Biomedical subjects

F Rojo

Publications and source records attributed to F Rojo.

At least 55 records · Page 3Linked to original sources

PubMed1997

Mutational analysis of a site-specific recombinase: characterization of the catalytic and dimerization domains of the beta recombinase of pSM19035.

The beta recombinase encoded by the streptococcal plasmid pSM19035, which shows 28 to 34% identity with DNA resolvases and DNA invertases, can catalyze formation of deletions or inversions between properly oriented target sites. We have constructed a number of site-directed mutations at residues that are conserved between the beta protein and other DNA recombinases of the resolvase/invertase family. The analysis of the recombination and DNA-binding ability of each mutant protein shows that the mutations affect the catalytic activity and, in two cases, the dimerization of the protein. The results suggest that the beta protein probably mediates recombination by a catalytic mechanism similar to that proposed for the resolvase/invertase family. Since the beta recombinase differs from DNA resolvases and DNA invertases in its lack of bias towards either of these reactions, the results presented support the hypothesis that its unique properties might depend on details of the architecture or assembly of the recombination complex. In addition, two beta protein mutants that can no longer form dimers in solution have provided new insights into the way the protein binds to DNA.

Amino Acid Sequence↗

PubMed1997

Transcription activation or repression by phage psi 29 protein p4 depends on the strength of the RNA polymerase-promoter interactions.

Phage psi 29 protein p4 activates the late A3 promoter and represses the early A2c promoter, in both cases by binding upstream from RNA polymerase (RNAP) and interacting with the C-terminal domain of the RNAP alpha subunit. To investigate how this interaction leads to activation at PA3 and to repression at PA2c, mutant promoters were constructed. We show that the position of protein p4 relative to that of RNAP, which is different at each promoter, does not dictate the outcome of the interaction. Rather, in the absence of a-35 consensus box for sigma A-RNAP activation was observed, while in its presence repression occurred. The results support the view that stabilization of RNAP at the promoter over a threshold level leads to repression.

Bacillus Phages↗

PubMed1997

The Bacillus subtilis chromatin-associated protein Hbsu is involved in DNA repair and recombination.

The Bacillus subtilis hbs gene encodes an essential chromatin-associated protein termed Hbsu. Hbsu, the counterpart of the Escherichia coli HU protein, binds DNA in a non-specific way but has a clear preference for bent, kinked or altered DNA sequences. To investigate the role of Hbsu in DNA repair and DNA recombination we have constructed a series of site-directed mutants in the hbs gene and used these mutant genes to substitute the wild-type chromosomal hbs gene. The hbs47 mutation, which codes for a mutant protein in which residue Phe-47 has been replaced by Trp, does not cause any discernible phenotype. Additional substitution of residue Arg-55 by Ala (hbs4755 mutation) rendered cells deficient in DNA repair, homologous recombination and beta protein-mediated site-specific recombination. We have also tested the effect on DNA repair of the hbs4755 mutation in combination with mutations in different functions of homologous DNA recombination (recA, recF, recG, recH and addAB). The hbs4755 mutation did not modify the sensitivity of recH and addAB cells to the DNA-damaging agents methylmethane sulphonate (MMS) or 4-nitroquinoline-1-oxide (4NQO), and it only marginally affected recF and recG cells. The hbs4755 mutation blocked intermolecular recombination in recH cells and markedly reduced it (20- to 50-fold) in recF and recG cells, but had no effect on addAB cells. Taken together, these data indicate that the Hbsu protein is required for DNA repair and for homologous DNA recombination.

Amino Acid Sequence↗

PubMed1996

Protein p4 represses phage phi 29 A2c promoter by interacting with the alpha subunit of Bacillus subtilis RNA polymerase.

Regulatory protein p4 from Bacillus subtilis phage phi 29 represses the strong viral A2c promoter (PA2c) by preventing promoter clearance; it allows RNA polymerase to bind to the promoter and form an initiated complex, but the elongation step is not reached. Protein p4 binds at PA2c immediately upstream from RNA polymerase; repression involves a contact between both proteins that holds the RNA polymerase at the promoter. This contact is held mainly through p4 residue Arg120, which is also required for activation of the phi 29 late A3 promoter. We have investigated which region of RNA polymerase contacts protein p4 at PA2c. Promoter repression was impaired when a reconstituted RNA polymerase lacking the 15 C-terminal residues of the alpha subunit C-terminal domain was used; this polymerase was otherwise competent for transcription. Binding cooperativity assays indicated that protein p4 cannot interact with this mutant RNA polymerase at PA2c. Protein p4 could form a complex at PA2c with purified wild-type alpha subunit, but not with a deletion mutant lacking the 15 C-terminal residues. Our results indicate that protein p4 represses PA2c by interacting with the C-terminal domain of the alpha subunit of RNA polymerase. Therefore, this domain of the alpha subunit can receive regulatory signals not only from transcriptional activators, but from repressors also.

Bacillus Phages↗

PubMed1996

Site-specific recombination in gram-positive theta-replicating plasmids.

This review summarises current information on the site-specific recombinases encoded by the plasmids of the Gram-positive bacteria that have low guanine and cytosine content in their DNA. It focuses on the peculiar biological features of the recombination systems encoded by the theta-replicating plasmids and compares them with the site-specific recombinases encoded by transposons or plasmids originally isolated from Gram-negative bacteria.

Amino Acid Sequence↗

PubMed1996

Site-specific recombination by the beta protein from the streptococcal plasmid pSM19035: minimal recombination sequences and crossing over site.

The beta recombinase from the broad host range Grampositive plasmid pSM19035 catalyzes intramolecular site-specific recombination between two directly or inversely oriented recombination sites in the presence of a chromatin-associated protein (Hbsu). The recombination site had been localized to a 447 bp DNA segment from pSM19035. This segment includes a 90 bp region that contains two adjacent binding sites (I and II) for beta protein dimers. Using in vitro recombination assays, we show that this 90 bp region is necessary and sufficient for beta protein-mediated recombination; this defines the six site as the region required for beta protein binding. The point of crossing over has been localized to the center of site I. Hbsu has a strong binding affinity for an unknown site located within the 447 bp segment containing the six site. We discuss the possibility that Hbsu recognizes an altered DNA structure, rather than a specific sequence, generated in the synaptic complex.

Bacterial Proteins↗

PubMed1996

Transcription activation by phage phi29 protein p4 is mediated by interaction with the alpha subunit of Bacillus subtilis RNA polymerase.

Regulatory protein p4 from Bacillus subtilis phage phi29 activates transcription from the viral late A3 promoter by stabilizing sigmaA-RNA polymerase at the promoter as a closed complex. Activation requires an interaction between protein p4 and RNA polymerase mediated by the protein p4 carboxyl-end, mainly through residue Arg-120. We have obtained derivatives of B. subtilis RNA polymerase alpha subunit with serial deletions at the carboxyl-end and reconstituted RNA polymerase holoenzymes harboring the mutant alpha subunits. Protein p4 promoted the binding of purified B. subtilis RNA polymerase alpha subunit to the A3 promoter in a cooperative way. Binding was abolished by deletion of the last 15 amino acids of the alpha subunit. Reconstituted RNA polymerases with deletions of 15 to 59 residues at the alpha subunit carboxyl-end could recognize and transcribe viral promoters not activated by protein p4, but they had lost their ability to recognize the A3 promoter in the presence of protein p4. In addition, these mutant reconstituted RNA polymerases could not interact with protein p4. We conclude that protein p4 activation of the viral A3 promoter requires an interaction between the carboxyl-end of protein p4 and the carboxyl-end of the alpha subunit of B. subtilis RNA polymerase that stabilizes the RNA polymerase at the promoter.

Amino Acid Sequence↗

PubMed1996

The Mfd protein of Bacillus subtilis 168 is involved in both transcription-coupled DNA repair and DNA recombination.

Inactivation of Bacillus subtilis orf1177 in an otherwise Rec+ strain reduced genetic exchange and DNA repair. When the mutation was transferred into a set of recombination-deficient and repair-deficient strains, the DNA repair and recombination ability of the double or triple mutant strains was drastically reduced. B. subtilis Orf1177 protein shares substantial homology with the Escherichia coli Mdf, RecG and UvrB proteins. In vivo analysis of UV-induced mutations suggests that Orf1177 is necessary for strand-specific DNA repair, as is the case for the E. coli MFD protein. Therefore, orf1177 and Orf1177 were termed mfd gene and Mfd protein, respectively. The purified Mfd protein has a native molecular mass of 140 kDa (expected molecular mass 133 kDa). The Mfd protein is a sequence-independent DNA binding protein with weak ATPase activity. The Mfd protein was able to displace in vitro B. subtilis or E. coli RNA polymerase stalled at a lesion. Therefore, Mfd protein appears to target the transcribed strand for repair by recognizing a stalled RNA polymerase and dissociating it from the DNA. In addition, the strong recombination-deficient phenotype of mfd- rec- strains suggest that Mfd protein is involved in homologous DNA recombination.

Amino Acid Sequence↗

PubMed1996

Activation and repression of transcription at two different phage phi29 promoters are mediated by interaction of the same residues of regulatory protein p4 with RNA polymerase.

Phage phi29 regulatory protein p4 activates transcription from the late A3 promoter and represses the main early promoters, named A2b and A2c. Activation involves stabilization of RNA polymerase (RNAP) at the A3 promoter as a closed complex and is mediated by interaction between RNAP and a small domain of protein p4 in which residue Arg120 plays an essential role. We show that protein p4 represses the A2c promoter by binding to DNA immediately upstream from RNAP in a way that does not hinder RNAP binding; rather, the two proteins bind cooperatively to DNA. In the presence of protein p4, RNAP can form an initiated complex at the A2c promoter that generates short abortive transcripts, but cannot leave the promoter. Mutation of protein p4 residue Arg120, which relieves the contact between the two proteins, leads to a loss of repression. Therefore, the contact between protein p4 and RNAP through the protein p4 domain containing Arg120 can activate or repress transcription, depending on the promoter. The relative position of protein p4 and RNAP, which is different at each promoter, together with the distinct characteristics of the two promoters, may determine whether protein p4 activates or represses transcription.

Amino Acid Sequence↗

PubMed1996

Transcriptional activator of phage phi 29 late promoter: mapping of residues involved in interaction with RNA polymerase and in DNA bending.

Phage phi 29 regulatory protein p4 activates transcription from the late A3 promoter by stabilizing sigma A-RNA polymerase at the promoter as a closed complex. Activation requires interaction between both proteins. Protein p4 bends the DNA upon binding. We have performed a detailed mutagenesis study of the carboxyl end of the protein, which is involved in both transcription activation and DNA bending. The results indicate that Arg-120 is the most critical residue for activation, probably mediating the interaction with RNA polymerase. Several basic residues have been identified, including Arg-120, that contribute to maintenance of the DNA bending, probably via electrostatic interactions with the DNA backbone. The degree or stability of the induced bend apparently relies on the additive contribution of all basic residues of the carboxyl end of the protein. Therefore, the activation and DNA bending surfaces overlap, and Arg-120 should interact with both DNA and RNA polymerase. As we show that protein p4 is a dimer in solution, and is bound to DNA as a tetramer, the results suggest a model in which two of the p4 subunits interact with the DNA, bending it, while the other two subunits remain accessible to interact with RNA polymerase.

Bacillus Phages↗

PubMed1995

The beta recombinase of plasmid pSM19035 binds to two adjacent sites, making different contacts at each of them.

The beta recombinase from plasmid pSM19035 catalyzes intramolecular site-specific recombination between two directly or inversely oriented six sites in the presence of a chromatin-associated protein (Hbsu, HU or HMG-1). The six site is a DNA segment containing two binding sites (I and II) for beta protein dimers. We show that beta recombinase binds sequentially to both sites, having a different affinity for each one. Hydroxyl radical footprints show a different protection pattern at each site. Positions critical for beta protein binding have been identified by methylation interference and missing nucleoside assays. The results indicate that the protein recognizes each site in a different way. Comparison of the beta protein recombination site with that of DNA resolvases and DNA invertases of the Tn3 family, to which it belongs, shows that these sequences can be divided into two regions. One corresponds to the crossover point and is similar for all recombinases of the family. The other region differs in the different subfamilies and seems to have an architectural role in aligning the crossover sites at the synaptic complex. The different ways to assemble this complex could explain why each system leads to a particular recombination event: DNA resolution (resolvases), inversion (invertases) or both (beta recombinase).

Bacterial Proteins↗

PubMed1995

Plasmid rolling circle replication and its control.

This review summarises current information on rolling circle replicating plasmids originally isolated from Gram-positive bacteria with a low guanine and cytosine content in their DNA. It focuses on the peculiar biological features of these small, high copy number plasmids that replicate via an asymmetric RC mechanism. The regulation of plasmid copy number is also discussed.

Bacterial Proteins↗

PubMed1995

The level of the pUB110 replication initiator protein is autoregulated, which provides an additional control for plasmid copy number.

Plasmids control their copy number by limiting the amount of the initiator for DNA replication. The plasmid pUB110 initiator protein is termed RepU. Expression of the pUB110 repU gene is controlled by two antisense RNAs that interfere with repU mRNA translation. Genetic evidence suggests that Rep protein levels may be regulated by additional uncharacterized mechanisms. The repU gene product was radiolabeled and purified by monitoring the radioactive label. RepU overproduction was performed in cells containing the plasmid leading strand replication origin (dso), to allow for a putative inactivation of RepU. Polypeptides with apparent molecular masses of 42 (RepU*) and 39 (RepU) kDa were purified, both having the N-terminal sequence expected for the repU gene. The RepU/RepU* protein mixture bound specifically to dso. At low protein concentrations, about six RepU/RepU* protomers bound to the dso region. At higher concentrations, an extended nucleoprotein complex was formed. The promoter for the repU gene was localized downstream of the dso region. The results suggest that the extended RepU/RepU*-dso DNA complex interferes with repU promoter utilization. This provides an additional copy number control by limiting RepU concentration. Our results suggest that during replication the RepU protein might be converted into an inactive RepU-RepU* hetero-oligomer, further limiting the amount of RepU protein available for replication initiation.

Amino Acid Sequence↗

PubMed1995

Transcription regulation in Bacillus subtilis phage phi 29: expression of the viral promoters throughout the infection cycle.

Transcription of the genome of Bacillus subtilis phage phi 29 is tightly controlled, taking place in two stages, early and late. We have analyzed the abundance of the transcripts produced from each viral promoter throughout the infection cycle. We compare the relative strength of each promoter, as well as get a better understanding of the regulatory events, finding a new promoter regulated by the viral protein p4. The two strong early promoters, A2b and A2c, responsible for the expression of genes 6 to 1, are coordinately repressed by the viral protein p4, although repression is not complete: both promoters are still active at late times of infection. Since repression by protein p4 was very efficient in vitro, and affects its own synthesis, it is likely that this protein is produced in limiting amounts, not being bound to all viral DNA molecules present in the cell at a given time. Protein p4, also known to activate the late promoter responsible for the expression of all the structural and morphogenetic genes, is the key regulator of phage phi 29 development.

Bacillus Phages↗

PubMed1995

The Bacillus subtilis histone-like protein Hbsu is required for DNA resolution and DNA inversion mediated by the beta recombinase of plasmid pSM19035.

The beta recombinase, encoded by the Gram-positive bacterial plasmid pSM19035, is unable to mediate DNA recombination in vitro unless a host factor is provided. The factor has now been identified as the Bacillus subtilis Hbsu protein. Hbsu is a nonspecific DNA-binding and DNA-bending protein. The beta recombinase, in the presence of highly purified Hbsu protein, is able to catalyze in vitro intramolecular recombination between two specific recombination sites on a supercoiled DNA molecule. DNA resolution was obtained when the two crossing over sites (six sites) were directly oriented, whereas DNA inversion was the product when the six sites were in inverse orientation. The ability of the Escherichia coli chromatin-associated proteins HU, IHF, Fis, and H-NS to substitute for Hbsu was investigated. HU efficiently stimulated beta-mediated recombination, while the effect of IHF was partial and that of Fis and H-NS was undetectable. In addition, the beta protein was able to mediate DNA recombination in both wild-type and IHF-deficient E. coli cells, but failed to do so in an HU-deficient strain. The data presented provide direct evidence that a chromatin-associated protein is strictly required for beta-mediated recombination.

Bacillus subtilis↗

PubMed1995

The role of chromatin-associated protein Hbsu in beta-mediated DNA recombination is to facilitate the joining of distant recombination sites.

The beta recombinase is unable to mediate in vitro DNA recombination between two directly oriented recombination sites unless a bacterial chromatin-associated protein (Bacillus subtilis Hbsu or Escherichia [correction of Eschrichia] coli HU] is provided. By electron microscopy, we show that the role of Hbsu is to help in joining the recombination sites to form a stable synaptic complex. Some evidence supports the fact that Hbsu works by recognizing and stabilizing a DNA structure at the recombination site, rather than by serving as a bridge between beta recombinase dimers through a protein-protein interaction. We show that the mammalian HMG1 protein, which shares neither sequence nor structural homology with Hbsu, can also stimulate beta-mediated recombination. These chromatin-associated proteins share the property of binding to DNA in a relatively non-specific fashion, bending it, and having a marked preference for altered DNA structures. Hbsu, HU or HMG1 proteins probably bind specifically at the crossing-over region, since at limiting protein-DNA molar ratios they could not be outcompeted by an excess of a DNA lacking the crossing over site. Distamycin, a minor groove binder that induces local distortions in DNA, did not affect the binding of beta protein to DNA, but inhibited the formation of the synaptic complex.

Bacterial Proteins↗

PubMed1994

The beta recombinase from the Streptococcal plasmid pSM 19035 represses its own transcription by holding the RNA polymerase at the promoter region.

The beta protein encoded by the Streptococcus pyogenes plasmid pSM19035 is a site-specific recombinase involved in both resolution of plasmid multimers into monomers and DNA inversion. It has been proposed that the DNA region to which the beta recombinase binds to mediate recombination includes a promoter from which orf alpha and the beta gene are transcribed. We have determined the sites at which transcription of the orf alpha and the beta gene initiates in vitro and we have demonstrated that highly purified beta recombinase acts as a repressor of its own synthesis. The promoters are located within the beta recombinase binding site, which we have defined previously. The binding of the beta recombinase to its target site does not seem to exclude RNA polymerase from the promoter, despite the overlapping of their binding sites. Therefore, it is likely that the beta recombinase does not repress transcription by a mere steric hindrance on RNA polymerase binding.

Base Sequence↗

PubMed1994

A novel site-specific recombinase encoded by the Streptococcus pyogenes plasmid pSM19035.

Genetic evidence suggests that the beta protein encoded by the Streptococcus pyogenes plasmid pSM19035 is involved both in the resolution of plasmid multimers into monomers and in DNA inversion. In this report we show that the highly purified beta protein is unable to mediate DNA recombination unless a host factor(s) is provided. In the presence of the host factor(s), the beta protein is able to catalyze in vitra intramolecular recombination between two specific sites on supercoiled templates: DNA resolution was obtained when the two recombination sites were directly oriented, whereas DNA inversion was the product if the recombination sites were in inverse orientation. In the absence of the host factor(s) the beta protein forms a specific complex with its target site. The beta protein binding site has been localized by DNase I footprinting to an 85 bp region that can be divided into two discrete sites (I and II). These sites are about 34 bp in length, they are separated by about 16 bp, and contain two 12 to 13 bp imperfectly conserved sequences (half-sites) with dyad axis symmetry. The protein binds co-operatively to sites I and II; between 3.6 and 4.2 beta protein protomers are required to saturate the DNA substrate. These data, together with gel retardation assays, suggest that the protein binds to DNA as two dimers, one to each discrete site, and that the dimers probably interact with each other. The beta protein binding site, though it resembles that of other DNA resolvases of the Tn3/gamma delta (Tn1000) family, differs in that only two adjacent sites are found (sites I and II), while those DNA resolvases normally bind to three adjacent sites. The results presented here suggest that a host factor(s) could work as an accessory effector to compensate for the absence of the third binding site.

Bacterial Proteins↗