J. Mol. Biol.
(1991)
219, 615-622
DNA Looping Alters Local DNA during Transcription Ha&Young
Conformation
Wu and Leroy F. Liut
Department The Johns Hopkins Baltimore, (Received 23 May
of Biological Chemistry University, School of Medicine MD 21205, U.S.A. 1990; accepted 26 February
1991)
The effect of protein-mediated DNA looping on local DNA conformation during active transcription was studied using the Zac repressor-operator system. Our results suggest that lac repressor-mediated DNA looping within a plasmid DNA molecule containing two lac repressor binding sequences in viva effectively separates plasmid DNA into two topological domains. Supercoils generated by transcription within each topological domain can be rapidly removed by DNA topoisomerase I. Keywords:
DNA
supercoiling;
DNA looping; DNA topoisomerases; RNA transcription
1. Introduction
from a number of observations. While the nature of the cellular components holding the loops is not completely known, each chromosomal DNA loop has been shown to be an independent topological domain (Stonington & Pettijohn, 1971; Worcel & Burgi, 1972; Benyajati & Worcel, 1976; Paulson & Laemmli, 1977; Marsden & Laemmli, 1979; Vogelstein et al., 1980; for a review, see Gross & Gerrard, 1988). The possibility that a looped DNA domain may co-ordinately regulate chromatin structure and gene expression in higher eukaryotes has also been speculated (Weintraub, 1985). According to the twin-supercoiled-domain model of RNA transcription, the chromosomal DNA loop attachment sites can effectively restrict the rotational diffusion of supercoils generated by RNA transcription (Liu & Wang, 1987). These chromosomal DNA loop attachment sites therefore may affect the local supercoiling state of chromosomal DNA in the vicinity of the attachment sites. In order to study the possible effect of chromosomal DXA loop attachment sites on local DNA conformation, we have modeled the chromosomal DNA loop attachment sites using the Zac repressoroperator system. lac repressor has been shown to loop DISA in vitro and probably in viwo as well (Besse et al., 1986; Hsieh et al., 1987; O’Gorman et al.: 1980; Whitson & Matthews, 1986). In the present communication, we show that transcription within each of the DNA loops closed by lac repressor-operator complex significantly alters the local supercoiling state of DNA.
Supercoiling has been recognized as an important structural determinant for many DXA functions (for a review, see Cozzarelli, 1980; Gellert, 1981; Rowe & Liu, 1984; Wang, 1985). Recent studies have suggested that in addition to DNA topoisomerase. RNA transcription can also significantly alter the supercoiling state of cellular DNA (Pruss & Drlica, 1986, 1989; Brill & Sternglanz, 1988). However, the mechanism through which RNA transcription, and possibly other cellular processes, effects DXA conformation is not completely understood. A model in which the process of RNA transcription generates positive and negative supercoils has been proposed (Liu & Wang, 1987), and additional support for the model has come from both in uivo and in vitro studies (Wu et al., 1988; Giaever & Wang, 1988; Tsao et al., 1989; Lodge et al., 1989; Koo et al., 1990). According to this model, local DNA supercoiling can be affected by a complex set of. parameters (Liu & Wang, 1987; Wu et al., 1988). One such parameter relates to the rotational diffusion pathway of supercoils generated by transcription (Liu & Wang, 1987). Protein (or other factor)enclosed DNA loop is expected to block such a rotational diffusion pathway of supercoils generated by transcription. The presence of chromosomal DNA loops in both prokaryotic and eukaryotic cells has been suggested t Butor addressed.
to whom
0022-2836/91/12061&08
all correspondence
$03.00/O
should
Zac repressor;
be 615
0
1991 Academic
Press Limited
H.-Y.
616
Wu and L. F. Liu
- IPTG
l
IPTG
put 19 bp)
(2686
PAO (2232
bp)
pAO-SLO bp)
(2274
Figure 1. Preferential supercoiling of multimeric~ plasmid DIVA requires the essential Inc repressor binding I)NA sequence in cis. IPTG (1 my) was added to E. coli AS19 harboring various plasmids (A: pL’(‘19; 1%:pA0: and (‘: pAOSLO) 15 min prior to the addition of novobiocin (100 rig/ml). Treatment was cont,inued for another 30 min before plasmid DXAs were isolated and analyzed by 2-dimensional gel electrophoresis. Plasmid DXAs isolated from E. coli AS19 with and without IPTG treatment prior to novobiocin treatment are shown on the left and right column of tht P-dimensional gel panels. respectively. Streak a and a’ marks the position of negatively supercoiled monomeric3 and dimeric plasmid DSA. respectively. Streak b and b’ marks the position of positively supercoiled monomrric~ and dimeric, plasmid DKA. respectively.
2. Materials and Methods (a)
Chemicals,
enzymes
and
plasmid
DiV.-ls
Chloroquine diphosphate. novobiocin. rifampicin. IPTGt and chloramphenicol were purchased from Sigma. Enzymes were purchased from various commercial sources. pA0 plasmid DKA was constructed by blunt-end ligation of large EcoRIIAvaI (2232 bp) fragment of pAT1.53 DPU’A (Twigg & Sherratt. 1980). pAO-SLO plasmid DKA was constructed by inserting a single copy of a 42 bp DKA oligomer which contains a 21 bp essential luc repressor binding sequence (Gilbert & Maxam. 1973) in the middle of a polylinker sequence. The following plast Abbreviations used: IPTG. isopropyl-8-n thiogalactoside; bp. base-pair(s); kb. lo3 base-pairs.
mids are generous gifts from St~efan Orhlrr and I~IIIIO Miiller-Hill of Institute of Genetics, University of Kiiln, Germany; pSOlOO0 and pSOlO0 are low copy number plasmids (pACYC based), which carry the wild-type lac/ gene and the mutated EacI gene (iadi) (Lehming et al.. 1987), respectively. Roth genes are expressed via the iq promoter (Oehler et al., 1990). (b)
Ba,ctrria,t
strains
amf
crll
yrowfh
Escherichia coli AS19 is a 13 strain isolated for its D and other antibiotics. permeability to actinomycin including novobiocin (Sekiguchi & Iida. 1969). E. w/i MC1060 {A(lacI- Y)74. galEl.5. galKl6. i-. rdA I, rpsLl50. spoTI. hsdR2) a la,c repressor deletion strain ((‘asadaban & Cohen. 1980), was obtained from I)r Hamilton Smith (Johns Hopkins University). Transformants \~rre grown
Local
DNA
Conformation
in LB to early log phase(A,,, = @15)prior to inhibitor treatments. (c) Tluo-dinzensional gel electrophoresis Plasmid Dru’As were isolated by the alkaline lysis method as described(Wu et al.. 1988).Topoisomerdistributions of DNA sampleswere then analyzed by d-dimensional gel electrophoresis(Wu et al., 1988). The second dimension was carried out in gel electrophoresisbuffer containing 3 PM-chloroquine. Dried slab gels were then prepared for in situ Southern hybridization as described (Wu et al.. 1988).
3. Results The effect of transcription on local DNA conformation can be studied by monitoring the linking number of a plasmid DNA in bacterial cells treated with gyrase inhibitors (Wu et al., 1988). When DKA topoisomerase II (gyrase) is not available to relax positive supercoils generated ahead of the transcription complex and/or to introduce negative supercoils, positive supercoils on a transcribing plasmid DKA accumulate due to selective relaxation of transcription-generated negative supercoils by topoisomerase I (o protein). Thus, the rate of accumulation of positive supercoils on a plasmid DNA may reflect various parameters that affect transcriptiongenerated supercoils. (a) The accelerated rate of accumulation of positive .supercoilaon plasmid DNAs containing lac repressor
binding
sequences
The effect of protein-mediated DXA looping on DNA conformation during transcription was initia,lly suggested from studies of the rate of accumulation of positive supercoils on a number of pBR322 derivatives (Ll’u et al.. 1988). A pRR32Z derivative, pA0 DiVA, which contains only the h/n gene as the major transcription unit (see Fig. 1). accumulated positive supercoils at a much lower rate than did pBR322 DNA in E. coli AS19 cells treated with novobiocin. After a 30 minute novobiocin treatment, pA0 DNA was isolated as a of topoisomers as heterogeneous population revealed by two-dimensional gel electrophoresis (Fig. 1; see a streak of topoisomers in panel B, -1PTG). Only a very small population (less than 20%) of pA0 DNA was slightly positively supercoiled whereas under identical conditions, nearly all pBR322 DNA was highly positively supercoiled (Wu et al., 1988). The reduced rate of accumulation of positive supercoils on pA0 DNA is presumably due to the lack of the stronger transcription unit, the tetA gene. When the rate of accumulation of positive supercoils on pUC19 DNA, which is identical with pA0 DNA except for a 445 bp insertion containing the lac promoter, was compared with that of pA0 DNA, two surprising findings were noted; first monomeric plasmid DNA populations of pUC19 accumulated positive supercoils at a faster
Changes
617
rate than the corresponding monomeric populations of pA0 DNA (Fig. 1; seea streak of topoisomers in panel A, -1PTG). (This effect will be referred to as the “monomer effect”.) Second, the rate of accumulation of positive supercoils on dimeric and multimerit pUCl9 DNA populations (marked by b’ for the dimer population) was even faster than that of the monomeric pUCl9 DNA (marked by b; Fig. lA, - IPTG); this effect will be referred to as the “dimer or multimer effect”). Under our experimental conditions, only 40% of the monomeric pUCl9 DNA topoisomers were positively supercoiled, while nearly all the dimeric and multimeric pUCl9 DNA topoisomers were positively supercoiled (Fig. lA, - IPTG) . Both the monomer effect and the dimer (or multimer) effect were sensitive to TI’TC: treatment. The faster rate of accumulation of positive supercoils on dimeric pc’Cl9 DSA was c*ompletel> abolished by IPTG treatment (compare 1)’ in Fig. 1A (-IPTG) and b’ in Fig. 1A (+IPTG)). A decrease in the positively supercoiled population of monomeric pUCl9 DNA topoisomers upon IPTG treatment was also reproducibly observed (Fig. IA; compare streak b (-IPTG) with streak b ( + IPTG)). In addition, the relative proportion of positively supercoiled DNA population appeared to be about the same for both monomeric and dimeric pUCl9 DNA upon IPTG treatment (Fig. 1A (IPTG)). In contrast, IPTG treatment had no effect on the relative distribution of pA0 DNA topoisomer populations (Fig. 1B; compare topoisomer streaks with and without IPTG treatment). In fact, upon IPTG treatment, pUCl9 DNA accumulated positive supercoils at the same rate as did pA0 DNA. The effect of IPTG on the accumulation of positive supercoils in the dimeric and monomeric pUCl9 DNA populations suggested that either the binding of the lac repressor to the operator or the state of RNA transcription from the lac promoter might be responsible. To distinguish between two possibilities, a 42 bp synthetic DXA containing a single lac repressor binding site (a 21 bp lac operator sequence, which is a near-perfect palindrome; Gilbert & Maxam, 1973) was inserted into the AatII site of pA0 DXA (see Fig. lC, pAO-SLO DNA). The new plasmid DNA, pAO-SLO DXA, which does not contain the lac promoter but does contain the lac repressor binding sequence: exhibited a similar response in E. coli AS19 cells treated with novobiocin. First, the rate of accumulation of positively supercoiled monomeric pAO-SLO DXA increased relative to that of pA0 DSA (Fig. 1, compare streak b in C ( - TPTG) with streak b in B (-IPTG)). Second, dimeric (or multimerit) pAO-SLO DXA accumulated positive supercoils significantly faster than its monomeric counterpart (Fig. 1C; compare b’/a’ with b/a). Both the monomer effect and the dimer effect were again effectively eliminated by IPTG treatment (compare b’/a’ in Fig. 1C (-IPTG) and Fig. 1C (+IPTG)). These results suggest that both the monomer effect and the multimer effect are mediated by the binding
618
H.-Y.
Wu and L. F. Liu
2nd D
Figure
2. Lack of preferential supercoiling of multimeric plasmid I)N;As in a /UC repressor deletion strain of’ /!‘. co/i. MC1060. a luc repressor deletion strain (Casadaban & Cohen. 1980). harboring pAO-SLO was treated bvith novobiocin (500 pg/ml) and oxolinir acid (100 pg/ml) for 30 min. DNA was isolated and analyzed by 2-dimensional gel elertrophoresis. A: no IPTG treatment. B: TPTG (1 mM) (see Fig. 1 for IPTG treatment). Each group of USA topoisomers is labeled with a number near its upper apex of the triangle patt,ern. 1. 11. TIT and IV indicate the monomeric. dimeric. trimerir and tetrameric topoisomer populations of pAG-ST,0 I)NA. respectively. E. coli
of the lac repressor transcription from
to the operator but not by RNA the lac promoter.
(b) The accelerated rate of accumulation of positive supercoils on plasmid DNAs requires lac repressor in trans To strengthen our argument, E. coli MC1060 {A(lacI-Y)74, galE15, GalK16, I-, relAl, rpsLl50, spoT1, hsdR2) (a lac repressor deletion strain; (‘asadaban & Cohen, 1980) was used as a host As expected, pAO-SLO instead of E. coli ASl9. DNA isolated from E. coli MC1060 treated with novobiocin showed neither the monomer effect (compare the topoisomer distribution in Fig. 2A with that in Fig. 2B) nor the multimer effect (compare b/a of topoisomer group I, II, III and IV in Fig. 2A). These results suggested that the presence of lac repressor in cells was necessary both for the increased rate of accumulation of positive supercoils on monomeric plasmid DNA populations of pAO-SLO (the monomer effect) and for the preferential accumulation of positive supercoils in the multimeric plasmid DNA populations (the multimer effect). We have also introduced lac repressor into E. coli MC1060 via a pACYC-based plasmid, pSO1000, which is compatible with pAO-SLO. pSOlOO0
expresses wild-type lac repressor fin iq promoter (Oehler et al.. 1990). The presence of pSOlOO0 in E. coli MCI060 enhanced the extent of positive supercoiling of pAOSLO DNA (Fig. 3A, marked I (monomer), II (dimer) and TIT (trimer)). The effect of pSOlOO0 on the extent of positive supercoiling of the trimeric form of pAO-SLO DNA was most prominent. However, the effect of pS01000 on extent of positive supercoiling of the dimeric form of in pAO-SLO DNA was less prominent than that We do not quite understand this E. coli ASl9. phenomenon. Tt is possible that overexpression of lac repressor as in E. coli MCI060 harboring pSOlOO0 may inhibit the extent of positive supercoiling of the plasmid DNA. We have also expressed tnutated Inc repressor. which is defective in tet,ramerization but retains DNA binding activity (Lehming Pt a/.. 1987). pSOlO0, which expresses the rnutat’ed (dimeric, active) luc repressor via iq promoter, is also pA(‘Y(‘based. E. coli MC1060 harboring both pHO100 and pAO-SLO did not show any preferential positive supercoiling of the multimeric form of pAO-SLO in novobiocin-treated cells. These resuits toget)hcr is suggest that, the lac repressor binding to operator insufficient to cause preferential positive superof plasmid 1)NA. coiling of multimeric forms lo Simultaneous binding of t,etrameric lac repressor two operator sites is necessary to effect this process.
Local
DNA
Conformation
619
Changes
2nd D
2nd D
\
C Figure 3. Preferential supercoiling of multimeric plesmid DNA requires wild-type Zac repressor in. trans. E. coli MC1060, a Zac repressor deletion strain (Casadaban & Cohen, 1980), harboring both pAO-SLO and 1 of the 2 plasmids pSOlO60 (A) or pSOl66 (B), was treated with novobiocin (500 pg/ml) and oxolinic acid (166 pg/ml) for 30 min. DNAs were then isolated and analyzed by 2-dimensional gel electrophoresis. Each group of DNA topoisomers is labeled with a number near its upper apex of the triangle pattern. I, II and III indicate the monomeric, dimeric and trimeric topoisomer populations of pAO-SLO DNA, respectively. Ie and IIe indicate the monomeric and dimeric topoisomer populations of plasmid DNAs expressing wild-type lac repressor, pSOl660 DNA (A) or mutated Zac repressor (defective in tetramerization), pSOl66 DNA (B). C is a schematic diagram illustrating a typical distribution of DNA topoisomers in this 2-dimensional gel electrophoresis system. In each triangular pattern of topoisomer distribution, (a) marks the topoisomers with lowest linking number (high degree of negative superhelicity). (b) marks the topoisomers with highest linking number (high degree of positive supercoiling). (c) marks the topoisomers with intermediate linking number (relaxed and moderately supercoiled DNA).
(c) The preferential accumulation of positive supercoils on multimeric DNA is dependent on coupled transcription/translation Previous studies have suggested that transcription is responsible for the accumulation of positive supercoils on plasmid DNA in cells treated with gyrase inhibitors (Wu et al., 1988). Furthermore, coupled transcription/translation is expected to have an even stronger effect on template supercoiling (Koo et al., 1990; Liu & Wang, 1987; Lodge et al., 1989). To test whether the observed monomer and multimer effects on plasmid DXAs containing lac repressor binding sequences required coupled transcription/translation, an inhibitor of either RNA or protein synthesis was simultaneously added to E. coli AS19 cells during novobiocin treatment. As shown in Figure 4, either rifampicin (Fig. 4B) or chloramphenicol (Fig. 4C) treatment reduced the rate of accumulation of positively supercoiled mono merit populations of pAO-SLO DNA. In addition. the preferential accumulation of positive supercoils on dimeric plasmid DNA populations was also abolished (compare b’/a’ with b/a in Fig. 4B and C). The effect of rifampicin (90 pg/ml) was stronger than that of chloramphenicol (206 pg/ml) compare b’/a’ in Fig. 4B and C). In the presence of rifampicin, the majority of the dimeric pAO-SLO DNA molecules were negatively rather than positively supercoiled. Tn the presence of chloramphenicol, roughly 60% of the dimeric pAO-SLO DNA remained positively supercoiled. These results
suggest that coupled transcription/translation responsible for the preferential accumulation positive supercoils on dimeric plasmid DiVAs.
is of
4. Discussion (a) lac repressor-mediated DNA looping responsible for the preferential accwnulation positive supercoils on multimerir DNA (the multimer effect)
is of
A model that can explain our results is shown in Figure 5. In this model, a tetrameric lac repressor complex binds simultaneously and co-operatively to two operator sites that are diametrically opposed on a dimeric pAO-SLO DNA molecule (Hsieh et aE., 1987; O’Gorman et al., 1980; Whitson & Matthews, 1986). This simultaneous and c-o-operative binding to two operator sites intramolecularly results in the formation of two stable D;I’A loops. each of which contains a single bla transcription unit (ori-related transcripts may also contribute). The faster rate of accumulation of positive supercoils on multimeric 1)X,4 in gyrase-inhibited cells mav be due to one or both of the following two possibihties. (1) The rotational diffusion pathway for the fusion of positive and negative supercoils generated by caoupled transcription/translation of each bin gene is restricted due to the formation of this ternary cornpIes. To fuse the positive and negative super(oils generated by transcript,ion, the entire DSX loop with thr transcribing RNA polymerase c$omplrxes has to
If.- Y. Wu and L. F. Liu
2nd D B
>
C
Figure 4. Active RSA and protein synthesis are required for preferential awumulation of positive supercoils on /C/C(200 pg/ml) was added to E. co/i ASI9 oE)erator-containirlg plasmid DSAs. Rifampicin (90 pg/ml) or chloramphenicol harboring pBO-ST,0 10 min prior to the addition of novobiocin (100 pg/ml). T rratment was cwntinurcl for another 30 min before plasmid DSAs were isolated and analyzed by d-dimensional gel rlwtrophoresis. A: cw>trol. nowbiwitl treatment only: B: vo-treatment with rifamyicin: and C”: co-t.reatment wit.h chloramphenicol. Spot (3 marks thr position (ii’ monomeric pA@SLO DSA that had been irreversibly denatured during I)K;A isolation. Streak d’ denotes the Iwsitioll 01’ the interlocked USA population.
rotate and pass through the enclosed space within the other DSA loop. Due to the difficulties of such a rotational diffusion process. positive and negative supercoils accumulate within each DSA loop. The preferential
E.
coli
removal
I>SA
of
negative
supewoils
topoisomerase 1 rwults
1))
in rapid
accumulation of positive supercoils on the I1S.A template of dimeric and multimeric plasmitl I1S.A. (2) The stable binding of lac repressor to DSA dur
mediated intermolecular binding Iwt\vtwl two monomeric IIXA molewles also restricts the rot:Itional diffusion of transcription-get~et,ate(l supwcoils. However, becauw of the faster rwtr of’ intramolecular binding rela,tive to intel~molrc~iil;~~~ binding. dimeric or multimerica pAO-SLO I)SA molecules. 1vhic.h (San br stably Imund 1)). lrrt repressor’ intramolecularl~~. are mow likeI>. t0
accumulate supercoils than are monomeric pAO-SLO
to the formation of a ternary complex (operator-lnr repressor
tetramer-operator)
may
create
new sites
for DSA topoisomerase 1 and hence increases the rate of relaxation of negative supercoils generated by transcription. For example. the formation of the ternary complex may partially unwind the I)SA near t.he operator site and allow entr\. of 1)S.A topoisomerase T. (11) lac repressor-mediated intermoleculw may be responsible fcr thr prrjerentinl of positiw supercoils on monomrric (the monomer qffect)
nyyruyntio,t rtrcu mzrlntiots ll)sA1
When the tetramerica Inr repressor hinds to the single operator site on monomericDSA, the bound tetrameric lac repressor can only bind intermolwularly to another operator site. The lac repressor-
Figure 5. A model for lwal DSA super-coiling changes due to RX.4 transcription within Znc repressor-closed DSA loop domains. A tetrameric lrcc repressor hinds simultanrousl,v to 2 operator sites on a dimeric- I)?r’A molecule. Binding results in formation of 2 1)X23 loops. each of which cwntains a single bla. transcript.ion unit. These 2 stable DSA loops restrict fusion of positive and negative supercoils generated by RSA transcription from each of the bin genes.
Local
DNA
Conformation
D?r’A molecules. which can only be stably bound 1)) lnc repressor intermolecularly. The preferential accumulation of positive supercoils on dimeric pAOSLO l>KA in gyrase-inhibited cells is therefore due to the relatively higher population of dimeric l)SA in the hound form. Our recent studies of interlocked dimeric plasmid DSA populations in I’$. co/i provide further support that lac repressor can mediate aggregation of two plasmid DNA molecules inter molecularly (H.-Y. Wu & L. F. Liu, unpublished results).
Changes
\Ve are grateful to Drs Annette Bodley. Peter. D’Arpa. Hyeon-sook Koo. Erasmus Schneider and Mr Kawai Lau for critical readings of the manuscript. Stefan Oehler and Benno Miiller-Hill for providing their plasmid constructs. This work was supported by an PIU’IH grant GM27731. L.F.L. is a recipient of the AC’S Faculty Research Award.
References Brnyajati. C. & \Vorcel. A. (1976). Isolation characterization and structure of the folded interphase genome of Drosophila
((2) .3 new methodology for drtecting protein-mediated DXA looping in vivo Evidence that the lac repressor mediates DNA loop formation by simultaneous and co-operative binding to two lac operators has been amply documented from in vitro studies (Hsieh et al., 1987; O’Gorman rt al., 1980; Whitson & Matthews, 1986). Studies in GPO have also suggested that a DSA loop is formed by a stable lac operator-repressoroperator (pseudo-operator) complex (Besse et al., 1986). Our present studies provide further evidence that the lac repressor can mediate DSA loop formation over a long distance (about 2-3 kb) in viva. The multimer effect has also been observed in different plasmid constructs in which the lac repressor binding site is inserted into various locations (either 5 or 3’ end) relative to the transcription unit (either the /J-lactamase or the tetA gene transcription unit; H.-Y. Wu & L. F. Liu, unpublished results). A weaker multimer effect has also been observed for pBR322 DNA derivatives containing the tetA transcription unit (Wu et al., 1988) and for a 2 pm based plasmid DXA in yeast (Giaever & Wang. 1988). Whether these plasmid 1)X& also form DXA loops in their multimeric plasmid D?r’A populations remains to be tested. Our present studies suggest a possible new methodology for detecting protein-mediated DSA looping in vivo. (d)
DSA
looping
affects
local
DNA
supercoiling
Recent studies have suggested possible local variations in the degree of DSA supercoiling on cellular DXAs during active R?r’A transcription (Wu et aE., 1988: Brill & Sternglanz, 1988; Giaever & \Vang. 1988: Rahmouni & Wells, 1989; Lodge et al., 1989; Koo et al.. 1990). Our present studies indicate that protein-mediated DXA looping, which presumably mimics chromosomal DXA loops anchored at the attachment sites, can be an important parameter affecting the local supercoiling state of cellular DXA. Although the multimer effect was observed in gyrase-inhibited cells, studies in E. coli AS1 9 not treated with gyrase inhibitors also showed a slight relative increase in linking number of the multimeric pAO-Sl,O DSA relative to that of the monomeric pAO-SLO DXA (unpublished results). \Vhether altered local supercoiling due to DXA looping affects the physiological functions of DNA remains to be tested.
621
melanogaster.
Cell,
9. 393-407.
Besse, M., v. Wilcken-Bergmann, B. & Miiller-Hill, B. (1986). Synthetic Zuc operator mediates repression through Zac repressor when introduced upstream and downstream from Zac promoter. EMBO J. 5, 1377-1381. Brill, S. .J. & Sternglanz. R. (1988). Transcriptiondependent DEA superroiling in yeast DXA toJ)oisomerase mutants. Cell, 54. 403-41 I. Casadaban. M. J. & (‘ohen, S. N. (1980). Analysis of gene control signals by DKA fusion and c*loning in Escherichia coli. .I. Mol. Biol. 138. 179.-207. Cozzarelli. S. R. (1980). DNA topoisomrrasr. (‘~11. 22. 327.-328. Gellert. M. (1981). DXA topoisomrrase. .-lnn~. Krv. Biocham. 50. 879-910. Giaever, G. K;. & b’ang. ?J. (‘. (1988). Supercoiling of intracellular DXA can oprur in eukaryotiv cells. (‘rll. 55. 849-856. Gilbert. W. & Maxam. A. (1973). The nuc*leotidr sequencv of the lac operator. Proc. Sat. Acad. Sri.. I’.N.A. 70. 3!%&3Fi84. Gross. I). S. & Gerrard. W. T. (1988). Xuclease hypersensitive sites in chromatin Annu. JZw. Biochem. 57. 1,59-197. Hsieh. W.-T., Whitson. P. A.. Matthews. K. S. & Wells. R. I). (1987). Influence of sequrnc~~ and distance between two operators on interaction with the la,c rrprrssar. .J. Biol. (‘hrnz. 262. 14583~14.‘91. Koo. H.-S.. Wu. H.-Y. & Liu. F. 1,. ( 1990). Effect of transcription and translation on gyrase-mediated DNA cleavage in Escherichiu coli. .J. Bid. (‘hem. 265. l2300-12305. Lehming, X.. Sartorius.
(1991)
219, 615-622
DNA Looping Alters Local DNA during Transcription Ha&Young
Conformation
Wu and Leroy F. Liut
Department The Johns Hopkins Baltimore, (Received 23 May
of Biological Chemistry University, School of Medicine MD 21205, U.S.A. 1990; accepted 26 February
1991)
The effect of protein-mediated DNA looping on local DNA conformation during active transcription was studied using the Zac repressor-operator system. Our results suggest that lac repressor-mediated DNA looping within a plasmid DNA molecule containing two lac repressor binding sequences in viva effectively separates plasmid DNA into two topological domains. Supercoils generated by transcription within each topological domain can be rapidly removed by DNA topoisomerase I. Keywords:
DNA
supercoiling;
DNA looping; DNA topoisomerases; RNA transcription
1. Introduction
from a number of observations. While the nature of the cellular components holding the loops is not completely known, each chromosomal DNA loop has been shown to be an independent topological domain (Stonington & Pettijohn, 1971; Worcel & Burgi, 1972; Benyajati & Worcel, 1976; Paulson & Laemmli, 1977; Marsden & Laemmli, 1979; Vogelstein et al., 1980; for a review, see Gross & Gerrard, 1988). The possibility that a looped DNA domain may co-ordinately regulate chromatin structure and gene expression in higher eukaryotes has also been speculated (Weintraub, 1985). According to the twin-supercoiled-domain model of RNA transcription, the chromosomal DNA loop attachment sites can effectively restrict the rotational diffusion of supercoils generated by RNA transcription (Liu & Wang, 1987). These chromosomal DNA loop attachment sites therefore may affect the local supercoiling state of chromosomal DNA in the vicinity of the attachment sites. In order to study the possible effect of chromosomal DXA loop attachment sites on local DNA conformation, we have modeled the chromosomal DNA loop attachment sites using the Zac repressoroperator system. lac repressor has been shown to loop DISA in vitro and probably in viwo as well (Besse et al., 1986; Hsieh et al., 1987; O’Gorman et al.: 1980; Whitson & Matthews, 1986). In the present communication, we show that transcription within each of the DNA loops closed by lac repressor-operator complex significantly alters the local supercoiling state of DNA.
Supercoiling has been recognized as an important structural determinant for many DXA functions (for a review, see Cozzarelli, 1980; Gellert, 1981; Rowe & Liu, 1984; Wang, 1985). Recent studies have suggested that in addition to DNA topoisomerase. RNA transcription can also significantly alter the supercoiling state of cellular DNA (Pruss & Drlica, 1986, 1989; Brill & Sternglanz, 1988). However, the mechanism through which RNA transcription, and possibly other cellular processes, effects DXA conformation is not completely understood. A model in which the process of RNA transcription generates positive and negative supercoils has been proposed (Liu & Wang, 1987), and additional support for the model has come from both in uivo and in vitro studies (Wu et al., 1988; Giaever & Wang, 1988; Tsao et al., 1989; Lodge et al., 1989; Koo et al., 1990). According to this model, local DNA supercoiling can be affected by a complex set of. parameters (Liu & Wang, 1987; Wu et al., 1988). One such parameter relates to the rotational diffusion pathway of supercoils generated by transcription (Liu & Wang, 1987). Protein (or other factor)enclosed DNA loop is expected to block such a rotational diffusion pathway of supercoils generated by transcription. The presence of chromosomal DNA loops in both prokaryotic and eukaryotic cells has been suggested t Butor addressed.
to whom
0022-2836/91/12061&08
all correspondence
$03.00/O
should
Zac repressor;
be 615
0
1991 Academic
Press Limited
H.-Y.
616
Wu and L. F. Liu
- IPTG
l
IPTG
put 19 bp)
(2686
PAO (2232
bp)
pAO-SLO bp)
(2274
Figure 1. Preferential supercoiling of multimeric~ plasmid DIVA requires the essential Inc repressor binding I)NA sequence in cis. IPTG (1 my) was added to E. coli AS19 harboring various plasmids (A: pL’(‘19; 1%:pA0: and (‘: pAOSLO) 15 min prior to the addition of novobiocin (100 rig/ml). Treatment was cont,inued for another 30 min before plasmid DXAs were isolated and analyzed by 2-dimensional gel electrophoresis. Plasmid DXAs isolated from E. coli AS19 with and without IPTG treatment prior to novobiocin treatment are shown on the left and right column of tht P-dimensional gel panels. respectively. Streak a and a’ marks the position of negatively supercoiled monomeric3 and dimeric plasmid DSA. respectively. Streak b and b’ marks the position of positively supercoiled monomrric~ and dimeric, plasmid DKA. respectively.
2. Materials and Methods (a)
Chemicals,
enzymes
and
plasmid
DiV.-ls
Chloroquine diphosphate. novobiocin. rifampicin. IPTGt and chloramphenicol were purchased from Sigma. Enzymes were purchased from various commercial sources. pA0 plasmid DKA was constructed by blunt-end ligation of large EcoRIIAvaI (2232 bp) fragment of pAT1.53 DPU’A (Twigg & Sherratt. 1980). pAO-SLO plasmid DKA was constructed by inserting a single copy of a 42 bp DKA oligomer which contains a 21 bp essential luc repressor binding sequence (Gilbert & Maxam. 1973) in the middle of a polylinker sequence. The following plast Abbreviations used: IPTG. isopropyl-8-n thiogalactoside; bp. base-pair(s); kb. lo3 base-pairs.
mids are generous gifts from St~efan Orhlrr and I~IIIIO Miiller-Hill of Institute of Genetics, University of Kiiln, Germany; pSOlOO0 and pSOlO0 are low copy number plasmids (pACYC based), which carry the wild-type lac/ gene and the mutated EacI gene (iadi) (Lehming et al.. 1987), respectively. Roth genes are expressed via the iq promoter (Oehler et al., 1990). (b)
Ba,ctrria,t
strains
amf
crll
yrowfh
Escherichia coli AS19 is a 13 strain isolated for its D and other antibiotics. permeability to actinomycin including novobiocin (Sekiguchi & Iida. 1969). E. w/i MC1060 {A(lacI- Y)74. galEl.5. galKl6. i-. rdA I, rpsLl50. spoTI. hsdR2) a la,c repressor deletion strain ((‘asadaban & Cohen. 1980), was obtained from I)r Hamilton Smith (Johns Hopkins University). Transformants \~rre grown
Local
DNA
Conformation
in LB to early log phase(A,,, = @15)prior to inhibitor treatments. (c) Tluo-dinzensional gel electrophoresis Plasmid Dru’As were isolated by the alkaline lysis method as described(Wu et al.. 1988).Topoisomerdistributions of DNA sampleswere then analyzed by d-dimensional gel electrophoresis(Wu et al., 1988). The second dimension was carried out in gel electrophoresisbuffer containing 3 PM-chloroquine. Dried slab gels were then prepared for in situ Southern hybridization as described (Wu et al.. 1988).
3. Results The effect of transcription on local DNA conformation can be studied by monitoring the linking number of a plasmid DNA in bacterial cells treated with gyrase inhibitors (Wu et al., 1988). When DKA topoisomerase II (gyrase) is not available to relax positive supercoils generated ahead of the transcription complex and/or to introduce negative supercoils, positive supercoils on a transcribing plasmid DKA accumulate due to selective relaxation of transcription-generated negative supercoils by topoisomerase I (o protein). Thus, the rate of accumulation of positive supercoils on a plasmid DNA may reflect various parameters that affect transcriptiongenerated supercoils. (a) The accelerated rate of accumulation of positive .supercoilaon plasmid DNAs containing lac repressor
binding
sequences
The effect of protein-mediated DXA looping on DNA conformation during transcription was initia,lly suggested from studies of the rate of accumulation of positive supercoils on a number of pBR322 derivatives (Ll’u et al.. 1988). A pRR32Z derivative, pA0 DiVA, which contains only the h/n gene as the major transcription unit (see Fig. 1). accumulated positive supercoils at a much lower rate than did pBR322 DNA in E. coli AS19 cells treated with novobiocin. After a 30 minute novobiocin treatment, pA0 DNA was isolated as a of topoisomers as heterogeneous population revealed by two-dimensional gel electrophoresis (Fig. 1; see a streak of topoisomers in panel B, -1PTG). Only a very small population (less than 20%) of pA0 DNA was slightly positively supercoiled whereas under identical conditions, nearly all pBR322 DNA was highly positively supercoiled (Wu et al., 1988). The reduced rate of accumulation of positive supercoils on pA0 DNA is presumably due to the lack of the stronger transcription unit, the tetA gene. When the rate of accumulation of positive supercoils on pUC19 DNA, which is identical with pA0 DNA except for a 445 bp insertion containing the lac promoter, was compared with that of pA0 DNA, two surprising findings were noted; first monomeric plasmid DNA populations of pUC19 accumulated positive supercoils at a faster
Changes
617
rate than the corresponding monomeric populations of pA0 DNA (Fig. 1; seea streak of topoisomers in panel A, -1PTG). (This effect will be referred to as the “monomer effect”.) Second, the rate of accumulation of positive supercoils on dimeric and multimerit pUCl9 DNA populations (marked by b’ for the dimer population) was even faster than that of the monomeric pUCl9 DNA (marked by b; Fig. lA, - IPTG); this effect will be referred to as the “dimer or multimer effect”). Under our experimental conditions, only 40% of the monomeric pUCl9 DNA topoisomers were positively supercoiled, while nearly all the dimeric and multimeric pUCl9 DNA topoisomers were positively supercoiled (Fig. lA, - IPTG) . Both the monomer effect and the dimer (or multimer) effect were sensitive to TI’TC: treatment. The faster rate of accumulation of positive supercoils on dimeric pc’Cl9 DSA was c*ompletel> abolished by IPTG treatment (compare 1)’ in Fig. 1A (-IPTG) and b’ in Fig. 1A (+IPTG)). A decrease in the positively supercoiled population of monomeric pUCl9 DNA topoisomers upon IPTG treatment was also reproducibly observed (Fig. IA; compare streak b (-IPTG) with streak b ( + IPTG)). In addition, the relative proportion of positively supercoiled DNA population appeared to be about the same for both monomeric and dimeric pUCl9 DNA upon IPTG treatment (Fig. 1A (IPTG)). In contrast, IPTG treatment had no effect on the relative distribution of pA0 DNA topoisomer populations (Fig. 1B; compare topoisomer streaks with and without IPTG treatment). In fact, upon IPTG treatment, pUCl9 DNA accumulated positive supercoils at the same rate as did pA0 DNA. The effect of IPTG on the accumulation of positive supercoils in the dimeric and monomeric pUCl9 DNA populations suggested that either the binding of the lac repressor to the operator or the state of RNA transcription from the lac promoter might be responsible. To distinguish between two possibilities, a 42 bp synthetic DXA containing a single lac repressor binding site (a 21 bp lac operator sequence, which is a near-perfect palindrome; Gilbert & Maxam, 1973) was inserted into the AatII site of pA0 DXA (see Fig. lC, pAO-SLO DNA). The new plasmid DNA, pAO-SLO DXA, which does not contain the lac promoter but does contain the lac repressor binding sequence: exhibited a similar response in E. coli AS19 cells treated with novobiocin. First, the rate of accumulation of positively supercoiled monomeric pAO-SLO DXA increased relative to that of pA0 DSA (Fig. 1, compare streak b in C ( - TPTG) with streak b in B (-IPTG)). Second, dimeric (or multimerit) pAO-SLO DXA accumulated positive supercoils significantly faster than its monomeric counterpart (Fig. 1C; compare b’/a’ with b/a). Both the monomer effect and the dimer effect were again effectively eliminated by IPTG treatment (compare b’/a’ in Fig. 1C (-IPTG) and Fig. 1C (+IPTG)). These results suggest that both the monomer effect and the multimer effect are mediated by the binding
618
H.-Y.
Wu and L. F. Liu
2nd D
Figure
2. Lack of preferential supercoiling of multimeric plasmid I)N;As in a /UC repressor deletion strain of’ /!‘. co/i. MC1060. a luc repressor deletion strain (Casadaban & Cohen. 1980). harboring pAO-SLO was treated bvith novobiocin (500 pg/ml) and oxolinir acid (100 pg/ml) for 30 min. DNA was isolated and analyzed by 2-dimensional gel elertrophoresis. A: no IPTG treatment. B: TPTG (1 mM) (see Fig. 1 for IPTG treatment). Each group of USA topoisomers is labeled with a number near its upper apex of the triangle patt,ern. 1. 11. TIT and IV indicate the monomeric. dimeric. trimerir and tetrameric topoisomer populations of pAG-ST,0 I)NA. respectively. E. coli
of the lac repressor transcription from
to the operator but not by RNA the lac promoter.
(b) The accelerated rate of accumulation of positive supercoils on plasmid DNAs requires lac repressor in trans To strengthen our argument, E. coli MC1060 {A(lacI-Y)74, galE15, GalK16, I-, relAl, rpsLl50, spoT1, hsdR2) (a lac repressor deletion strain; (‘asadaban & Cohen, 1980) was used as a host As expected, pAO-SLO instead of E. coli ASl9. DNA isolated from E. coli MC1060 treated with novobiocin showed neither the monomer effect (compare the topoisomer distribution in Fig. 2A with that in Fig. 2B) nor the multimer effect (compare b/a of topoisomer group I, II, III and IV in Fig. 2A). These results suggested that the presence of lac repressor in cells was necessary both for the increased rate of accumulation of positive supercoils on monomeric plasmid DNA populations of pAO-SLO (the monomer effect) and for the preferential accumulation of positive supercoils in the multimeric plasmid DNA populations (the multimer effect). We have also introduced lac repressor into E. coli MC1060 via a pACYC-based plasmid, pSO1000, which is compatible with pAO-SLO. pSOlOO0
expresses wild-type lac repressor fin iq promoter (Oehler et al.. 1990). The presence of pSOlOO0 in E. coli MCI060 enhanced the extent of positive supercoiling of pAOSLO DNA (Fig. 3A, marked I (monomer), II (dimer) and TIT (trimer)). The effect of pSOlOO0 on the extent of positive supercoiling of the trimeric form of pAO-SLO DNA was most prominent. However, the effect of pS01000 on extent of positive supercoiling of the dimeric form of in pAO-SLO DNA was less prominent than that We do not quite understand this E. coli ASl9. phenomenon. Tt is possible that overexpression of lac repressor as in E. coli MCI060 harboring pSOlOO0 may inhibit the extent of positive supercoiling of the plasmid DNA. We have also expressed tnutated Inc repressor. which is defective in tet,ramerization but retains DNA binding activity (Lehming Pt a/.. 1987). pSOlO0, which expresses the rnutat’ed (dimeric, active) luc repressor via iq promoter, is also pA(‘Y(‘based. E. coli MC1060 harboring both pHO100 and pAO-SLO did not show any preferential positive supercoiling of the multimeric form of pAO-SLO in novobiocin-treated cells. These resuits toget)hcr is suggest that, the lac repressor binding to operator insufficient to cause preferential positive superof plasmid 1)NA. coiling of multimeric forms lo Simultaneous binding of t,etrameric lac repressor two operator sites is necessary to effect this process.
Local
DNA
Conformation
619
Changes
2nd D
2nd D
\
C Figure 3. Preferential supercoiling of multimeric plesmid DNA requires wild-type Zac repressor in. trans. E. coli MC1060, a Zac repressor deletion strain (Casadaban & Cohen, 1980), harboring both pAO-SLO and 1 of the 2 plasmids pSOlO60 (A) or pSOl66 (B), was treated with novobiocin (500 pg/ml) and oxolinic acid (166 pg/ml) for 30 min. DNAs were then isolated and analyzed by 2-dimensional gel electrophoresis. Each group of DNA topoisomers is labeled with a number near its upper apex of the triangle pattern. I, II and III indicate the monomeric, dimeric and trimeric topoisomer populations of pAO-SLO DNA, respectively. Ie and IIe indicate the monomeric and dimeric topoisomer populations of plasmid DNAs expressing wild-type lac repressor, pSOl660 DNA (A) or mutated Zac repressor (defective in tetramerization), pSOl66 DNA (B). C is a schematic diagram illustrating a typical distribution of DNA topoisomers in this 2-dimensional gel electrophoresis system. In each triangular pattern of topoisomer distribution, (a) marks the topoisomers with lowest linking number (high degree of negative superhelicity). (b) marks the topoisomers with highest linking number (high degree of positive supercoiling). (c) marks the topoisomers with intermediate linking number (relaxed and moderately supercoiled DNA).
(c) The preferential accumulation of positive supercoils on multimeric DNA is dependent on coupled transcription/translation Previous studies have suggested that transcription is responsible for the accumulation of positive supercoils on plasmid DNA in cells treated with gyrase inhibitors (Wu et al., 1988). Furthermore, coupled transcription/translation is expected to have an even stronger effect on template supercoiling (Koo et al., 1990; Liu & Wang, 1987; Lodge et al., 1989). To test whether the observed monomer and multimer effects on plasmid DXAs containing lac repressor binding sequences required coupled transcription/translation, an inhibitor of either RNA or protein synthesis was simultaneously added to E. coli AS19 cells during novobiocin treatment. As shown in Figure 4, either rifampicin (Fig. 4B) or chloramphenicol (Fig. 4C) treatment reduced the rate of accumulation of positively supercoiled mono merit populations of pAO-SLO DNA. In addition. the preferential accumulation of positive supercoils on dimeric plasmid DNA populations was also abolished (compare b’/a’ with b/a in Fig. 4B and C). The effect of rifampicin (90 pg/ml) was stronger than that of chloramphenicol (206 pg/ml) compare b’/a’ in Fig. 4B and C). In the presence of rifampicin, the majority of the dimeric pAO-SLO DNA molecules were negatively rather than positively supercoiled. Tn the presence of chloramphenicol, roughly 60% of the dimeric pAO-SLO DNA remained positively supercoiled. These results
suggest that coupled transcription/translation responsible for the preferential accumulation positive supercoils on dimeric plasmid DiVAs.
is of
4. Discussion (a) lac repressor-mediated DNA looping responsible for the preferential accwnulation positive supercoils on multimerir DNA (the multimer effect)
is of
A model that can explain our results is shown in Figure 5. In this model, a tetrameric lac repressor complex binds simultaneously and co-operatively to two operator sites that are diametrically opposed on a dimeric pAO-SLO DNA molecule (Hsieh et aE., 1987; O’Gorman et al., 1980; Whitson & Matthews, 1986). This simultaneous and c-o-operative binding to two operator sites intramolecularly results in the formation of two stable D;I’A loops. each of which contains a single bla transcription unit (ori-related transcripts may also contribute). The faster rate of accumulation of positive supercoils on multimeric 1)X,4 in gyrase-inhibited cells mav be due to one or both of the following two possibihties. (1) The rotational diffusion pathway for the fusion of positive and negative supercoils generated by caoupled transcription/translation of each bin gene is restricted due to the formation of this ternary cornpIes. To fuse the positive and negative super(oils generated by transcript,ion, the entire DSX loop with thr transcribing RNA polymerase c$omplrxes has to
If.- Y. Wu and L. F. Liu
2nd D B
>
C
Figure 4. Active RSA and protein synthesis are required for preferential awumulation of positive supercoils on /C/C(200 pg/ml) was added to E. co/i ASI9 oE)erator-containirlg plasmid DSAs. Rifampicin (90 pg/ml) or chloramphenicol harboring pBO-ST,0 10 min prior to the addition of novobiocin (100 pg/ml). T rratment was cwntinurcl for another 30 min before plasmid DSAs were isolated and analyzed by d-dimensional gel rlwtrophoresis. A: cw>trol. nowbiwitl treatment only: B: vo-treatment with rifamyicin: and C”: co-t.reatment wit.h chloramphenicol. Spot (3 marks thr position (ii’ monomeric pA@SLO DSA that had been irreversibly denatured during I)K;A isolation. Streak d’ denotes the Iwsitioll 01’ the interlocked USA population.
rotate and pass through the enclosed space within the other DSA loop. Due to the difficulties of such a rotational diffusion process. positive and negative supercoils accumulate within each DSA loop. The preferential
E.
coli
removal
I>SA
of
negative
supewoils
topoisomerase 1 rwults
1))
in rapid
accumulation of positive supercoils on the I1S.A template of dimeric and multimeric plasmitl I1S.A. (2) The stable binding of lac repressor to DSA dur
mediated intermolecular binding Iwt\vtwl two monomeric IIXA molewles also restricts the rot:Itional diffusion of transcription-get~et,ate(l supwcoils. However, becauw of the faster rwtr of’ intramolecular binding rela,tive to intel~molrc~iil;~~~ binding. dimeric or multimerica pAO-SLO I)SA molecules. 1vhic.h (San br stably Imund 1)). lrrt repressor’ intramolecularl~~. are mow likeI>. t0
accumulate supercoils than are monomeric pAO-SLO
to the formation of a ternary complex (operator-lnr repressor
tetramer-operator)
may
create
new sites
for DSA topoisomerase 1 and hence increases the rate of relaxation of negative supercoils generated by transcription. For example. the formation of the ternary complex may partially unwind the I)SA near t.he operator site and allow entr\. of 1)S.A topoisomerase T. (11) lac repressor-mediated intermoleculw may be responsible fcr thr prrjerentinl of positiw supercoils on monomrric (the monomer qffect)
nyyruyntio,t rtrcu mzrlntiots ll)sA1
When the tetramerica Inr repressor hinds to the single operator site on monomericDSA, the bound tetrameric lac repressor can only bind intermolwularly to another operator site. The lac repressor-
Figure 5. A model for lwal DSA super-coiling changes due to RX.4 transcription within Znc repressor-closed DSA loop domains. A tetrameric lrcc repressor hinds simultanrousl,v to 2 operator sites on a dimeric- I)?r’A molecule. Binding results in formation of 2 1)X23 loops. each of which cwntains a single bla. transcript.ion unit. These 2 stable DSA loops restrict fusion of positive and negative supercoils generated by RSA transcription from each of the bin genes.
Local
DNA
Conformation
D?r’A molecules. which can only be stably bound 1)) lnc repressor intermolecularly. The preferential accumulation of positive supercoils on dimeric pAOSLO l>KA in gyrase-inhibited cells is therefore due to the relatively higher population of dimeric l)SA in the hound form. Our recent studies of interlocked dimeric plasmid DSA populations in I’$. co/i provide further support that lac repressor can mediate aggregation of two plasmid DNA molecules inter molecularly (H.-Y. Wu & L. F. Liu, unpublished results).
Changes
\Ve are grateful to Drs Annette Bodley. Peter. D’Arpa. Hyeon-sook Koo. Erasmus Schneider and Mr Kawai Lau for critical readings of the manuscript. Stefan Oehler and Benno Miiller-Hill for providing their plasmid constructs. This work was supported by an PIU’IH grant GM27731. L.F.L. is a recipient of the AC’S Faculty Research Award.
References Brnyajati. C. & \Vorcel. A. (1976). Isolation characterization and structure of the folded interphase genome of Drosophila
((2) .3 new methodology for drtecting protein-mediated DXA looping in vivo Evidence that the lac repressor mediates DNA loop formation by simultaneous and co-operative binding to two lac operators has been amply documented from in vitro studies (Hsieh et al., 1987; O’Gorman rt al., 1980; Whitson & Matthews, 1986). Studies in GPO have also suggested that a DSA loop is formed by a stable lac operator-repressoroperator (pseudo-operator) complex (Besse et al., 1986). Our present studies provide further evidence that the lac repressor can mediate DSA loop formation over a long distance (about 2-3 kb) in viva. The multimer effect has also been observed in different plasmid constructs in which the lac repressor binding site is inserted into various locations (either 5 or 3’ end) relative to the transcription unit (either the /J-lactamase or the tetA gene transcription unit; H.-Y. Wu & L. F. Liu, unpublished results). A weaker multimer effect has also been observed for pBR322 DNA derivatives containing the tetA transcription unit (Wu et al., 1988) and for a 2 pm based plasmid DXA in yeast (Giaever & Wang. 1988). Whether these plasmid 1)X& also form DXA loops in their multimeric plasmid D?r’A populations remains to be tested. Our present studies suggest a possible new methodology for detecting protein-mediated DSA looping in vivo. (d)
DSA
looping
affects
local
DNA
supercoiling
Recent studies have suggested possible local variations in the degree of DSA supercoiling on cellular DXAs during active R?r’A transcription (Wu et aE., 1988: Brill & Sternglanz, 1988; Giaever & \Vang. 1988: Rahmouni & Wells, 1989; Lodge et al., 1989; Koo et al.. 1990). Our present studies indicate that protein-mediated DXA looping, which presumably mimics chromosomal DXA loops anchored at the attachment sites, can be an important parameter affecting the local supercoiling state of cellular DXA. Although the multimer effect was observed in gyrase-inhibited cells, studies in E. coli AS1 9 not treated with gyrase inhibitors also showed a slight relative increase in linking number of the multimeric pAO-Sl,O DSA relative to that of the monomeric pAO-SLO DXA (unpublished results). \Vhether altered local supercoiling due to DXA looping affects the physiological functions of DNA remains to be tested.
621
melanogaster.
Cell,
9. 393-407.
Besse, M., v. Wilcken-Bergmann, B. & Miiller-Hill, B. (1986). Synthetic Zuc operator mediates repression through Zac repressor when introduced upstream and downstream from Zac promoter. EMBO J. 5, 1377-1381. Brill, S. .J. & Sternglanz. R. (1988). Transcriptiondependent DEA superroiling in yeast DXA toJ)oisomerase mutants. Cell, 54. 403-41 I. Casadaban. M. J. & (‘ohen, S. N. (1980). Analysis of gene control signals by DKA fusion and c*loning in Escherichia coli. .I. Mol. Biol. 138. 179.-207. Cozzarelli. S. R. (1980). DNA topoisomrrasr. (‘~11. 22. 327.-328. Gellert. M. (1981). DXA topoisomrrase. .-lnn~. Krv. Biocham. 50. 879-910. Giaever, G. K;. & b’ang. ?J. (‘. (1988). Supercoiling of intracellular DXA can oprur in eukaryotiv cells. (‘rll. 55. 849-856. Gilbert. W. & Maxam. A. (1973). The nuc*leotidr sequencv of the lac operator. Proc. Sat. Acad. Sri.. I’.N.A. 70. 3!%&3Fi84. Gross. I). S. & Gerrard. W. T. (1988). Xuclease hypersensitive sites in chromatin Annu. JZw. Biochem. 57. 1,59-197. Hsieh. W.-T., Whitson. P. A.. Matthews. K. S. & Wells. R. I). (1987). Influence of sequrnc~~ and distance between two operators on interaction with the la,c rrprrssar. .J. Biol. (‘hrnz. 262. 14583~14.‘91. Koo. H.-S.. Wu. H.-Y. & Liu. F. 1,. ( 1990). Effect of transcription and translation on gyrase-mediated DNA cleavage in Escherichiu coli. .J. Bid. (‘hem. 265. l2300-12305. Lehming, X.. Sartorius.
