Nature Vol. 292 23 July 1981
`
`8. Nelbach. M. E., Piglet, V. P., Gerhardt, J. C. & Schachman, H. K. Biochemistry 11.
`315-327 (1972).
`9. Cohlberg, J. A., Pigiet, V. P. & Schachman, H. K. Biochemistry 11, 3396-3411 (1972).
`10. Monaco, H. L.. Crawford, J. L. & Lipscomb, W. N. Proc. nam. Acad. Sci. U.S.A. 75,
`5276-5280 (1978).
`11. Beckwitz, J. R., Pardee, A. B., Austrian, R. & Jacob, F. J. molec. Biol. 5, 618-634 (1962).
`12. Bachmann, B. J. & Low, K. B. Microbiol. Rev. 44, 1-56 (1980).
`13. Perbal, B. & Hervé. G. J. molec. Biol. 70, 511-529 (1972).
`14. Perbal, B., Guegen, P. & Hervé, G. J. molec. Biol. 110, 319-340 (1977).
`15. O'Donovan, G. A., Holoubek, H. & Gerhart, J. C. Nature new Biol. 238, 264-266 (1972).
`16. Syvanen, J. M. & Roth, J. R. 1. moles. Biol. 76. 363-378 (1973).
`17. Wild, J. R., Foltermann, K. F., Roof, W. D. & O’Donovan, G. A. Nature 292, 373-375
`(1981).
`18. Legrain, C., Staloln. V. & Glansdorff, N. J. Bact. 128, 35-38 (1976).
`19. Feller, A., Lissens, W., Glansdorfi, N. & Piérard. A. Arch: int. Physiol. Binchim. 86,
`941-942 (1978).
`20. Gerhart, J. C. & Holoubeck, H. J. biol. Chem. 242. 2886-2892 (1967).
`21. Glansdorfi, N. Genetics 51, 167-179 (1965).
`22. Mergeay, M., Gigot, D., Beckrnann. J., Glansdortf, N. & Piérard, A. Malec. gen. Genet.
`133, 299-316 (1974).
`23. Legrain, C. at al. Eur. J. Biachem. 80, 401-409 (1977).
`24. Wargnies. B., Lauwers, N. & Stalon, V. Eur. J. Biochem. 101, 143-152 (1979).
`
`
`A mutation in the catalytic cistron of
`aspartate carbamoyltransferase
`affecting catalysis, regulatory
`response and holoenzyme assembly
`
`James R. Wild, Karen F. Foltermann*,
`William D. Roof"‘ & Gerard A. 0’Donovan"‘
`
`373
`
`Table l ATCase holoenzymes from wild-type E. coli strain E63 and mutant
`E. coli strain WR38-h204
`
`[S],,,, (mM aspartate)
`% Activity + CTP
`% Activity+ATP
`
`£I_°_‘*_S‘flVf';“£fl
`ATCase activity at pH 8.4
`Shape of velocity-substrate curve
`
`Wild-type
`5.5 mM
`21%
`166%
`
`Mutant
`2.0-2.5 mM
`97%
`105%
`
`3,,
`
`0,5,
`
`Sigmoidal
`
`Hyperbolic
`
`ATCase assay mixtures of 2.0 ml volume contained 40 mM potassium phos-
`phate, pH 7.0, 3.6 mM dilithium carbamoyl phosphate, pH 7.0, 5 mM potassium
`aspartate, pH 7.0, for the wild-type and 2.5 mM for the mutant (approximate [S}0_5
`values), and enzyme (holoenzyme fractions of G-200 eluate, Fig. 2). CTP or ATP
`was absent from control tubes and present at a concentration of 2 mM when
`inhibition (CTP) or activation (ATP) was measured. The control activity, that is,
`the activity in the absence of effector, is set at 100%. ATCase activity was assayed
`by measuring the amount of carbamoyl aspartate formed in 30 min at 30°C as
`previously described“. Carbamoyl aspartate production was determined at
`466 nm. Specific activity is expressed as nmol carbamoyl aspartate formed per min
`pep mg protein. Specific activity of ATCase was determined in conditions in which
`carbamoyl aspartate formation was proportional to extract concentration and
`time. The value obtained for the ratio of ATCase specific activity at pH
`7.0/ATCase specific activity at pH 8.4 may be used as an index for the presence or
`absence of cooperativity between catalytic sites for aspartate binding according to
`the methods of Kerbiriou and l-fervé". A ratio of < 1.0 signifies the absence of
`cooperativity; one of 22.0 reflects cooperative homotropic interactions. Frac-
`tions from beneath the holoenzyme peak (Fig. 2) were used to estimate this ratio.
`
`Genetics Section and *Department of Biochemistry and Biophysics,
`Texas A & M University, College Station, Texas 77843, USA
`
`
`positive homotropic interactions between catalytic sites as evi-
`denced by the sigmoidal dependence of activity on substrate
`concentrations°"°, (2) substrate binding is subject to positive
`heterotropic interactions between catalytic and regulatory sites
`in the presence of ATP and negative heterotropic interactions
`We describe here a mutation in the gene encoding the catalytic
`with CTP“”“, and (3) the binding of CTP is subject to negative
`subunit of aspartate carbamoyltransferase (ATCase, pyrB)
`homotropic
`interactions between the
`regulatory sites”.
`which produces an enzyme retaining catalytic activity as holo-
`Moreover, ATCase is unusual among allosterically regulated
`enzyme (2C3:3R,) and catalytic trimer (C3) but which shows
`enzymes,
`in that, as with yeast phosphofructokinase“,
`the
`neither cooperative substrate kinetics nor nucleotide effector
`regulatory protein is distinct and can be physically dissociated
`response. Furthermore, the holoenzyme assembly seems quite
`from the catalytic subunit. The assembly of the enzyme is a
`fragile in that the enzymatic activity is recovered only partially as
`cytoplasmic event” which produces a dodecamer with six
`holoenzyme following growth of Escherichia coli
`in the
`regulatory and six catalytic polypeptides (race) associated as two
`presence of zinc. in contrast, the enzyme from wild-type E. coll
`separable catalytic trimers and three regulatory dimers““°. The
`strain E63 is recovered almost entirely in its dodecameric form
`holoenzyme may be reversibly dissociated by mild treatment
`in identical conditions. The gene encoding the mutant pyrB was
`with mercurials such as p-chloromercuribenzoate” or neo-
`isolated from a AdargI“pyrB* transducing phage (obtained
`hydrin“. The catalytic subunit (C3)
`is insensitive to allosteric
`from N. Glansdorfi). This mutation differs from other
`reviously
`effectors, possesses a half-saturation concentration ([S].,,5)
`reported mutations affecting the catalytic subunit '2 in that
`higher for aspartate than the holoenzyme (8-10 mM compared
`significant catalytic activity is retained but homotropic and
`with 5 mM) and produces a V,,,.,,, that is two- to fourfold higher“.
`heterotropic communication is lost’.
`The separate regulatory subunit (r2) has no catalytic activity
`A 6.0-kilobase fragment was isolated from purified ADNA‘,
`although ATP and CTP may still be bound”. On reassociation
`restricted with the endonuclease Pstl
`(Bethesda Research
`of the holoenzyme after removal of the mercurial by zinc
`Laboratory)5, and cloned on to plasmid pBR322 (ref. 6). The
`replacement dialysis in the presence of dithiothreitol, the orig-
`recombinant
`plasmid
`pPB-h204
`(plasmid—pyrimidine-B
`inal catalytic and regulatory properties of the native enzyme
`cistron-holoenzyme-strain 204) was transformed into E. coli
`are re-established”.
`WR38, which contains a Mu insertion’ in the chromosomal pyrB
`ATCase was prepared from E. colt’ wild-type strain E63 and
`and does not produce catalytic or regulatory polypeptides as
`the transformed mutant strain WR38-h204 by the methods of
`determined by immunoassay with specific antisera (W.D.R.,
`Wild et (11.23. Several pertinent properties of the wild-type and
`unpublished observations). This plasmid encodes both the
`mutant ATCases are compared in Table 1. We must emphasize
`catalytic (pyrB) and the regulatory (pyrl) polypeptides of
`three points regarding these data. (1) The mutant ATCase is not
`ATCase. [A mutation in the cistron encoding the regulatory
`subject to allosteric regulation by either ATP or CTP at subsat-
`polypeptide is described in the accompanying report by Feller et
`urating concentrations of aspartate (1-5 mM). Minimal effector
`al.8. The cistron is designated pyrl in agreement with Bachmann
`response was observed when velocity—substrate plots were
`(personal communication).] The synthesis of ATCase in the
`examined over the range of 0.5 mM to 50 mM aspartate (<5°/o
`transformed strain, WR38-h204,
`is repressed by growth in
`variation throughout the range). As noted by Gerhart“, such
`uracil, so that
`the cloned fragment contains the operator-
`effects are due only to the direct competition for any phosphate-
`promoter region as well as both ATCase cistrons. The catalytic
`containing compound. (2) The apparent [S]9,5 for aspartate of
`activity recovered from this strain is distributed as ~40% holo-
`the mutant enzyme is significantly lower (2.0-2.5 mM) than the
`enzyme with a molecular weight (M,) of 300,000 and 60% as the
`wild-type requirements (5.5 mM). (3) The homotropic kinetic
`catalytic trimer with a M, of 100,000. Both forms of ATCase
`responses of the mutant enzyme are dramatically reduced or
`from this mutant lack cooperative homotropic kinetics for
`abolished in the mutant enzyme as shown by the lack of sig-
`aspartate, do not respond to the allosteric effectors ATP and
`moidal dependence of activity on aspartate concentration (Fig.
`CTP, and seem to have altered affinities for aspartate.
`1) even when plotted according to Eadie-Hofstee” (see insert
`ATCase is the archetype of allosteric enzymes because: (1)
`Fig. 1). Thus, there is no apparent cooperativity from 0.5 to
`both its substrates, carbamoyl phosphate and aspartate, produce
`10 mM (20% to fivefold [S]o_5, respectively). In addition, the
`0028-0836/81/300373-03301.00
`
`© 1981 Macmillan Journals Ltd
`
`Merck Ex. 1051, pg 1319
`
`Merck Ex. 1051, pg 1319
`
`

`
`
`
`© Nature Publishing Group1981
`
`Merck Ex. 1051, pg 1320
`
`

`
`""""““
`mm , , ,ETecA
`
`Pstl site
`
`“GT9, , ,
`
`PLPH9
`
`===81EiEi111=======lll=========11l8i9===
`
`PU’1*6°
`
`===Eli8i11i111====i1l========1lliEii===
`
`PLPWJ
`
`===819Ei1i1=======111=========1118iE===
`
`PLP219
`
`=
`
`= =ElEE1E8E= =
`
`=
`
`=
`
`=
`
`= =E8E= = = =
`
`= = =8EE1EEii= =
`
`=
`
`pLP222
`
`===El€EiEEE= = = = = = =8E£= = = = = = =E88lEEiE= ==
`
`M225
`
`===8l€8EE€========E9E=========EEE8ii===
`
`375
`
`Total
`ier?-éiiatbpt
`0
`
`19
`
`"0
`
`‘W
`
`19
`
`22
`
`25
`
`3“
`===819EEEE=====:==EEE=========EE‘éE1E===
`PLP234
`Fig. 1 Sequences of homopolymer tract inserts. Clones were
`initially screened for insert size by following the restriction
`endonuclease Haelll digest on a 6% polyacrylamide gel. For DNA
`sequencing, the insert containing the Alul-generated restriction
`fragment was isolated, end-labelled, and sequenced after strand
`separation according to the procedures of Maxam and Gilbert“. In
`most cases both strands were sequenced. No changes in plasmid
`sequence were observed outside the Pstl site.
`
`The principle of the band-shift method has been reported
`elsewhere’. Note that the helical repeat, h°, obtained from the
`band-shift method is the number of base pairs of the sequence
`inserted that will increase the average linking number of the
`DNA in its relaxed state by one. In cases where the insert does
`not have an intrinsic spatial writhe, h° is also identical to the
`number of base pairs that forms a complete helical turn.
`In addition to the absolute magnitudes of the helical repeats of
`different sequences, the band-shift method gives the relative
`handedness of the helices. We shall make the generally accepted
`assumption that a double—stranded DNA of typical sequence is a
`right-handed helix in solution. The band-shift method then
`provides directly the handedness of the inserted helical seg-
`ments as well.
`To extend the band-shift method to the determination of the
`helical repeats of DNAs of defined sequences, families of
`covalent closed circular DNAs containing inserts of these
`sequences of known lengths are needed. The method used to
`insert homopolymer tracts of various lengths into the Pstl site of
`pBR322, a plasmid with known nucleotide sequence”, involved
`tailing with terminal transferase and one of the deoxynucleoside
`triphosphates“. On digestion with restriction endonuclease
`BamHI, preparative gel electrophoresis allowed isolation of the
`desired fragments. The appropriate pairs of DNA fragments
`thus obtained were ligated together with T4 polynucleotide
`ligase and competent Escherichia coli cells were transformed.
`The nucleotide sequences in the regions containing the inserts
`are given in Fig. 1. In several cases, at the junctions between the
`plasmid DNA and the homopolymer inserts, a few unexpected
`base changes occurred. These changes were probably due to a
`low level of 3’—> 5‘ exonuclease activity in the calf thymus
`terminal transferase used in the construction of these plasmids.
`An example of the gel electrophoretic patterns of several
`DNA samples relaxed in identical conditions is shown in Fig. 2a ;
`lane 9 illustrates the band pattern observed when a single DNA,
`that of plasmid pLP222, is present. In lane 8, a second DNA
`(pLP219) shorter than pLP222 by three GC base pairs (bp) is
`mixed in. It can be readily seen that the band pattern of the
`longer plasmids shows a relative upward shift of ~ 0.3 times the
`interband spacing between topoisomers.
`The set of covalently closed DNA samples prepared are all
`positively supercoiled in electrophoresis conditions”, thus the
`faster migrating topoisomers have higher linking numbers than
`the slower migrating ones. Therefore an upward shift (that is, a
`reduction in the distance migrated) by 0.3 times the interband
`
`© 1981 Macmillan Journals Ltd
`
`Merck Ex. 1051, pg 1321
`
`Nature Vol. 292 23 July 1981
`
`catalytic capabilities of the enzyme. This mutation should be
`compared with that affecting the regulatory polypeptide
`described by Feller et (11.8 in the accompanying report. It is
`striking that the holoenzyme assembly is apparently deficient in
`both mutants, one affecting the catalytic sununit and the other
`the regulatory subunit.
`One final observation regarding the catalytic mutation seems
`pertinent. Examination of the kinetic characteristics (Fig. 1) of
`the mutant (with or without ATP added) revealed that they
`approximate the wild-type enzyme in the presence of ATP.
`Indeed, the high aflinity exhibited by the mutant holoenzyme for
`aspartate suggests that the mutant ATCase may be frozen in its
`activated R state“. The present data suggest that strikingly
`similar characteristics can exist for two independent mutations
`in either the catalytic or regulatory cistrons of ATCase.
`This research was supported in part by a grant from the
`National
`Institute of General Medical Sciences,
`1R01
`GM291S2-01, the US Department of Agriculture (CRGO 59-
`2485-0-1-463-01), the Robert A. Welch Foundation, and the
`Texas Agriculture Experiment Station, I-I-1670 and H-6458.
`Received 30 December 1980; accepted 2 June 1981.
`
`I-2., Gibbons, I 8t. Schachman, H. K. J. blol. Chem. 254.
`
`1. Wall. K. A., Flntgnard, J.
`11910-11916 (1979).
`2. Beckwith, J. 1?... Pardee, A. B., Austrian, R. 8: Jacob, R. J. molec. Biol. 5, 618-634 (1962).
`3. Kantrowitz, E. R., Pastra-Landis, S. C. & Lipscomb, W. N. Trends biachem. Sci. 5, 124-128
`(1980).
`Davis. R. W., Botstein, D. & Roth, J. R. Manual for Genetic Engineering. 80-82 (Cold
`Spring Harbor Laboratory, New York. 1980).
`Smith, D. E.. Blattner, F. R. & Davies, J. Nucleic acid Res. 3. 343-353 (1976).
`Bolivar, F. er al. Gene 2. 95-113 (1977).
`Taylor, A. L. Prac. natn. Acad. Sci. U.S.A. 50. 1043-1051 (1963).
`. Feller, A., Piérard, A., Glansdorff, N.. Charlier, D. & Crabeel, M. Nature 292, 370-373
`(1981).
`9. Gerhart, J. C. dc Schachman, H. K. Biochemistry 7. 538-552 (1968).
`I0. Bethell, M. 1?... Smith, K. E., White. J. S. & Jones, M. E. Prac. natn. Acad. Sci. U.S.A. 60,
`1442-1449 (1968).
`. Kerbiriou, D. & Hervé, G. J. malec. Biol. 64, 379-392 (1972).
`. Kerbiriou, D. & Herve, G. J. malec. Biol. 78, 687-702 (1973).
`...._...5-Ho-
`Kantrowitz, E. R.. Jacobsberg, L. B., Landfear, S. M. & Lipscomb, W. N. Proc. natn. Acad.
`Sci. U.S.A. 74, 111-114 (1977).
`14. Kerbiriou. D., Herve, G. & Griflin, J. H. J. biol. Chem. 252. 2881-2890 (1977).
`15. Winlund. C. C. 8: Chamberlin. M. J. Biochem. biophys. Res. Cammun. 40, 43-49 (1970).
`16. Laurent, M., Chsfiotte. A. I-‘.,Tenu, J. P. Raucous, C. & Seydoux, F. Biochem. biaphys. Res.
`Commun. 80, 646-652 (1978).
`17. Thiry. 1.. Gt Herve, G. 1. make. Biol. 125, 515-534 (1978).
`18. Weber, K. Nature 218, 1116-1119 (1968).
`19. Wiley, D. C. at Lipscomb, W. N. Nature 218, 1119-1121 (1968).
`20. Gerhart, J. C. & Schachman, H. K. Biochemistry 4. 1054-1062 (1965).
`21. Yang, Y. R.. Kirachner, M. W. & Schachman, H. K. Meth. Enzym. 51, 35-41 (1978).
`22. Nelbach, M. E., Piglet. V. P.. Gerhart, J. C. & Schachman, H. K. Biochemistry 11, 315-327
`(1972).
`23. Wild. 1. R., l-‘oltermann, K. F. & O'Donovan, G. A. Archs Biochcm. Biophys. 201, 506-517
`(1980).
`24. Gerhart, J. C. Curr. Topics cell. Regulation 2, 275-325 (1970).
`25. Eadie. G. S. J. biol. Chem. 146, 85-93 (1942).
`26. Monod. J.. Wyman, J. & Changeux, J.-P. J. malec. Biol. 12. 88-118 (1965).
`
`
`9 °
`
`".‘‘?‘!*''
`
`Sequence dependence of the
`helical repeat of DNA in solution
`
`Lawrence J. Peck & James C. Wang
`
`Department of Biochemistry and Molecular Biology,
`Harvard University, Cambridge, Massachusetts 02138, USA
`
`Considerable progress has recently been made on the fine
`tructure of DNA. X-ray diffraction studies of crystals of oliga-
`nucleotides of defined sequences have already provided several
`structures at atomic resolution”. Whereas all
`the crystals
`studied so far show bihelical structures with antiparallel chains
`and Watson-Crick-type base pairing, there is a striking range
`of structural variability, from the right-handed B-type to the
`left-handed Z-type helices. In contrast to the well developed
`methology for crystal structure determination, few methods of
`high precision and low ambiguity are available for studies of
`DNA in solution. Here we report the application of the band-
`shift method previously developed in this laboratory’ to the
`determination of the dependence of the DNA helical repeat in
`solution on its nucleotide sequence.
`0028-0836/81/300375—04$01.00
`
`Merck Ex. 1051, pg 1321