Biomedical subjects

P Soultanas

Publications and source records attributed to P Soultanas.

17 recordsLinked to original sources

PubMed2003

Clamp-loader-helicase interaction in Bacillus. Leucine 381 is critical for pentamerization and helicase binding of the Bacillus tau protein.

Recently, we revealed the architecture of the clamp-loader-helicase (tau-DnaB) complex in Bacillus by atomic force microscopy imaging and constructed a structural model, whereby a pentameric clamp-loader interacts with the hexameric helicase. Crucial to this model is the assumption that the clamp-loader forms a pentamer in the absence of other components of the clamp-loader complex such as deltadelta'. Here, we show that the Bacillus subtilis tau protein, even in the absence of deltadelta', interacts as a pentamer with the hexameric DnaB and that the L381 of tau is critical for the integrity of the tau oligomer and interaction with DnaB. The effects of the L381A mutation were confirmed by gel filtration, ultracentrifugation, circular dichroism, cross-linking studies, and genetic replacement of the dnaX gene with a mutant L381A dnaX gene in vivo. The L381A protein is able to support growth in vivo only when expressed in high quantities. Finally, despite the fact that a mutation at P465 has been reported to result in a thermosensitive gene in vivo, a P465L mutant protein interacts with DnaB in vitro suggesting that this defect is not a result of a defective tau-DnaB interaction.

Amino Acid Sequence↗

PubMed2002

Site-directed mutagenesis reveals roles for conserved amino acid residues in the hexameric DNA helicase DnaB from Bacillus stearothermophilus.

Site-directed mutagenesis studies on conserved amino acid residues within motifs H1, H1a, H2 and H3 of the hexameric replicative helicase DnaB from Bacillus stearothermophilus revealed specific functions associated with these residues. In particular, residues that coordinate a bound Mg2+ in the active site (T217 and D320) are important for the function of the enzyme but are not required for the formation of stable hexamers. A conserved glutamic acid (E241) in motif H1a is likely to be involved in the activation of a water molecule for in line attack on the gamma-phosphate of the bound nucleotide during catalysis. A conserved glutamine (Q362) in motif H3 acts as a gamma-phosphate sensor and mediates the conformational coupling of nucleotide- and DNA-binding sites. The nature of the residue at this position is also important for the primase-mediated activation of DnaB, suggesting that primase uses the same conformational coupling pathway to induce its stimulatory effect on the activity of DnaB. Together, these mutations reveal a conservation of many aspects of biochemical activity in the active sites of monomeric and hexameric helicases.

Adenosine Triphosphatases↗

PubMed2002

The beta-propeller protein YxaL increases the processivity of the PcrA helicase.

The DNA helicase PcrA is found in gram-positive bacteria and belongs to the superfamily 1 (SF1) of helicases, together with Rep and UvrD helicases from Escherichia coli. These helicases have been extensively studied in vitro and their mode of unwinding are well characterised. However, little is known about the putative cellular partners of such helicases. To identify PcrA-interacting factors, PcrA was used as a bait in a genome-wide yeast two-hybrid screen of a Bacillus subtilis library. Three proteins with unknown functions - YxaL, YwhK and YerB - were found to interact specifically with PcrA. The yxaL gene was cloned, the product was overexpressed and purified, and its effect on the PcrA activity was investigated in vitro. YxaL enhanced the processivity of the PcrA helicase. A comparison of the amino acid sequence of YxaL with other proteins from data banks suggests that YxaL belongs to a family of proteins with a repeated domain, which adopt a typical three-dimensional structure designated as a "beta-propeller". This raises the possibility that YxaL acts as a connector protein between PcrA and another cellular component.

Amino Acid Sequence↗

PubMed2002

A functional interaction between the putative primosomal protein DnaI and the main replicative DNA helicase DnaB in Bacillus.

In Gram negative Escherichia coli there are two well-characterised primosomal assembly processes, the PriA- and DnaA-mediated cascades. The presence of PriA and DnaA proteins in Gram positive Bacillus spp. supports the assumption that both the PriA- and DnaA-mediated primosomal assembly cascades also operate in these organisms. However, the lack of sequence homology between the rest of the primosomal proteins indicates significant differences between these two bacterial species. Central to the process of primosomal assembly is the loading of the main hexameric replicative helicase (DnaB in E.coli and DnaC in Bacillus subtilis) on the DNA. This loading is achieved by specialised proteins known as 'helicase loaders'. In E.coli DnaT and DnaC are responsible for loading DnaB onto the DNA during primosome assembly, in the PriA- and DnaA-mediated cascades, respectively. In Bacillus the identity of the helicase loader is still not established unequivocally. In this paper we provide evidence for a functional interaction between the primosomal protein DnaI from B.subtilis and the main hexameric replicative helicase DnaB from Bacillus stearothermophilus. Our results are consistent with the putative role of DnaI as the 'helicase loader' in the Gram positive Bacillus spp.

Adenosine Triphosphatases↗

PubMed2001

Defining the roles of individual residues in the single-stranded DNA binding site of PcrA helicase.

Crystal structures and biochemical analyses of PcrA helicase provide evidence for a model for processive DNA unwinding that involves coupling of single-stranded DNA (ssDNA) tracking to a duplex destabilization activity. The DNA tracking model invokes ATP-dependent flipping of bases between several pockets on the enzyme formed by conserved aromatic amino acid residues. We have used site-directed mutagenesis to confirm the requirement of all of these residues for helicase activity. We also demonstrate that the duplex unwinding defects correlate with an inability of certain mutant proteins to translocate effectively on ssDNA. Moreover, the results define an essential triad of residues within the ssDNA binding site that comprise the ATP-driven DNA motor itself.

Adenosine Triphosphate↗

PubMed2001

Unwinding the 'Gordian knot' of helicase action.

Helicases are enzymes involved in every aspect of nucleic acid metabolism. Recent structural and biochemical evidence is beginning to provide details of their molecular mechanism of action. Crystal structures of helicases have revealed an underlying common structural fold. However, although there are many similarities between the mechanisms of different classes of helicase, not all aspects of the helicase activity are the same in all members of this enzyme family.

Adenosine Triphosphate↗

PubMed2000

Uncoupling DNA translocation and helicase activity in PcrA: direct evidence for an active mechanism.

DNA footprinting and nuclease protection studies of PcrA helicase complexed with a 3'-tailed DNA duplex reveal a contact region that covers a significant region of the substrate both in the presence and absence of a non-hydrolysable analogue of ATP, ADPNP. However, details of the interactions of the enzyme with the duplex region are altered upon binding of nucleotide. By combining this information with that obtained from crystal structures of PcrA complexed with a similar DNA substrate, we have designed mutant proteins that are defective in helicase activity but that leave the ATPase and single-stranded DNA translocation activities intact. These mutants are all located in domains 1B and 2B, which interact with the duplex portion of the DNA substrate. Taken together with the crystal structures, these data support an 'active' mechanism for PcrA that involves two distinct ATP-dependent processes: destabilization of the duplex DNA ahead of the enzyme that is coupled to DNA translocation along the single strand product.

Adenosine Triphosphatases↗

PubMed2000

Mapping protein-protein interactions within a stable complex of DNA primase and DnaB helicase from Bacillus stearothermophilus.

For the first time, we demonstrate directly a stable complex between a bacterial DnaG (primase) and DnaB (helicase). Utilizing fragments of both proteins, we are able to dissect interactions within this complex and provide direct evidence that it is the C-terminal domain of primase that interacts with DnaB. Furthermore, this C-terminal domain is sufficient to induce maximal stimulation of the helicase and ATPase activities of DnaB. However, the region of DnaB that interacts with the C-terminal domain of primase appears to comprise a surface on DnaB that includes regions from both of the previously identified N- and C-terminal domains. Using a combination of biochemical and physical techniques, we show that the helicase-primase complex comprises one DnaB hexamer and either two or three molecules of DnaG. Our results show that in Bacillus stearothermophilus the helicase-primase interaction at the replication fork may not be transient, as was shown to be the case in Escherichia coli. Instead, primase appears to interact with the helicase forming a tighter complex with enhanced ATPase and helicase activities.

Bacterial Proteins↗

PubMed2000

DNA helicases: 'inching forward'.

Recently determined crystal structures of PcrA helicase complexed with a DNA substrate have revealed details of the helicase mechanism. PcrA and UvrD helicases have been shown to be functional as monomers, challenging previous suggestions that all helicases are required to be oligomeric. Crystal structures of the hexameric helicases RepA and T7 gene 4 explain the formation of hexameric assemblies from identical monomers with RecA-like folds, but their molecular mechanism remains elusive.

Bacterial Proteins↗

PubMed1999

Site-directed mutagenesis of motif III in PcrA helicase reveals a role in coupling ATP hydrolysis to strand separation.

Motif III is one of the seven protein motifs that are characteristic of superfamily I helicases. To investigate its role in the helicase mechanism we have introduced a variety of mutations at three of the most conserved amino acid residues (Q254, W259 and R260). Biochemical characterisation of the resulting proteins shows that mutation of motif III affects both ATP hydrolysis and single-stranded DNA binding. We propose that amino acid residue Q254 acts as a gamma-phosphate sensor at the nucleotide binding pocket transmitting conformational changes to the DNA binding site, since the nature of the charge on this residue appears to control the degree of coupling between ATPase and helicase activities. Residues W259 and R260 both participate in direct DNA binding interactions that are critical for helicase activity.

Adenosine Triphosphatases↗

PubMed1999

DNA binding mediates conformational changes and metal ion coordination in the active site of PcrA helicase.

Based upon the crystal structures of PcrA helicase, we have made and characterised mutations in a number of conserved helicase signature motifs around the ATPase active site. We have also determined structures of complexes of wild-type PcrA with ADPNP and of a mutant PcrA complexed with ADPNP and Mn2+. The kinetic and structural data define roles for a number of different residues in and around the ATP binding site. More importantly, our results also show that there are two functionally distinct conformations of ATP in the active site. In one conformation, ATP is hydrolysed poorly whereas in the other (activated) conformation, ATP is hydrolysed much more rapidly. We propose a mechanism to explain how the stimulation of ATPase activity afforded by binding of single-stranded DNA stabilises the activated conformation favouring Mg2+binding and a consequent repositioning of the gamma-phosphate group which promotes ATP hydrolysis. A part of the associated conformational change in the protein forces the side-chain of K37 to vacate the Mg2+binding site, allowing the cation to bind and interact with ATP.

Adenosine Diphosphate↗

PubMed1999

Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism.

We have determined two different structures of PcrA DNA helicase complexed with the same single strand tailed DNA duplex, providing snapshots of different steps on the catalytic pathway. One of the structures is of a complex with a nonhydrolyzable analog of ATP and is thus a "substrate" complex. The other structure contains a bound sulphate ion that sits in a position equivalent to that occupied by the phosphate ion produced after ATP hydrolysis, thereby mimicking a "product" complex. In both complexes, the protein is monomeric. Large and distinct conformational changes occur on binding DNA and the nucleotide cofactor. Taken together, these structures provide evidence against an "active rolling" model for helicase action but are instead consistent with an "inchworm" mechanism.

Adenylyl Imidodiphosphate↗

PubMed1999

Plasmid replication initiator protein RepD increases the processivity of PcrA DNA helicase.

The replication initiator protein RepD encoded by the Staphylococcus chloramphenicol resistance plasmid pC221 stimulates the helicase activity of the Bacillus stearothermophilus PcrA DNA helicase in vitro. This stimulatory effect seems to be specific for PcrA and differs from the stimulatory effect of the Escherichia coli ribosomal protein L3. Whereas L3 stimulates the PcrA helicase activity by promoting co-operative PcrA binding onto its DNA substrate, RepD stimulates the PcrA helicase activity by increasing the processivity of the enzyme and enables PcrA to displace DNA from a nicked substrate. The implication of these results is that PcrA is the helicase recruited into the replisome by RepD during rolling circle replication of plasmids of the pT181 family.

Adenosine Triphosphatases↗

PubMed1998

Escherichia coli ribosomal protein L3 stimulates the helicase activity of the Bacillus stearothermophilus PcrA helicase.

Escherichia coli ribosomal protein L3 stimulates the in vitro helicase activity of Bacillus stearothermophilus PcrA helicase upon a variety of different substrates. L3 has no intrinsic helicase or ATPase activity nor is it able to stimulate the ATPase activity of PcrA. Gel mobility shift assays revealed that the affinity of PcrA for a variety of different DNA species (single-stranded, nicked and 3'-tailed) was enhanced in the presence of L3. We suggest that the stimulatory effect of L3 upon the helicase activity of PcrA is mediated via a protein-protein interaction which promotes cooperative binding of PcrA to its DNA substrate. This activity of L3 appears to be specific for PcrA helicase.

Adenosine Triphosphatases↗

PubMed1995

Site-specific recombination at res sites containing DNA-binding sequences for both Tn21 and Tn3 resolvases.

Tn3 and gamma delta resolvases catalyse site-specific recombination at res sites from Tn3 but not at Tn21 res sites. Tn21 resolvase has no activity at Tn3 sites and acts only at Tn21 sites. In both Tn3 and Tn21, res had three binding sites for the cognate resolvases; the cross-over site, I; and the accessory sites II and III, from which the bound proteins may stabilize the synaptic complex by protein-protein interactions. In this study hybrid res sites were made by replacing either II or III in the Tn21 res site with the equivalent sequence from Tn3. Plasmids containing either a hybrid and a wild-type Tn21 res site, or two hybrid sites, were tested for recombination. Relative to the reaction with two wild-type sites, recombination by Tn21 resolvase was reduced by replacing II at one res site and it was reduced further by replacing II at both loci but, in both cases, Tn21 recombination was enhanced by Tn3 or gamma delta resolvases. Very few of the amino acid on the external surface of gamma delta resolvases are conserved in Tn21. Moreover, mutants of gamma delta resolvase with defective protein-protein interactions also enhanced Tn21 recombination at this hybrid site. The resolvase at II thus seems not to be involved in protein-protein interactions and its main role may be to bend the DNA to the required structure. The replacement of III in the Tn21 site with Tn3 sequence also reduced recombination by Tn21 resolvase, especially when both loci carried the alteration but, in contrast to before, Tn3 or gamma delta resolvases now inhibited the Tn21 reaction. Recombination thus seems to require identical proteins at I and III, perhaps to allow for protein-protein interactions.

Base Sequence↗

PubMed1995

Site-specific recombination at res sites containing DNA-binding sequences for both Tn21 resolvase and CAP.

The res sites, the loci for site-specific recombination by resolvase, contain three binding sites for the protein; the cross-over site, I, and accessory sites II and III. The role of DNA bending by resolvase was examined by replacing either II or III in the res site from Tn21 with the recognition sequence for a heterologous DNA-bending protein, CAP (the catabolite-gene activator protein from Escherichia coli). The CAP sequence was placed at either the same position as the target sequence for Tn21 resolvase or a different position along the DNA. The activity of Tn21 resolvase for recombination between each hybrid and a wild-type res site was measured in the presence of CAP and cyclic AMP. When III was substituted, CAP inhibited Tn21 recombination, except when the CAP sequence was placed sufficiently far away from site II to allow resolvase to bind non-specifically to the DNA between II and the CAP site. With the substitutions at II, the extent of Tn21 recombination in the presence of CAP varied with the position of the CAP sequence: more recombination was observed when it superimposed the target sequence for resolvase than when it was displaced by five base-pairs. Efficient recombination by Tn21 resolvase thus seems to demand the cognate protein at site III in res, presumably for protein-protein interactions in the synaptic complex, while the function of resolvase at site II can be fulfilled, at least in part, by a heterologous DNA bend.

Base Sequence↗

PubMed1993

Modulation of human DNA methyltransferase activity and mRNA levels in the monoblast cell line U937 induced to differentiate with dibutyryl cyclic AMP and phorbol ester.

The regulation of DNA (cytosine-5) methyltransferase (DNA MeTase) enzyme activity and gene expression was examined in the monoblastoid U937 cell line induced to differentiate with either dibutyryl cyclic AMP (dbcAMP) or phorbol ester. dbcAMP treatment was found to cause the rapid (< 4 h) suppression of DNA MeTase specific activity, with no DNA MeTase activity detectable after 10 h. Equally, no DNA MeTase activity was detectable in nuclear extracts of fresh peripheral blood monocytes. Using both a U937 DNA MeTase cDNA and a mouse DNA MeTase cDNA as probes, steady-state levels of DNA MeTase mRNA were found to decline sharply between 4 and 15 h after dbcAMP treatment. No DNA MeTase mRNA was detectable after 20 h of dbcAMP treatment. Nuclear run-on analysis showed there to be only a small (40%) suppression of DNA MeTase gene transcription in cells treated with dbcAMP for 24 h, implying a role for post-transcriptional processes in the regulation of DNA MeTase mRNA levels. The observed decline in DNA MeTase activity/mRNA levels appeared to precede the dbcAMP-induced arrest in DNA replication, as judged by the incorporation of tritiated thymidine into DNA. In contrast to the effect of dbcAMP, treatment of U937 cells with the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) led to an overall stimulation of DNA MeTase specific activity. The TPA response was found to be complex and broadly consisted of an early (0-15 h) burst of DNA MeTase activity followed by a more gradual sustained increase in DNA MeTase activity after prolonged (16-40 h) TPA treatment. The early phase of high DNA MeTase activity was not mirrored by an increase in steady-state levels of DNA MeTase mRNA, as judged by Northern blot analysis. However, a substantial induction of DNA MeTase mRNA levels was observed after 20-24 h of TPA treatment. Nuclear run-on analysis showed this not to be due to any significant increase in DNA MeTase gene transcription. The observed increases in DNA MeTase activity/mRNA levels were observed whilst cells were undergoing deproliferation. Interestingly, the addition of TPA and more physiological protein kinase C (PKC) activators, such as diacylglycerol and phosphatidylserine, to DNA MeTase-enriched nuclear extracts generated a 4.5-fold and a 1.5-fold increase in DNA MeTase specific activity respectively. The TPA-induced stimulation of DNA MeTase activity could be inhibited by the PKC inhibitor H-9, implicating a role for PKC in the regulation of DNA MeTase activity in vivo.

Bucladesine↗