DNA Recombination

Abstract

Holliday junctions are critical intermediates for homologous, site-specific recombination, DNA repair, and replication. A wealth of structural information is available for immobile four-way junctions, but the controversy on the mechanism of branch migration of Holliday junctions remains unsolved. Two models for the mechanism of branch migration were suggested. According to the early model of Alberts-Meselson-Sigal (Sigal, N., and Alberts, B. (1972) J. Mol. Biol. 71, 789–793 and Meselson, M. (1972) J. Mol. Biol. 71, 795–798), exchanging DNA strands around the junction remain parallel during branch migration. Kinetic studies of branch migration (Panyutin, I. G., and Hsieh, P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2021–2025) suggest an alternative model in which the junction adopts an extended conformation. We tested these models using a Holliday junction undergoing branch migration and time-lapse atomic force microscopy, an imaging technique capable of imaging DNA dynamics. The single molecule atomic force microscopy experiments performed in the presence and in the absence of divalent cations show that mobile Holliday junctions adopt an unfolded conformation during branch migration that is retained despite a broad range of motion in the arms of the junction. This conformation of the junction remains unchanged until strand separation. The data obtained support the model for branch migration having the extended conformation of the Holliday junction. Holliday junctions are critical intermediates for homologous, site-specific recombination, DNA repair, and replication. A wealth of structural information is available for immobile four-way junctions, but the controversy on the mechanism of branch migration of Holliday junctions remains unsolved. Two models for the mechanism of branch migration were suggested. According to the early model of Alberts-Meselson-Sigal (Sigal, N., and Alberts, B. (1972) J. Mol. Biol. 71, 789–793 and Meselson, M. (1972) J. Mol. Biol. 71, 795–798), exchanging DNA strands around the junction remain parallel during branch migration. Kinetic studies of branch migration (Panyutin, I. G., and Hsieh, P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2021–2025) suggest an alternative model in which the junction adopts an extended conformation. We tested these models using a Holliday junction undergoing branch migration and time-lapse atomic force microscopy, an imaging technique capable of imaging DNA dynamics. The single molecule atomic force microscopy experiments performed in the presence and in the absence of divalent cations show that mobile Holliday junctions adopt an unfolded conformation during branch migration that is retained despite a broad range of motion in the arms of the junction. This conformation of the junction remains unchanged until strand separation. The data obtained support the model for branch migration having the extended conformation of the Holliday junction. The Holliday junction (HJ) 1The abbreviations used are: HJ, Holliday junction; AFM, atomic force microscopy; APS-mica, mica functionalized with aminopropyl silatrane. suggested in 1964 by Robin Holliday (6Holliday R. Genet. Res. Camb. 1964; 5: 282-304Crossref Scopus (1267) Google Scholar) is a central intermediate in homologous and site-specific recombination (7Leach D.R.F. Genetic Recombination. Blackwell Science, Oxford1996Google Scholar). This type of DNA structure is also involved in double-stranded break repair (7Leach D.R.F. Genetic Recombination. Blackwell Science, Oxford1996Google Scholar, 8Cromie G.A. Connelly J.C. Leach D.R. Mol. Cell. 2001; 8: 1163-1174Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Recent data show that HJs are critical intermediates in replication fork stalling leading to subsequent correction of the corrupting template lesion (9McGlynn P. Lloyd R.G. Marians K.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8235-8240Crossref PubMed Scopus (141) Google Scholar, 10Postow L. Ullsperger C. Keller R.W. Bustamante C. Vologodskii A.V. Cozzarelli N.R. J. Biol. Chem. 2001; 276: 2790-2796Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 11Grompone G. Seigneur M. Ehrlich S.D. Michel B. Mol. Microbiol. 2002; 44: 1331-1339Crossref PubMed Scopus (58) Google Scholar). Movement of the crossover along DNA allowing for length extension of the heteroduplex is termed branch migration. If this process is not terminated by resolvases, branch migration leads to complete strand separation as shown in Fig. 1A. Branch migration, whether spontaneous or mediated by proteins, is a key step in various genetic processes involving the Holliday junction. Various models for the Holliday junction intermediate have been utilized to unravel the structural basis for branch migration and resolution of the junction. The immobile four-way junction was a primary model system for the Holliday junction, and a great deal of information on the structure of HJs was obtained from their study. Numerous techniques (12Churchill M.E. Tullius T.D. Kallenbach N.R. Seeman N.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4653-4656Crossref PubMed Scopus (191) Google Scholar, 13Lilley D.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9513-9515Crossref PubMed Scopus (30) Google Scholar, 14Lilley D.M. Norman D.G. Nat. Struct. Biol. 1999; 6: 897-899Crossref PubMed Scopus (23) Google Scholar, 15Sha R. Liu F. Seeman N.C. Biochemistry. 2002; 41: 5950-5955Crossref PubMed Scopus (37) Google Scholar, 16Sha R. Liu F. Seeman N.C. Biochemistry. 2000; 39: 11514-11522Crossref PubMed Scopus (18) Google Scholar, 17Fogg J.M. Schofield M.J. Declais A.C. Lilley D.M. Biochemistry. 2000; 39: 4082-4089Crossref PubMed Scopus (39) Google Scholar), including very recent x-ray crystallography analysis (18Ho P.S. Eichman B.F. Curr. Opin. Struct. Biol. 2001; 11: 302-308Crossref PubMed Scopus (46) Google Scholar, 19Eichman B.F. Ortiz-Lombardia M. Aymami J. Coll M. Ho P.S. J. Mol. Biol. 2002; 320: 1037-1051Crossref PubMed Scopus (40) Google Scholar), show that in the presence of multivalent cations, the junction adopts an antiparallel orientation in which the four helices stack in pairs to form two double-helical domains. Immobile HJs can adopt two conformational states undergoing transition between them via extended conformation as a transient state (20McKinney S.A. Declais A.C. Lilley D.M. Ha T. Nat. Struct. Biol. 2003; 10: 93-97Crossref PubMed Scopus (274) Google Scholar). However, these findings are in contrast to data obtained for HJs formed by inverted repeats (2-fold sequence symmetry), which allow branch migration to occur. Atomic force microscopy (AFM) imaging of a supercoil-stabilized cruciform (21Shlyakhtenko L.S. Potaman V.N. Sinden R.R. Lyubchenko Y.L. J. Mol. Biol. 1998; 280: 61-72Crossref PubMed Scopus (120) Google Scholar) shows that the cruciform adopts various conformations and that parallel orientation of the arms is the predominant HJ structure in the presence of Mg cations (22Shlyakhtenko L.S. Hsieh P. Grigoriev M. Potaman V.N. Sinden R.R. Lyubchenko Y.L. J. Mol. Biol. 2000; 296: 1169-1173Crossref PubMed Scopus (73) Google Scholar). This finding is in line with results on the structure of the 2-fold symmetry intermediate for the Flp recombination reaction even after the removal of the protein (23Huffman K.E. Levene S.D. J. Mol. Biol. 1999; 286: 1-13Crossref PubMed Scopus (17) Google Scholar). It has been hypothesized that DNA supercoiling and the mobility of the junction are important factors contributing to HJ conformation (21Shlyakhtenko L.S. Potaman V.N. Sinden R.R. Lyubchenko Y.L. J. Mol. Biol. 1998; 280: 61-72Crossref PubMed Scopus (120) Google Scholar, 22Shlyakhtenko L.S. Hsieh P. Grigoriev M. Potaman V.N. Sinden R.R. Lyubchenko Y.L. J. Mol. Biol. 2000; 296: 1169-1173Crossref PubMed Scopus (73) Google Scholar). Little is known as to how the structural features of the HJ relate to the mechanism of branch migration. A textbook model suggested 30 years ago (1Sigal N. Alberts B. J. Mol. Biol. 1972; 71: 789-793Crossref PubMed Scopus (167) Google Scholar) utilizes the parallel orientation of exchanging strands (Path I in Fig. 1A). In this model, hybrid DNA molecules are formed by rotatory diffusion of the arms (2Meselson M. J. Mol. Biol. 1972; 71: 795-798Crossref PubMed Scopus (59) Google Scholar). This view has been challenged with an alternative model suggesting that an extended configuration of the junction (Path II in Fig. 1A) is more appropriate for spontaneous movement of the HJ (3Panyutin I.G. Hsieh P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2021-2025Crossref PubMed Scopus (233) Google Scholar, 4Panyutin I.G. Biswas I. Hsieh P. EMBO J. 1995; 14: 1819-1826Crossref PubMed Scopus (84) Google Scholar, 5Biswas I. Yamamoto A. Hsieh P. J. Mol. Biol. 1998; 279: 795-806Crossref PubMed Scopus (39) Google Scholar). However, there is no direct experimental evidence for either of these models. In this study, we have used single molecule AFM (21Shlyakhtenko L.S. Potaman V.N. Sinden R.R. Lyubchenko Y.L. J. Mol. Biol. 1998; 280: 61-72Crossref PubMed Scopus (120) Google Scholar, 24Lyubchenko Y.L. Shlyakhtenko L.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 496-501Crossref PubMed Scopus (351) Google Scholar, 25Lyubchenko Y.L. Shlyakhtenko L.S. Potaman V.N. Sinden R.R. Microsc. Microanal. 2002; 8: 170-171Crossref PubMed Scopus (13) Google Scholar, 26Shlyakhtenko L.S. Potaman V.N. Sinden R.R. Gall A.A. Lyubchenko Y.L. Nucleic Acids Res. 2000; 28: 3472-3477Crossref PubMed Scopus (78) Google Scholar) and a mobile HJ capable of spontaneous branch migration to test the models and to observe the conformations and dynamics of this biologically relevant model. We show that the mobile junction adopts an extended conformation during branch migration supporting the Path II model for branch migration (Fig. 1A). The Design of a Symmetric (Mobile) Holliday Junction—Four synthetic oligonucleotides, 1) 5′-AGCTTGCATGCATCGATAT AATACGTGAGGCCTAGGATC-3′; 2) 5′-ACCATGCTCGAGATTACGAG ATATCGATGCATGCA-3′; 3) 5′-AGCTTGCATGCATCGATAT CTCGTAATCTCGAGCATGGT-3′; and 4) 5′-GATCCTAGGCCTCACGTATT ATATCGATGCATGCA-3′, were pair-wise (1 + 2 and 3 + 4) annealed to form the hemi-junctions. 235 and 300 bp linear DNA duplexes were obtained by HindIII (New England Biolabs) digestion of a 535-bp PCR fragment, which was generated using the primers for positions 97–116 and 614– 632 of plasmid pUC18. The hemi-junction synthetic duplexes (1 + 2 and 3 + 4) were ligated with the 300-bp Hind III fragments using the procedure described earlier (24Lyubchenko Y.L. Shlyakhtenko L.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 496-501Crossref PubMed Scopus (351) Google Scholar) to form hemi-junctions L and R (Fig. 1B). The hemi-junctions were separated in 6% polyacrylamide and purified using the QIAquick DNA purification kit (Qiagen Inc.). Equimolar amounts of hemi-junctions L and R (30–50 ng) were mixed in 5 μl of TNM buffer (10 mm Tris-HCl, pH 7.9, 50 mm NaCl, 10 mm MgCl2). The mixture was incubated for 4 min at 50 °C. The reaction was stopped by adding 5 μl of ice-cold TNM buffer containing 1 μg/ml ethidium bromide. Electrophoresis in 1.2% agarose was performed at 4 °C in TAE buffer (40 mm Tris-acetate, 1 mm EDTA) containing 10 mm MgCl2. The slow moving HJ band was excised from the gel and purified by filtration on an UltraFree UFC3 0HV column (Millipore) at 4 °C. The filtrate was immediately used for deposition on pretreated mica. AFM Procedures—Mica functionalized with aminopropyl silatrane (APS-mica) was used as an AFM substrate (26Shlyakhtenko L.S. Potaman V.N. Sinden R.R. Gall A.A. Lyubchenko Y.L. Nucleic Acids Res. 2000; 28: 3472-3477Crossref PubMed Scopus (78) Google Scholar). Briefly, DNA samples (3–5 μl) were placed onto APS-mica for 2 mins, and the mica was rinsed with deionized water (Continental Water System Co., San Antonio, TX) and dried with argon. Images were acquired in air using a MultiMode SPM NanoScope III system (Veeco/Digital Instruments, Santa Barbara, CA) operating in tapping mode using OTESPA probes (Digital Instruments, Inc.). The length, height, and angle measurements were performed using Femtoscan software (Advanced Technologies Center, Moscow, Russia). For imaging in aqueous solutions, the sample was diluted in TNM buffer right before application to APS-mica to make the final DNA concentration 0.8 ng/μl. The sample was placed onto APS-mica mounted on the scanning stage of the MultiMode SPM NanoScope III system. The optical head with mounted liquid cell was placed on the sample, and the tip was brought into contact with the surface. TNM buffer was added to fill up the space between the cell and mica surface as needed. Images were acquired in tapping mode using standard silicon nitride probes (Veeco/Digital Instruments). Buffer was changed by injection of TE buffer (10 mm Tris-HCl, pH 7.9, and 5 mm EDTA) with 1 ml plastic syringes attached to the inlet and outlet holes of the flow cell. The schematic illustrating the procedure for preparation of the mobile HJ is shown in Fig. 1B. The HJ was obtained by hybridization of left and right hemi-junctions having homologous duplex regions via annealing of the single-stranded ends (3Panyutin I.G. Hsieh P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2021-2025Crossref PubMed Scopus (233) Google Scholar, 4Panyutin I.G. Biswas I. Hsieh P. EMBO J. 1995; 14: 1819-1826Crossref PubMed Scopus (84) Google Scholar). The annealing was performed in TNM buffer to slow branch migration (3Panyutin I.G. Hsieh P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2021-2025Crossref PubMed Scopus (233) Google Scholar, 4Panyutin I.G. Biswas I. Hsieh P. EMBO J. 1995; 14: 1819-1826Crossref PubMed Scopus (84) Google Scholar). A sequence of the of the junction illustrating the migration is shown in Fig. A complete sequence for synthetic the mobile HJ is in the of up branch migration, which leads to the of two DNA duplexes as shown at the of Fig. 1A. analysis that more of the HJs were into linear duplexes after a in TE buffer at 50 °C not The HJs were separated from hemi-junctions by agarose gel and from the cations in a 10 mm concentration were at of the sample purification to the of branch migration. AFM data for the sample onto APS-mica and are shown in Fig. of the molecules in Fig. 2 are four-way DNA The of the arms for the molecules This is the for HJs undergoing branch migration as by the model (3Panyutin I.G. Hsieh P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2021-2025Crossref PubMed Scopus (233) Google Scholar, 5Biswas I. Yamamoto A. Hsieh P. J. Mol. Biol. 1998; 279: 795-806Crossref PubMed Scopus (39) Google Scholar). The conformations of the HJs were in the The HJ conformations in which the arms form an strand and with the arms on of the are to as in the conformation 1 in Fig. The extended in which the arms are to a is a of the conformation. The conformations of the HJs having arms to are to as in the conformation 2 in Fig. The parallel having an angle of between homologous arms is a of the conformation. The antiparallel conformation is by between a and the and can or conformations N.C. Kallenbach N.R. Struct. 1994; PubMed Scopus Google Scholar). at the dynamics of mobile HJs and to test the models of branch migration, we the of AFM to of the In this time-lapse imaging AFM scanning a allowing the of dynamics at the single molecule (21Shlyakhtenko L.S. Potaman V.N. Sinden R.R. Lyubchenko Y.L. J. Mol. Biol. 1998; 280: 61-72Crossref PubMed Scopus (120) Google Scholar, 24Lyubchenko Y.L. Shlyakhtenko L.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 496-501Crossref PubMed Scopus (351) Google Scholar, 26Shlyakhtenko L.S. Potaman V.N. Sinden R.R. Gall A.A. Lyubchenko Y.L. Nucleic Acids Res. 2000; 28: 3472-3477Crossref PubMed Scopus (78) Google Scholar). In imaging in aqueous sample preparation by of the This AFM imaging mode has been very for the single molecule of the dynamics of DNA (26Shlyakhtenko L.S. Potaman V.N. Sinden R.R. Gall A.A. Lyubchenko Y.L. Nucleic Acids Res. 2000; 28: 3472-3477Crossref PubMed Scopus (78) Google Scholar), DNA (21Shlyakhtenko L.S. Potaman V.N. Sinden R.R. Lyubchenko Y.L. J. Mol. Biol. 1998; 280: 61-72Crossref PubMed Scopus (120) Google Scholar), and the to transition Y.L. Shlyakhtenko L.S. Potaman V.N. Sinden R.R. Microsc. Microanal. 2002; 8: 170-171Crossref PubMed Scopus (13) Google Scholar). In the time-lapse the sample was onto APS-mica L.S. Gall A.A. A. A. Lyubchenko Y.L. 2003; PubMed Scopus Google Scholar) and with AFM immediately after the AFM tip the surface. The results of experiments performed in TNM buffer are shown in Fig. 1 is in the conformation in Fig. but adopts the conformation in Fig. is molecule (2Meselson M. J. Mol. Biol. 1972; 71: 795-798Crossref PubMed Scopus (59) Google Scholar) on the scanning that conformational This molecule is in the conformation with between the and arms in A and but the junction adopts the conformation in Fig. The molecule and the conformation in Fig. is an intermediate state of the HJ to the conformation. For the conformational for these two molecules are shown in are of the molecules on a In this and and and are the of molecules 1 and We the time-lapse in an to the process of strand separation. branch migration, TNM buffer in the AFM cell was with TE buffer the of The of 4 after of the buffer is shown in Fig. The molecule with the is in the conformation in the (Fig. The arms to an extended conformation (Fig. of the arms is in Fig. to branch migration in the of the molecule into two linear strands as in Fig. the dynamics of this HJ, the of the molecule were onto a from a The containing of the data is shown in Fig. This data illustrating the dynamics of the junction in TNM buffer in which the HJ conformation from to in Fig. shown in on a The of the of this data show the dynamics of the HJ after the removal of Mg cations molecules on a show that the mobility of the arms is in TE buffer in TNM mobility of the arms in the presence of is by of the DNA molecules to the mica surface in to of the DNA with of aminopropyl (24Lyubchenko Y.L. Shlyakhtenko L.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 496-501Crossref PubMed Scopus (351) Google Scholar, 26Shlyakhtenko L.S. Potaman V.N. Sinden R.R. Gall A.A. Lyubchenko Y.L. Nucleic Acids Res. 2000; 28: 3472-3477Crossref PubMed Scopus (78) Google Scholar). despite an mobility in TE the HJ not from the conformation into the the transition to the extended conformation to strand was in acquired suggest that the extended the conformation is for branch migration. We the of the arms of the molecules in the in Fig. and the data are in Fig. The data show that the of the arms during the Branch migration leading to the of the junction is as a process between and in Fig. However, this is not a The between the two imaging is and to I.G. Hsieh P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2021-2025Crossref PubMed Scopus (233) Google and 4Panyutin I.G. Biswas I. Hsieh P. EMBO J. 1995; 14: 1819-1826Crossref PubMed Scopus (84) Google Scholar, the for strand via spontaneous branch migration by a mechanism is The of the molecule with the surface slow the a for molecules and to the surface is In spontaneous branch migration is a the length of the arms can in either of HJ dynamics is shown in Fig. on the and show the dynamics and and the strand separation and the of the dynamics on including the two in TNM to the data in Fig. the of the molecules placed on a are shown for in the dynamics. this junction the conformation even after the buffer and after the conformation. molecules after strand separation The in Fig. shows the in the of the arms during the is a in the of the arms in after the transition from the to the conformation The arms after and in length before the of the junction The of the arms in an as for arms during branch migration. The strand also and as we for the in Fig. The complete of for experiments as can in the attached to the and We and of the arms to strand The data in I show that the of arms A and the of arms and of the of the arms with in a analysis was to the dynamics of HJs in TNM The results for two molecules in the are shown in Fig. The data in Fig. shows the dynamics of the molecule in the conformation. The shows the arms for and arms are and arms are The dynamics of the molecule in the conformation is in Fig. and is no in the of the arms for this to data (Fig. we have strand of the junctions in TNM suggesting that cations slow the of branch migration. This is in line with the branch migration results of Hsieh and (3Panyutin I.G. Hsieh P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2021-2025Crossref PubMed Scopus (233) Google Scholar, 4Panyutin I.G. Biswas I. Hsieh P. EMBO J. 1995; 14: 1819-1826Crossref PubMed Scopus (84) Google Scholar, 5Biswas I. Yamamoto A. Hsieh P. J. Mol. Biol. 1998; 279: 795-806Crossref PubMed Scopus (39) Google Scholar) a in branch migration in the presence of data also show that HJs in the presence of cations are very various conformations including extended and parallel and antiparallel The dynamics of two molecules can in and in the to this broad dynamics of HJs is in line with recent findings (20McKinney S.A. Declais A.C. Lilley D.M. Ha T. Nat. Struct. Biol. 2003; 10: 93-97Crossref PubMed Scopus (274) Google Scholar) single molecule was to immobile HJs undergoing between two crossover conformations via the extended conformations as an intermediate The from these data is that the extended conformation is the predominant of the HJ during branch migration. the transition of HJs from the to the extended is for branch migration to suggesting that the of HJs branch migration. are with the branch migration model of I.G. Hsieh P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2021-2025Crossref PubMed Scopus (233) Google Scholar, 4Panyutin I.G. Biswas I. Hsieh P. EMBO J. 1995; 14: 1819-1826Crossref PubMed Scopus (84) Google Scholar, 5Biswas I. Yamamoto A. Hsieh P. J. Mol. Biol. 1998; 279: 795-806Crossref PubMed Scopus (39) Google Scholar, suggesting that the of HJs from the conformation by was for branch migration. data show that conformation of HJs is involved in branch migration. the of the junction into an extended conformation is for branch migration to occur. data show that the extended conformation is a state for mobile HJs even in the presence of cations and there is a spontaneous transition between conformations suggesting that the between various conformations is with the This is with analysis of the various models of HJs performed Biochemistry. 1994; PubMed Scopus Google Scholar). In branch migration is the process in which the extended conformation of the junction is by protein even in the presence of cations (22Shlyakhtenko L.S. Hsieh P. Grigoriev M. Potaman V.N. Sinden R.R. Lyubchenko Y.L. J. Mol. Biol. 2000; 296: 1169-1173Crossref PubMed Scopus (73) Google Scholar, A. A.A. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). conformations of HJs are in a of site-specific recombination Struct. 2001; PubMed Scopus Google Scholar). this evidence that an extended conformation of mobile Holliday junctions is their conformation during branch migration, and that are to the unfolded conformation of the junction and this conformation for branch migration. molecule AFM was used in this to the dynamics of HJs allowing branch migration. The of time-lapse AFM imaging is that this technique to observe the dynamics of an molecule with range molecules in the scanning can and allowing the of the dynamics of molecules at However, the dynamics of molecules to the space of a and with the dynamics of molecules in to the of the allowing the of dynamics of HJs including the separation of but the of branch migration is of in AFM experiments for molecules the single molecule AFM is a technique that information on the range of conformational of the molecules the of these which can by single molecule in this that a broad dynamics of the arms of mobile HJs at for branch migration the presence of is with the recent findings (20McKinney S.A. Declais A.C. Lilley D.M. Ha T. Nat. Struct. Biol. 2003; 10: 93-97Crossref PubMed Scopus (274) Google Scholar) the arms dynamics of immobile HJs was with at the single molecule We L. C. and M. for at of the and P. Hsieh, M. S. and Potaman for critical of the with