs and likely results from their unstable conformation. The constitutional t(11;22)(q23;q11) is the only known recurrent non-Robertsonian translocation in humans. Its recurrent nature implicates a specific genomic structure at the t(11;22) breakpoints. Analyses of numerous independent t(11;22) cases have localized the breakpoints within palindromic AT-rich repeats (PATRRs) 1The abbreviations used are: PATRR, palindromic AT-rich repeat; TBE, Tris/borate/EDTA buffer; EMSA, electrophoretic mobility shift assay; AFM, atomic force microscopy. on 11q23 and 22q11 (1Kurahashi H. Shaikh T.H. Hu P. Roe B.A. Emanuel B.S. Budarf M.L. Hum. Mol. Genet. 2000; 9: 1665-1670Crossref PubMed Scopus (124) Google Scholar, 2Kurahashi H. Shaikh T.H. Zackai E.H. Celle L. Driscoll D.A. Budarf M.L. Emanuel B.S. Am. J. Hum. Genet. 2000; 67: 763-768Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 3Edelmann L. Spiteri E. Koren K. Pulijaal V. Bialer M.G. Shanske A. Goldberg R. Morrow B.E. Am. J. Hum. Genet. 2001; 68: 1-13Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 4Tapia-Paez I. Kost-Alimova M. Hu P. Roe B.A. Blennow E. Fedorova L. Imreh S. Dumanski J.P. Hum. Genet. 2001; 109: 167-177Crossref PubMed Scopus (49) Google Scholar). Most 11;22 translocations show breakpoints at the center of the PATRRs, suggesting that the center of the palindrome is susceptible to double-strand breaks, leading to the translocation (5Kurahashi H. Emanuel B.S. Hum. Mol. Genet. 2001; 10: 2605-2617Crossref PubMed Scopus (126) Google Scholar, 6Emanuel B.S. Shaikh T.H. Nat. Rev. Genet. 2001; 2: 791-800Crossref PubMed Scopus (217) Google Scholar). Indeed, translocation-specific PCR detects a high frequency of de novo t(11;22)s in normal sperm samples (7Kurahashi H. Emanuel B.S. Nat. Genet. 2001; 29: 139-140Crossref PubMed Scopus (67) Google Scholar). The breakpoints on 22q11 are located within one of the unclonable gaps in the human genome (8Dunham I. Shimizu N. Roe B.A. Chissoe S. Hunt A.R. Collins J.E. Bruskiewich R. Beare D.M. Clamp M. Smink L.J. Ainscough R. Almeida J.P. Babbage A. Bagguley C. Bailey J. Barlow K. Bates K.N. Beasley O. Bird C.P. Blakey S. Bridgeman A.M. Buck D. Burgess J. Burrill W.D. O'Brien K.P. et al.Nature. 1999; 402: 489-495Crossref PubMed Scopus (921) Google Scholar, 9Shaikh T.H. Kurahashi H. Saitta S.C. O'Hare A.M. Hu P. Roe B.A. Driscoll D.A. McDonald-McGinn D.M. Zackai E.H. Budarf M.L. Emanuel B.S. Hum. Mol. Genet. 2000; 9: 489-501Crossref PubMed Scopus (432) Google Scholar). Extensive screening of YAC/BAC/PAC libraries has not been successful in cloning this breakpoint region. However, experimentally derived sequences from numerous t(11;22) junction fragments demonstrate that the 22q11 breakpoints reside within a larger PATRR. The breakpoints of a variety of translocations involving 22q11 cluster within this region, suggesting that the 22q11 PATRR is highly unstable (10Kehrer-Sawatzki H. Haussler J. Krone W. Bode H. Jenne D.E. Mehnert K.U. Tummers U. Assum G. Hum. Genet. 1997; 99: 237-247Crossref PubMed Scopus (77) Google Scholar, 11Rhodes C.H. Call K.M. Budarf M.L. Barnoski B.L. Bell C.J. Emanuel B.S. Bigner S.H. Park J.P. Mohandas T.K. Cytogenet. Cell Genet. 1997; 78: 247-252Crossref PubMed Scopus (27) Google Scholar, 12Debeer P. Mols R. Huysmans C. Devriendt K. Van de Ven W.J. Fryns J.P. Clin. Genet. 2002; 62: 410-414Crossref PubMed Scopus (21) Google Scholar, 13Spiteri E. Babcock M. Kashork C.D. Wakui K. Gogineni S. Lewis D.A. Williams K.M. Minoshima S. Sasaki T. Shimizu N. Potocki L. Pulijaal V. Shanske A. Shaffer L.G. Morrow B.E. Hum. Mol. Genet. 2003; 12: 1823-1837Crossref PubMed Scopus (46) Google Scholar). More recently, molecular cloning of translocation breakpoints has demonstrated similar palindromic sequences on partner chromosomes, such as 17q11, 4q35.1, and 1p21.2 (14Kurahashi H. Shaikh T. Takata M. Toda T. Emanuel B.S. Am. J. Hum. Genet. 2003; 72: 733-738Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 15Nimmakayalu M.A. Gotter A.L. Shaikh T.H. Emanuel B.S. Hum. Mol. Genet. 2003; 12: 2817-2825Crossref PubMed Scopus (49) Google Scholar, 16Gotter A.L. Shaikh T.H. Chieffo C. Budarf M.L. Rhodes C.H. Emanuel B.S. Hum. Mol. Genet. 2003; 13: 103-115Crossref PubMed Scopus (80) Google Scholar). It has been suggested that for palindromic sequences in double-stranded DNA, the interstrand base pairs might convert to intrastrand pairs, producing a set of hairpin structures described as a cruciform (see Fig. 1A). In appropriate conditions, these structures can be generated from palindromic DNA in vitro and visualized directly by electron microscopy (17Mizuuchi K. Mizuuchi M. Gellert M. J. Mol. Biol. 1982; 156: 229-243Crossref PubMed Scopus (178) Google Scholar). Under physiological conditions, cruciform extrusion is usually kinetically blocked; it appears to take place only in response to heating, suggesting that a considerable amount of energy is required for cruciform extrusion (18Courey A.J. Wang J.C. Cell. 1983; 33: 817-829Abstract Full Text PDF PubMed Scopus (159) Google Scholar). However, under appropriate conditions, palindromic DNA prefers to extrude cruciform arms even at physiological temperatures (19Panyutin I. Klishko V. Lyamichev V. J. Biomol. Struct. Dyn. 1984; 1: 1311-1324Crossref PubMed Scopus (34) Google Scholar). In a previous report, we demonstrated that the PATRRs on 11q23, 17q11, and 22q11 are all comprised of a long AT-rich region with relatively GC-rich ends (5Kurahashi H. Emanuel B.S. Hum. Mol. Genet. 2001; 10: 2605-2617Crossref PubMed Scopus (126) Google Scholar, 14Kurahashi H. Shaikh T. Takata M. Toda T. Emanuel B.S. Am. J. Hum. Genet. 2003; 72: 733-738Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). We have proposed that the AT richness of the PATRRs contributes to strand separation at physiological temperatures, whereas the relatively GC-rich ends contribute to the stable intrastrand complementary interaction of the PATRR. These characteristics are likely to induce or favor the formation of a cruciform structure. In this study, we have investigated the in vitro tertiary structure of the 11q23 PATRR and demonstrated that it forms a cruciform structure at physiological conditions. Our data suggest that PATRRs may form unstable structures that lead to chromosomal translocations in humans. These data also implicate a biological role for the temperature-sensitive conformational change characteristic of palindromic DNA. PATRR Plasmid—A plasmid containing the chromosome 11 PATRR (204 bp) was constructed by PCR and TA cloning as described previously (psPATRR11) (1Kurahashi H. Shaikh T.H. Hu P. Roe B.A. Emanuel B.S. Budarf M.L. Hum. Mol. Genet. 2000; 9: 1665-1670Crossref PubMed Scopus (124) Google Scholar). The control plasmid that deletes the PATRR region was constructed using BAC 442e11, which deletes almost the entire PATRR region (pΔPATRR11). Plasmid DNA was isolated by means of alkaline lysis (denaturing) or Triton lysis (non-denaturing) methods and purified using ion-exchange columns (Qiagen) without the use of phenol. Isopropanol-precipitated DNA was dissolved in phosphate-buffered saline/1 mm EDTA. All of the procedures were performed at 4 °C in a cold room to avoid artifactual cruciform extrusion during the procedure. Isolated plasmid DNA was divided into small aliquots and immediately frozen until used in an experiment. The plasmid was quickly thawed, incubated at a given temperature, and then cooled on ice before electrophoresis. DNA topoisomers containing different linking numbers were prepared by relaxation of the DNA with topoisomerase I (Invitrogen) in the presence of varied concentrations of ethidium bromide as described (20Peck L.J. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 6206-6210Crossref PubMed Scopus (269) Google Scholar). Several samples with different ranges of linking numbers were pooled to produce a mixture with a broader distribution of topoisomers. Two-dimensional Gel Electrophoresis—Standard agarose gel electrophoresis was performed in horizontal gel slabs immersed in 1× Tris/borate/EDTA (TBE) electrophoresis buffer. The 0.7% agarose gel was prepared in TBE in a square gel tray. Electrophoresis was performed at 45 V for 20 h at 4 °C. The gel was stained in water plus ethidium bromide at 1 μg/ml for 2 h followed by extensive destaining in several changes of water before photography. For two-dimensional gel electrophoresis, the sample was applied at the left upper corner. Electrophoresis in the first dimension was performed using the same conditions as for standard electrophoresis. The gel was then soaked in TBE plus chloroquine (0.75 μg/ml) (Sigma) for 8 h with gentle agitation for equilibration. After soaking, the gel was rotated clockwise 90° from the original position. Electrophoresis in the second dimension was performed in TBE plus chloroquine at 45 V for 20 h. The gel was then soaked in several changes of deionized water to remove the chloroquine. The gel was stained in water plus ethidium bromide in similar conditions as for standard agarose gel electrophoresis. To stabilize the cruciform structure, two-dimensional gel electrophoresis was also performed in Mg2+-containing buffer (0.5× TBE, 10 mm MgCl2) for the first dimension (21Dai X. Greizerstein M.B. Nadas-Chinni K. Rothman-Denes L.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2174-2179Crossref PubMed Scopus (82) Google Scholar). Electrophoresis was carried out at 45 V for 30 h at 4 °C, replacing the electrophoresis buffer every 6 h (22Vologodskaia M.Y. Vologodskii A.V. J. Mol. Biol. 1999; 289: 851-859Crossref PubMed Scopus (18) Google Scholar). endonuclease S1 nuclease and nuclease were performed at room temperature for 1 h in the appropriate 10 of T7 1 of S1 and of nuclease were used for of 1 of After the plasmid DNA was with and then to 0.7% agarose gel electrophoresis. For of the the were with or and then to agarose gel electrophoresis. The fragments were purified and using was performed as described previously H. PubMed Scopus (27) Google using a antibody cruciform DNA, L. M. J. Mol. Biol. PubMed Scopus Google Scholar). To the DNA and were with and and into of plasmid DNA was incubated at 4 °C for of buffer mm mm 1 mm and mm MgCl2) with or without second plasmid was into the for After the of 2 of the plasmid was for 30 at room temperature with of both and 1 of and 10 of were to the and for 10 The fragments were on at 4 °C at were and for an appropriate plasmid DNA sample was a with with 1 of and with was carried out using a in the at room temperature in of in with a of were from of these were The were at a of The of the DNA were using To the PATRR plasmid into the cruciform structure, DNA was performed by a similar to that described previously Biol. 1983; PubMed Google Scholar). In plasmid DNA was dissolved in a of μg/ml) and to at for The DNA was with and then to PATRR Plasmid to in Its have that the PATRR forms a cruciform structure even in physiological conditions, leading to genomic in humans. To test this hypothesis, we have the tertiary structure of a cloned 11q23 PATRR. PATRR is unstable to be cloned into a plasmid in The 11q23 BAC the t(11;22) breakpoint region deletes the PATRR during that this region also the PATRR, suggesting that it is unstable even when is used as the we a of the PATRR that can be as a plasmid in (5Kurahashi H. Emanuel B.S. Hum. Mol. Genet. 2001; 10: 2605-2617Crossref PubMed Scopus (126) Google Scholar). We prepared the PATRR-containing plasmid as as a plasmid that deletes the PATRR in a buffer at physiological salt and used for the we the prepared by the standard alkaline lysis standard agarose gel electrophoresis, a plasmid usually which to and DNA. In to the expected the a at the for DNA. The not such a of the to a containing different linking topoisomers in the The observed that the PATRR-containing the plasmid without the PATRR, prefers to be in in formation of the complementary and reduces the interstrand For DNA, a in reduces the of and a in electrophoretic The observed is with cruciform formation of the PATRR DNA is known to be by S1 nuclease or T7 endonuclease D.E. 13: PubMed Scopus Google Scholar). To cruciform extrusion of the and the of conformational we the nuclease of the PATRR plasmid prepared by the alkaline lysis S1 which is known to cut DNA at the of the cruciform structure, most of the into a linear form only a small amount of the or TA cloning 6 and the for the S1 nuclease buffer is it may the plasmid and the we also incubated the plasmid with which in not which can at physiological similar results not To the that S1 nuclease DNA of the only of the AT-rich nature of the we used T7 which can cut the junction of the cruciform structure. incubated at room temperature, a considerable amount of the was by T7 endonuclease into a linear form In the endonuclease not cut or the TA cloning and Two-dimensional Gel cruciform extrusion of the we performed two-dimensional gel electrophoresis. The first in the of the topoisomers on the of in the to are in the presence of a second dimension in the presence of this the topoisomers on the of linking of prepared by the standard alkaline lysis on the of an we with topoisomerase I in the presence of of ethidium bromide as to with broader After topoisomerase I an the original to topoisomers generated by topoisomerase I In the first the electrophoretic of topoisomers are in with the same topoisomers without the whereas these forms in the second is the of the of cruciform structures in the presence of which to a of is known to cruciform extrusion (17Mizuuchi K. Mizuuchi M. Gellert M. J. Mol. Biol. 1982; 156: 229-243Crossref PubMed Scopus (178) Google Scholar, A.J. Wang J.C. Cell. 1983; 33: 817-829Abstract Full Text PDF PubMed Scopus (159) Google the on this gel are likely to from cruciform DNA. We topoisomerase with S1 nuclease or T7 endonuclease prior to the two-dimensional gel electrophoresis. As the as a of nuclease whereas the and spots of and linear DNA These results demonstrate that from cruciform DNA and with the data derived from the of when isolated by the alkaline lysis forms a cruciform is known to stabilize a cruciform structure (21Dai X. Greizerstein M.B. Nadas-Chinni K. Rothman-Denes L.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2174-2179Crossref PubMed Scopus (82) Google Scholar, M.Y. Vologodskii A.V. J. Mol. Biol. 1999; 289: 851-859Crossref PubMed Scopus (18) Google Scholar). We the two-dimensional gel electrophoresis in Mg2+-containing buffer for the first The results were similar to for standard TBE buffer not of in the presence of on the two-dimensional gel indicates that the of cruciform To the of the PATRR for cruciform we nuclease using and T7 endonuclease and S1 nuclease several fragments that were that of the PATRR-containing whereas the of the original was by with the The of cruciform extrusion of the PATRR region of the To the we performed of fragment. endonuclease fragments were and we localized the at we that forms and small cruciform extrusions as was from the two-dimensional gel The small cruciform extrusion from the region at the center of the PATRR. the PATRR has a relatively GC-rich region at both ends (1Kurahashi H. Shaikh T.H. Hu P. Roe B.A. Emanuel B.S. Budarf M.L. Hum. Mol. Genet. 2000; 9: 1665-1670Crossref PubMed Scopus (124) Google Scholar, H. Emanuel B.S. Hum. Mol. Genet. 2001; 10: 2605-2617Crossref PubMed Scopus (126) Google Scholar). the cruciform from the region that this GC-rich region. The of S1 nuclease were of the with the center of the PATRR, the of the cruciform structure The of the of S1 nuclease were at the the and arms of the PATRR. These results suggest intrastrand within the PATRR, which is also with cruciform formation. of the the PATRR plasmid cruciform in E. we have prepared the plasmid by a Triton lysis of the PATRR can induce artifactual cruciform formation. isolated by the alkaline lysis not a characteristic in standard agarose gel electrophoresis that the not form a cruciform in E. we incubated the at temperatures for 1 h prior to agarose gel electrophoresis. of the plasmid was observed as a characteristic in samples incubated at temperatures and °C shift be the of a from to a cruciform conformation. mobility was most prominent in the plasmid incubated at room The as as 1 the of at room temperature, and the of with of the of the sample 1 h of was similar to that prepared by the alkaline lysis at room temperature for 1 h to be for a of the plasmid to form the cruciform The sample incubated for 1 h at room temperature was to two-dimensional gel producing similar results to that prepared by the alkaline lysis not performed using the anti-cruciform antibody which cruciform DNA L. M. J. Mol. Biol. PubMed Scopus Google Scholar). To the size of the PATRR we fragments into a plasmid The of the from and are and Plasmid DNA samples prepared by the Triton lysis were used for of the the of the antibody not in a shift for the from and or from not However, when the were incubated at room temperature for prior to the to the cruciform from demonstrated a shift in an antibody The from not show a shift even at room temperature for of was not observed with control and this shift indicates that the antibody to the PATRR plasmid incubated at room used atomic force microscopy to the tertiary structure of the PATRR plasmid and to directly the cruciform extrusion J. Mol. Biol. PubMed Scopus Google Scholar). We prepared by the Triton lysis cruciform extrusion However, of the DNA samples at room temperature for of 45 cruciform extrusions and The of extrusions was the size of the PATRR is the size for the cruciform is the GC-rich region at both ends of the PATRR was the size of the cruciform is which to the observed of extrusions are with or with of the cruciform However, all of the observed with a cruciform of The small the size of which was to be from size was small to be by To the cruciform extrusion of the within the PATRR region, we the the PATRR from the plasmid was with and prior to extrusion was observed at the center of the not in the that the PATRR a cruciform In this study, we have demonstrated that the 11q23 PATRR located at the t(11;22) breakpoint region can form a cruciform structure in Most a PATRR with an AT of The the and arms is a palindromic structure (5Kurahashi H. Emanuel B.S. Hum. Mol. Genet. 2001; 10: 2605-2617Crossref PubMed Scopus (126) Google Scholar). we a of the PATRR a of The size of the PATRR is with an AT of The the and arms is with a it is to DNA containing the the plasmid we used for this of the 11q23 PATRR. Our results demonstrate that the 11q23 PATRR forms a cruciform at physiological conditions. is evidence that the PATRR is in of the one t(11;22) appears to have from a PATRR (5Kurahashi H. Emanuel B.S. Hum. Mol. Genet. 2001; 10: 2605-2617Crossref PubMed Scopus (126) Google Scholar). it likely that the PATRR to the long PATRR. It is to that the long PATRR, as as PATRRs in chromosomal may also form cruciform structures that induce genomic instability. It has been suggested that palindromic DNA of unstable cruciform structures both and leading to The data this proposed the mechanism of double-strand it is that the cruciform structure of the ends that be PubMed Scopus Google Scholar, D.A. M.A. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). a endonuclease may at the center of the PATRR. However, to this has been structures that a junction which may be a for junction a might be by for are at the center of the PATRR, and is the sequences of the translocation A.L. Shaikh T.H. Chieffo C. Budarf M.L. Rhodes C.H. Emanuel B.S. Hum. Mol. Genet. 2003; 13: 103-115Crossref PubMed Scopus (80) Google Scholar). In this report, we have demonstrated that cruciform extrusion is cruciform formation is most at room temperature, with previous by et (19Panyutin I. Klishko V. Lyamichev V. J. Biomol. Struct. Dyn. 1984; 1: 1311-1324Crossref PubMed Scopus (34) Google Scholar). We have observed strand separation of the PATRR even at °C. S. Emanuel and H. The in cruciform formation might be to the for energy place cruciform and within the We previously that only this translocation (7Kurahashi H. Emanuel B.S. Nat. Genet. 2001; 29: 139-140Crossref PubMed Scopus (67) Google Scholar). the that translocations may represent in and that the double-strand in might a specific DNA conformation. might be to such temperature-sensitive cruciform and we have previously de novo t(11;22)s in a similar not to We have only one of a de novo and it an in the S. Emanuel and H. it is not to that translocations might only in generated at a In cruciform extrusion is has directly demonstrated in cruciform structures in palindromic DNA. previous cruciform DNA in E. have the (18Courey A.J. Wang J.C. Cell. 1983; 33: 817-829Abstract Full Text PDF PubMed Scopus (159) Google Scholar, D.E. 13: PubMed Scopus Google Scholar, V. I. S. J. Biomol. Struct. Dyn. 1984; 2: PubMed Scopus (21) Google Scholar). on the that PATRR prepared by the not demonstrate cruciform the PATRR cruciform is likely to be in E. extrusion is known to The of a plasmid in E. may not it the to a cruciform the of a plasmid may be for a to a cruciform to take this may be the the of cruciform is in In the is long to palindromic DNA to cruciform extrusion a cruciform is to be results that the PATRR prefers to a cruciform even at physiological conditions. have demonstrated that induce of DNA, leading to cruciform extrusion of palindromic DNA K. A. M. R. D.M. T. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). that palindromic DNA may a cruciform within the The conformational of genomic DNA the of DNA or H. PubMed Scopus (27) Google Scholar, O. D. M. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). the of an in cruciform structure may lead to a of the of such The M. and G. for anti-cruciform DNA were by R. and of the of