Aug 9, 1993 - specific initiator, RepA, and the host initiator, DnaA. Here we show that DnaA .... Sequence of the top strand of P1 origin. Binding sites for DnaA ...
The EMBO Journal vol.12 no.12 pp.4547-4554, 1993
Open-complex formation by the host initiator, DnaA, at the origin of P1 plasmid replication
Gauranga Mukhopadhyay, Kevin M.Carr1, Jon M.Kaguni' and Dhruba K.Chattoraj2 Laboratory of Biochemistry, National Cancer Institute, NIH, Bethesda, MD 20892 and 'Department of Biochemistry, Michigan State University, East Lansing, MI 48824, USA 2Corresponding author Communicated by W.Messer
Replication of P1 plasmid requires both the plasmidspecific initiator, RepA, and the host initiator, DnaA. Here we show that DnaA can make the P1 origin reactive to the single-strand specific reagents KMnO4 and mung bean nuclease. Addition of RepA further increased the KMnO4 reactivity of the origin, although RepA alone did not influence the reaction. The increased reactivity implies that the two initiators interact in some way to alter the origin conformation. The KMnO4 reactivity was restricted to one strand of the origin. We suggest that the roles of DnaA in P1 plasmid and bacterial replication are similar: origin opening and loading of the DnaB helicase. The strand-bias in chemical reactivity at the P1 origin most likely indicates that only one of the strands is used for the loading of DnaB, a scenario consistent with the unidirectional replication of the plasmid. Key words: DnaA/DNA-protein interactions/DNA replication/plasmid/RepA
Introduction P1 plasmid belongs to a distinct class of replicons whose initiation and control of initiation are mediated by iterons (Nordstrom, 1990). The plasmid is maintained at low copy numbers characteristic of the bacterial chromosome (Prentki et al., 1977; Pal et al., 1986; Austin and Eichorn, 1992). Understanding the mechanism of this stringent mode of replication control has been the primary interest in studying the replication of the plasmid. There are five tandemly repeated iterons in the P1 origin, each 19 bp long (Abeles et al., 1984). These iterons are the binding sites for the plasmid-encoded initiator, RepA, both in vivo and in vitro (Abeles, 1986; Sozhamannan and Chattoraj, 1993). It has been postulated that the iterated sequences help to form a specialized nucleoprotein structure that has several advantages over single DNA-protein complexes (Echols, 1990). For events like DNA replication, the structure can provide increased site specificity, a signal for initiation utilizing both the tertiary structure of proteins and the threedimensional conformation of DNA and, finally, more opportunities for control. RepA binding bends the iterons and binding to all five iterons absorbs one positive superhelical turn of DNA, indicating that the DNA is wrapped around RepA (Mukhopadhyay and Chattoraj, 1993). How the information in the structure is used in
initiation and its control is not known, but it must serve to open the strands of the origin. DNA opening is an essential step in the initiation of the theta mode of replication, as it sets the stage for loading of the helicase, DnaB (Bramhill and Kornberg, 1988b). The subsequent events in initiation are not considered relevant to the control of initiation. From the right-handed wrapping of the origin DNA in RepA-DNA complexes, a compensatory turn of unwinding was expected, but the complexes did not show enhanced reactivity to a single-strand specific reagent, KMnO4 (Mukhopadhyay and Chattoraj, 1993). The failure to observe unwinding by RepA alone drew our attention to the bacterial initiator, DnaA. The protein is required for replication of P1 plasmid in vivo and in vitro (Hansen and Yarmolinsky, 1986; Wickner and Chattoraj, 1987). The P1 origin has two sets of 9 bp sequences that are highly homologous to the boxes that bind DnaA (Hansen and Yarmolinsky, 1986; Abeles et al., 1990; Stenzel et al., 1991; see Holz et al., 1992 for a review). One set of two tandem boxes maps at the left end of the origin and a second set of three tandem boxes maps at the right end. Either set allows replication, although optimal replication requires both of the sets (Abeles et al., 1990). The origin of replication of Escherichia coli, oriC, has four discretely spaced DnaA boxes and binding of DnaA leads to opening of the origin DNA (Bramhill and Kornberg, 1988a). In the case of the plasmids Rl and pSC 101, the plasmid-encoded initiator is known to stimulate the origin binding activity of DnaA, suggesting that the two proteins could interact with each other (Masai and Arai, 1987; Stenzel et al., 1991; Ortega-Jimenez et al., 1992). In this study, we show that the DnaA protein stimulates the specific DNA binding activity of RepA and can open the strands of P1 origin. The DNA-opening reaction is stimulated when RepA protein was added together with DnaA. The initiators are thus used for two important functions: origin selection and DNA opening. These studies thus provide a rationale for the essential role of DnaA in P1 plasmid replication.
Results Binding of RepA and DnaA proteins to P1 origin The positions of the iterons and the DnaA boxes of the P1 origin are shown in Figure 1. RepA binding to the iterons has been characterized in detail (Abeles, 1986; Sozhamannan and Chattoraj, 1993). In comparison, little is known about the nature of binding of DnaA to the P1 origin. In this work, DnaA binding was studied on supercoiled DNA and assayed by DNase I footprinting. Specific protection of the DnaA boxes (coordinates 387-404) could be demonstrated on both strands of the DNA (Figure 2, lanes 3 and 7). In the presence of both RepA and DnaA, there was specific protection of both the iterons and the DnaA boxes (Figure 2, lanes 4 and 8). The two proteins could have influenced each other's 4547
G.Mukhopadhyay et al. 390 TTTCAC
TAT CCAC
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410 TATC
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CAC9GGaTAG aTCCAATAAT CAGGTCCATA 470
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CAGaTCCCAA
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TTAGaTCCAT ATAGaTCCCT GaTCGTTGCA GGCCGCGCCA CGTCTGGCTT AGAAGTGTAT 510
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CGCGaTGTGT GCTGGAGGGA-AAACGaTGTG TGCTGGAGGG
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CGGAAAAAAG TAATATGAAT
Fig. 1. Sequence of the top strand of P1 origin. Binding sites for DnaA protein are boxed; sites for Dam methylation are underlined. Arrows numbered from 10 to 14 indicate binding sites for P1-encoded RepA. Bold lower case letters represent bases whose complement in the bottom strand reacted to KMnO4 in the absence of proteins, while bold upper case letters represent bases whose complement in the bottom strand of pSP102 reacted preferentially to KMnO4 in the presence of DnaA and RepA (Figure 5, lanes 1-4). Asterisked bases preferentially reacted to DMS in the presence of RepA and DnaA (Figure 4).
binding as the protection was more complete. In the case of plasmid RI the protection by DnaA is also weak and improves dramatically upon addition of the plasmid initiator (Masai and Arai, 1987; Ortega-Jim6nez et al., 1992). Increased RepA binding to the iterons in the presence of DnaA was independently found by band-shift assay using linear DNA (Figure 3). DnaA binding could not be reliably demonstrated by this technique (Figure 3, lane 2) but the stimulation of DnaA binding by RepA was indicated by the appearance of hypersensitive sites within the DnaA boxes upon treatment with DMS (Figure 4). From these studies we conclude that RepA and DnaA can bind to specific sites on supercoiled DNA and can weakly influence each other's binding. KMnO4 reactivity of P1 origin In these experiments, supercoiled DNA and the two initiator proteins were used either singly or in combination and the complexes were probed with KMnO4 for unwinding of the origin region. KMnO4 reacts with pyrimidines (T > > C) in unstacked DNA and the reagent has been used widely to study open-complex formation in transcription (Sasse-Dwight and Gralla, 1989; Kainz and Roberts, 1992; Suh et al., 1993) and in replication (Gille and Messer, 1991). In a previous study, the bases that reacted in the absence of protein were primarily thymines in 5'-ATC-3' triplets (Mukhopadhyay and Chattoraj, 1993). In the presence of DnaA, the ATC triplet sites were no longer preferred and the reactive bases were distributed over the entire origin region (Figure 5, lane 3). More DNA had to be added to lanes 3 and 4 than to lanes 1 and 2 so that Ts of the ATC triplets appear identical in all four lanes. The change in the reaction pattern became more pronounced when RepA was added together with DnaA (lane 4). By itself RepA had an almost negligible influence on the cleavage pattern by KMnO4, as was found earlier (Mukhopadhyay and Chattoraj, 1993). pSP102, the plasmid used above, carries both sets of DnaA boxes that flank the origin. Either set suffices for initiation in vivo (Abeles et al., 1990). In order to determine the requirement of the boxes for the observed increase in KMnO4 reactivity in vitro, we examined pALA63 1, pALA657 and pALA646. Increased reactivity was most pronounced in plasmid pSP102, followed by the plasmids 4548
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