Missing-base and ethylation interference footprinting of P1 plasmid replication initiator.

RepA, the replication initiator protein of plasmid P1, binds to specific 19 bp sequences on the plasmid DNA. Earlier footprinting studies with dimethylsulfate identified the guanines that contact RepA through the major groove of DNA. In this study, base elimination was used to identify the contribution of all four bases to the binding reaction. Depurination and depyrimidation of any base in the neighborhood of the contacting guanines was found to decrease RepA binding. These results are consistent with the notion that RepA contacts bases of two consecutive major grooves on the same face of DNA. We also observed that depurination but not methylation of three guanines (G3, G8 and G9) affected binding. We identified the DNA phosphate groups (3 in the top strand, one of which mapped between G8 and G9, and 4 in the bottom strand, one of which was adjacent to C3) that strongly interfered with RepA binding upon ethylation. These results indicate that certain bases (e.g. G3, G8 and G9) may not contact RepA directly but contribute to base and backbone contacts by maintaining proper structure of the binding site.     

Uracil interference, a rapid and general method for defining protein-DNA interactions involving the 5-methyl group of thymines: the GCN4-DNA complex.

We describe a novel uracil interference method for examining protein contacts with the 5-methyl group of thymines. The protein of interest is incubated with target DNA containing randomly distributed deoxyuracil substitutions that is generated by carrying out the polymerase chain reaction in the presence of a mixture of TTP and dUTP. After separating DNA-protein complexes away from unbound DNA, the locations of deoxyuracil residues that either do or do not interfere with DNA-binding are determined by cleavage with uracil-N-glycosylase followed by piperidine. Using this uracil interference assay, we show that the methyl groups of the four core thymines, but not the two peripheral thymines, of the optimal binding site (ATG-ACTCAT) are important for high affinity binding of GCN4. Similar, but not identical, results are obtained using KMnO4 interference, another method used for studying protein-DNA interactions involving thymine residues. These observations strongly suggest that GCN4 directly contacts the 5-methyl groups of the four core thymines that lie in the major groove of the target DNA. Besides providing specific structural information about protein-DNA complexes, uracil interference should also be useful for identifying DNA-binding proteins and their target sites in eukaryotic promoter regions.     

Plasmid P1 RepA is homologous to the F plasmid RepE class of initiators

DNA replication of plasmid P1 requires a plasmid-encoded origin DNA-binding protein, RepA. RepA is an inactive dimer and is converted by molecular chaperones into an active monomer that binds RepA binding sites. Although the sequence of RepA is not homologous to that of F plasmid RepE, we found by using fold-recognition programs that RepA shares structural homology with RepE and built a model based on the RepE crystal structure. We constructed mutants in the two predicted DNA binding domains to test the model. As expected, the mutants were defective in P1 DNA binding. The model predicted that RepA binds the first half of the binding site through interactions with the C-terminal DNA binding domain and the second half through interactions with the N-terminal domain. The experiments supported the prediction. The model was further supported by the observation that mutants defective in dimerization map to the predicted subunit interface region, based on the crystal structure of pPS10 RepA, a RepE family member. These results suggest P1 RepA is structurally homologous to plasmid initiators, including those of F, R6K, pSC101, pCU1, pPS10, pFA3, pGSH500, Rts1, RepHI1B, RepFIB, and RSF1010.     

Participation of the Escherichia coli heat shock proteins DnaJ, DnaK, and GrpE in autorepression of the P1 plasmid repA promoter

The replicon of the low copy number plasmid P1 uses the three Escherichia coli heat shock proteins DnaJ, DnaK, and GrpE for the efficient initiation of its DNA replication. The only P1-encoded protein required for plasmid replication is the initiator, RepA. Binding of RepA to the origin also represses the promoter for the repA gene, which is located within the origin. We found that repression is incomplete in E. coli strains with mutations in the dnaJ, dnaK, or grpE genes. Since there is no decrease in RepA concentration in the mutant strains, the mutations are likely to affect the protein-DNA or protein-protein reactions required for repression, thereby decreasing RepA binding at its promoter. We also showed that the deficit in repression can be overcome by providing excess RepA, implying that the mechanism of repression is not altered in the mutant strains. Since repression requires RepA binding to the origin, a binding deficit might account for the replication defect in the heat shock mutants.     

The P1 phage replication protein RepA contacts an otherwise inaccessible thymine N3 proton by DNA distortion or base flipping

The RepA protein from bacteriophage P1 binds DNA to initiate replication. RepA covers one face of the DNA and the binding site has a completely conserved T that directly faces RepA from the minor groove at position +7. Although all four bases can be distinguished through contacts in the major groove of B-form DNA, contacts in the minor groove cannot easily distinguish between A and T bases. Therefore the 100% conservation at this position cannot be accounted for by direct contacts approaching into the minor groove of B-form DNA. RepA binding sites with modified base pairs at position +7 were used to investigate contacts with RepA. The data show that RepA contacts the N3 proton of T at position +7 and that the T=A hydrogen bonds are already broken in the DNA before RepA binds. To accommodate the N3 proton contact the T₊₇ /A_+7^′ base pair must be distorted. One possibility is that T₊₇ is flipped out of the helix. The energetics of the contact allows RepA to distinguish between all four bases, accounting for the observed high sequence conservation. After protein binding, base pair distortion or base flipping could initiate DNA melting as the second step in DNA replication.     

Analysis of pCU1 replication origins: dependence of oriS on the plasmid-encoded replication initiation protein RepA

The broad-host-range replicon of the plasmid pCU1 has three origins of vegetative replication called oriB, oriS, and oriV. In the multi-origin replicon, individual origins can distinguish among replication factors provided by the host. It has been found that during replication in Escherichia coli polA(-) host, oriS was the only active origin of a mutant pCU1 derivative bearing a mutation in the gene encoding replication initiation protein RepA. To further investigate the capacity of oriS to function in an E. coli polA(-) host we constructed a number of clones of the basic replicon of pCU1 containing oriS as the only replication origin. An oriS construct created with pUC18 could transform the polA(-) strain when RepA was supplied in trans. When the oriS region (between nucleotides 290 and 832) was ligated to an antibiotic resistance Omega fragment, the construct could be recovered as a plasmid from polA(+) strain if functional RepA was provided in trans. Our results therefore indicate that the basic replicon of pCU1, containing oriS as the sole origin, does require RepA to initiate plasmid replication in E. coli     

Heat shock proteins DnaJ, DnaK, and GrpE stimulate P1 plasmid replication by promoting initiator binding to the origin.

Binding of the P1-encoded protein RepA to the origin of P1 plasmid replication is essential for initiation of DNA replication and for autoregulatory repression of the repA promoter. Previous studies have shown defects in both initiation and repression in hosts lacking heat shock proteins DnaJ, DnaK, and GrpE and have suggested that these proteins play a role in the RepA-DNA binding required for initiation and repression. In this study, using in vivo dimethyl sulfate footprinting, we have confirmed the roles of the three heat shock proteins in promoting RepA binding to the origin. The defects in both activities could be suppressed by increasing the concentration of wild-type RepA over the physiological level. We also isolated RepA mutants that were effective initiators and repressors without requiring the heat shock proteins. These data suggest that the heat shock proteins facilitate both repression and initiation by promoting only the DNA-binding activity of RepA. In a similar plasmid, F, initiator mutants that confer heat shock protein independence for replication were also found, but they were defective for repression. We propose that the initiator binding involved in repression and the initiator binding involved in initiation are similar in P1 but different in F.     

The DNA-binding domain of human c-Abl tyrosine kinase promotes the interaction of a HMG chromosomal protein with DNA.

The biological activity of the c-Abl protein is linked to its tyrosine kinase and DNA-binding activities. The protein, which plays a major role in the cell cycle response to DNA damage, interacts preferentially with sequences containing an AAC motif and exhibits a higher affinity for bent or bendable DNA, as is the case with high mobility group (HMG) proteins. We have compared the DNA-binding characteristics of the DNA-binding domain of human c-Abl and the HMG-D protein from Drosophila melanogaster. c-Abl binds tightly to circular DNA molecules and potentiates the interaction of DNA with HMG-D. In addition, we used a series of DNA molecules containing modified bases to determine how the exocyclic groups of DNA influence the binding of the two proteins. Interfering with the 2-amino group of purines affects the binding of the two proteins similarly. Adding a 2-amino group to adenines restricts the access of the proteins to the minor groove, whereas deleting this bulky substituent from guanines facilitates the protein-DNA interaction. In contrast, c-Abl and HMG-D respond very differently to deletion or addition of the 5-methyl group of pyrimidine bases in the major groove. Adding a methyl group to cytosines favours the binding of c-Abl to DNA but inhibits the binding of HMG-D. Conversely, deleting the methyl group from thymines promotes the interaction of the DNA with HMG-D but diminishes its interaction with c-Abl. The enhanced binding of c-Abl to DNA containing 5-methylcytosine residues may result from an increased propensity of the double helix to denature locally coupled with a protein-induced reduction in the base stacking interaction. The results show that c-Abl has unique DNA-binding properties, quite different from those of HMG-D, and suggest an additional role for the protein kinase.     

Protein domains and conformational changes in the activation of RepA, a DNA replication initiator.

RepA is the DNA replication initiator protein of the Pseudomonas plasmid pPS10. RepA has a dual function: as a dimer, it binds to an inversely-repeated sequence acting as a repressor of its own synthesis; as a monomer, RepA binds to four directly-repeated sequences to constitute a specialized nucleoprotein complex responsible for the initiation of DNA replication. We have previously shown that a Leucine Zipper-like motif (LZ) at the N-terminus of RepA is responsible for protein dimerization. In this paper we characterize the existence in RepA of two protein globular domains C-terminal to the LZ. We propose that dissociation of RepA dimers into monomers results in a conformational change from a compact arrangement of both domains, competent for binding to the operator, to an extended species that is suited for iteron binding. This model establishes the structural basis for the activation of DNA replication initiators in plasmids from Gram-negative bacteria.     

Critical base pairs and amino acid residues for protein-DNA interaction between the TyrR protein and tyrP operator of Escherichia coli.

In Escherichia coli K-12, the repression of tyrP requires the binding of the TyrR protein to the operator in the presence of coeffectors, tyrosine and ATP. This operator contains two 22-bp palindromic sequences which are termed TyrR boxes. Methylation, uracil, and ethylation interference experiments were used to identify the important sites in the TyrR boxes that make contacts with the TyrR protein. Methylation interference studies demonstrated that guanines at positions +8, -5, and -8 of the strong TyrR box and positions +8, -4, and -8 of the weak box are close to the TyrR protein. Uracil interference revealed that strong van der Waals contacts are made by the thymines at position -7 and +5 of the top strands of both strong and weak boxes and that weaker contacts are made by the thymines at positions +7 (strong box) and -5 and +7 (weak box) of the bottom strand. In addition, ethylation interference suggested that the phosphate backbone contacts are located at the end and central regions of the palindrome. These findings are supported by our results derived from studies of symmetrical mutations of the tyrP strong box. Overall, the results confirm the critical importance of the invariant (G x C)(C x G)8 base pairs for TyrR recognition and also indicate that interactions with (T x A)(A x T)7 are of major importance. In contrast, mutations in other positions result in weaker effects on the binding affinity of TyrR protein, indicating that these positions play a lesser role in TyrR protein recognition. Alanine scanning of both helices of the putative helix-turn-helix DNA-binding motif of TyrR protein has identified those amino acids whose side chains play an essential role in protein structure and DNA binding.     

Interaction of initiator proteins with the origin of replication of an IncL/M plasmid

The origin of replication of the IncL/M plasmid pMU604 was analyzed to identify sequences important for binding of initiator proteins and origin activity. A thrice repeated sequence motif 5'-NANCYGCAA-3' was identified as the binding site (RepA box) of the initiator protein, RepA. All three copies of the RepA box were required for in vivo activity and binding of RepA to these boxes appeared to be cooperative. A DnaA R box (box 1), located immediately upstream of the RepA boxes, was not required for recruitment of DnaA during initiation of replication by RepA of pMU604 unless a DnaA R box located at the distal end of the origin (box 3) had been inactivated. However, DnaA R box 1 was important for recruitment of DnaA to the origin of replication of pMU604 when the initiator RepA was that from a distantly related plasmid, pMU720. A mutation which scrambled DnaA R boxes 1 and 3 and one which scrambled DnaA R boxes 1, 3 and 4 had much more deleterious effects on initiation by RepA of pMU720 than on initiation by RepA of pMU604. Neither Rep protein could initiate replication from the origin of pMU604 in the absence of DnaA, suggesting that the difference between them might lie in the mechanism of recruitment of DnaA to this origin. DnaA protein enhanced the binding and origin unwinding activities of RepA of pMU604, but appeared unable to bind to a linear DNA fragment bearing the origin of replication of pMU604 in the absence of other proteins.     

Interactions of the RepA helicase hexamer of plasmid RSF1010 with the ssDNA. Quantitative analysis of stoichiometries, intrinsic affinities, cooperativities, and heterogeneity of the total ssDNA-binding site

Interactions between the replicative RepA helicase hexamer of plasmid RSF1010 with the single-stranded DNA (ssDNA) have been studied, using the quantitative fluorescence titration, analytical sedimentation velocity, and sedimentation equilibrium techniques. Experiments were performed with fluorescein-labeled ssDNA oligomers. Studies with unmodified ssDNA oligomers were accomplished using the macromolecular competition titration method. Analyses of RepA helicase interactions with a series of the ssDNA provide direct evidence that the total site-size of the RepA hexamer-ssDNA complex is 19 +/- 1 nucleotide residues. The total ssDNA-binding site of the hexamer has a heterogeneous structure. Part of the total binding site constitutes the proper ssDNA-binding site of the enzyme, an area that possesses strong ssDNA-binding capability and encompasses only 8 +/- 1 residues of the ssDNA. The statistical effect on the macroscopic binding constant for the proper ssDNA-binding site indicates that it is structurally separated from the remaining part of the total ssDNA-binding site. Engagement in interactions with the ssDNA is accompanied by net ion release. Moreover, the proper ssDNA-binding site shows little base specificity. On the other hand, with long ssDNA oligomers, the entire total ssDNA-binding site of the RepA hexamer engages in interactions with the ssDNA resulting in a dramatic change in the nature of interactions with the nucleic acid. The association includes an uptake of ions by the protein. Moreover, unlike the proper-ssDNA-binding site, the total binding site shows a significant preference for pyrimidine oligomers. In this aspect, the RepA helicase is different from the Escherichia coli DnaB hexamer that shows large preference for purine homo-oligomers. In similar solution conditions, the ssDNA intrinsic affinity of the RepA hexamer is similar to the intrinsic affinity of the DnaB helicase. The RepA helicase binds to ssDNA oligomers that can accept more than one RepA hexamer with significant positive cooperative interactions.     

Binding of sulphonated indigo derivatives to RepA-WH1 inhibits DNA-induced protein amyloidogenesis

The quest for inducers and inhibitors of protein amyloidogenesis is of utmost interest, since they are key tools to understand the molecular bases of proteinopathies such as Alzheimer, Parkinson, Huntington and Creutzfeldt–Jakob diseases. It is also expected that such molecules could lead to valid therapeutic agents. In common with the mammalian prion protein (PrP), the N-terminal Winged-Helix (WH1) domain of the pPS10 plasmid replication protein (RepA) assembles in vitro into a variety of amyloid nanostructures upon binding to different specific dsDNA sequences. Here we show that di- (S2) and tetra-sulphonated (S4) derivatives of indigo stain dock at the DNA recognition interface in the RepA-WH1 dimer. They compete binding of RepA to its natural target dsDNA repeats, found at the repA operator and at the origin of replication of the plasmid. Calorimetry points to the existence of a major site, with micromolar affinity, for S4-indigo in RepA-WH1 dimers. As revealed by electron microscopy, in the presence of inducer dsDNA, both S2/S4 stains inhibit the assembly of RepA-WH1 into fibres. These results validate the concept that DNA can promote protein assembly into amyloids and reveal that the binding sites of effector molecules can be targeted to inhibit amyloidogenesis.     

P1 plasmid replication. Purification and DNA-binding activity of the replication protein RepA

The minimal P1 replicon encompasses an open reading frame for the essential replication protein, RepA, bracketed by two sets of multiple 19-base pair repeated sequences, incA and incC. This study focused on the interaction of RepA with the incC and incA repeated sequences because earlier studies suggested that incA might control P1 copy number by titrating limiting amounts of RepA and because the incC repeats, which are part of the origin of replication, contain the promoter for repA. RepA is essential for origin function, autoregulates its own synthesis from the promoter, and, when overproduced, blocks origin function. In this study, RepA was overproduced from an expression vector and purified to 90% homogeneity. The binding of RepA to the DNA encompassing repeat sequences was assayed by monitoring the mobility of protein-DNA complexes on polyacrylamide gels. Distinct species of retarded bands were seen with the maximum number of bands corresponding to the number of repeats present in the target fragment. No evidence was found for RepA binding to fragments not containing the repeats. This suggests that the specific binding of RepA to the repeats may be involved in each of the diverse activities of RepA.     

The mechanism and control of DNA transfer by the conjugative relaxase of resistance plasmid pCU1

Bacteria expand their genetic diversity, spread antibiotic resistance genes, and obtain virulence factors through the highly coordinated process of conjugative plasmid transfer (CPT). A plasmid-encoded relaxase enzyme initiates and terminates CPT by nicking and religating the transferred plasmid in a sequence-specific manner. We solved the 2.3 Å crystal structure of the relaxase responsible for the spread of the resistance plasmid pCU1 and determined its DNA binding and nicking capabilities. The overall fold of the pCU1 relaxase is similar to that of the F plasmid and plasmid R388 relaxases. However, in the pCU1 structure, the conserved tyrosine residues (Y18,19,26,27) that are required for DNA nicking and religation were displaced up to 14 Å out of the relaxase active site, revealing a high degree of mobility in this region of the enzyme. In spite of this flexibility, the tyrosines still cleaved the nic site of the plasmid’s origin of transfer, and did so in a sequence-specific, metal-dependent manner. Unexpectedly, the pCU1 relaxase lacked the sequence-specific DNA binding previously reported for the homologous F and R388 relaxase enzymes, despite its high sequence and structural similarity with both proteins. In summary, our work outlines novel structural and functional aspects of the relaxase-mediated conjugative transfer of plasmid pCU1.     

Specific minor groove solvation is a crucial determinant of DNA binding site recognition

The DNA sequence preferences of nearly all sequence specific DNA binding proteins are influenced by the identities of bases that are not directly contacted by protein. Discrimination between non-contacted base sequences is commonly based on the differential abilities of DNA sequences to allow narrowing of the DNA minor groove. However, the factors that govern the propensity of minor groove narrowing are not completely understood. Here we show that the differential abilities of various DNA sequences to support formation of a highly ordered and stable minor groove solvation network are a key determinant of non-contacted base recognition by a sequence-specific binding protein. In addition, disrupting the solvent network in the non-contacted region of the binding site alters the protein''s ability to recognize contacted base sequences at positions 5–6 bases away. This observation suggests that DNA solvent interactions link contacted and non-contacted base recognition by the protein.     

A louse involved in the regulation of replication in plasmid pSC 101

The origin of replication of plasmid pSC101 contains three directly repeated sequences RS1, RS2, and RS3 separated by 22 bp from two palindromic sequences, IR1 and IR2, which are partially homologous to the direct repeats. These inverted repeat (IR) sequences overlap the promoter of the repA gene which encodes a protein essential for plasmid replication. We have shown that RepA binds to the RS sites as a monomer and to the IR sites as a dimer. The influence of the IR1 site, and of the DNA segment that separates it from RS3, on plasmid copy number control has been studied in detail. We show that the integrity of IR1 is essential for efficient replication and plasmid stability, the critical site extending to the left of IR1 proper. We also show that the presence of IR1 modifies profoundly the binding properties of purified RepA protein to a segment of DNA containing the RS sequences. IR1 is separated from its homologous site on RS3 by approximately four turns of the DNA helix. Replication is abolished if this distance is increased by half a turn of the helix but it is restored if the distance is increased by a whole turn. These results suggest a DNA looping interaction, in the initiation of replication, between the RepA dimer that binds iR1 and the RepA monomers that bind the RS sequences.