Pivotal roles for the receiver domain in the mechanism of action of the response regulator RamR of Streptomyces coelicolor

The response regulator RamR activates expression of the ramCSAB operon, the source of the morphogenetic peptide SapB, and is therefore important for morphogenesis of the bacterium Streptomyces coelicolor. Like most response regulators, RamR consists of an amino-terminal receiver domain and a carboxy-terminal DNA binding domain. Four of five highly conserved active site residues known to be important in other response regulators are present in RamR: D12, D56 (the predicted site of phosphorylation), T84 and K105. Here, we show that in spite of this, RamR did not demonstrate an ability to autophosphorylate in vitro in the presence of small molecule phosphodonors. The unphosphorylated protein behaved as a dimer and bound cooperatively to three sites in the ramC promoter, one with very high affinity and two with lower affinity. On its own, the RamR DNA binding domain could not bind DNA but was able to interfere with the action of full length RamR in a manner suggesting direct protein-protein contact. Surprisingly, substitution of residues D12 or T84 had no effect on RamR function in vivo. In contrast, D56A and K105A substitutions caused defects in both dimer formation and DNA binding while the more conservative substitution, D56N permitted dimer formation but not DNA binding. L102 in RamR corresponds to a well-conserved tyrosine (or aromatic) residue that is important for function in the other response regulators. While a L102Y variant, which introduced the aromatic side-chain usually found at this position, functioned normally, L102A and L102W substitutions blocked RamR function in vivo. We show that these substitutions specifically impaired cooperative DNA binding by RamR at the lower affinity recognition sequences. The biochemical properties of RamR therefore differ markedly from those of other well-characterized response regulators.     

An oriC-like distribution of GATC sites mediates full sequestration of non-origin sequences in Escherichia coli

Sequestration of newly replicated origins is one of the mechanisms required to limit initiation of Escherichia coli chromosome replication to once per generation. Origin sequestration lasts for a considerably longer period of time than the sequestration of other newly replicated regions of the chromosome. The reason for this may be the high number of GATC sites present in the origin. Alternatively, other sequence elements in the origin region may be important for its prolonged sequestration. To distinguish between these possibilities we constructed a DNA fragment containing ten GATC sites distributed with the same spacing as the ten GATC sites in the left half of oriC, but with random sequence between the GATC sites, and inserted it at a non-sequestered chromosome location. Sequestration of this GATC-cluster lasted as long as that of oriC, or even longer. The result shows that the presence of ten GATC sites, distributed as in oriC, is sufficient to cause full sequestration, and that other sequence elements most likely do not contribute to sequestration.     

Positive supercoiling is generated in the presence of Escherichia coli SeqA protein

In Escherichia coli, the SeqA protein is known as a negative regulator of chromosome replication. This protein is also suggested to have a role in chromosome organization. SeqA preferentially binds to hemi-methylated DNA and is by immunofluorescence microscopy seen as foci situated at the replication factories. Loss of SeqA leads to increased negative supercoiling of the DNA. We show that purified SeqA protein bound to fully methylated, covalently closed or nicked circular DNA generates positive supercoils in vitro in the presence of topoisomerase I or ligase respectively. This means that binding of SeqA changes either the twist or the writhe of the DNA. The ability to affect the topology of DNA suggests that SeqA may take part in the organization of the chromosome in vivo. The topology change performed by SeqA occurred also on unmethylated plasmids. It is, however, reasonable to suppose that in vivo the major part of such activity is performed on hemi-methylated DNA at the replication factories and presumably forms the basis for the characteristic SeqA foci observed by fluorescence microscopy.     

Termination Structures in the Escherichia coli Chromosome Replication Fork Trap

The Escherichia coli chromosome contains two opposed sets of unidirectional DNA replication pause (Ter) sites that, according to the replication fork trap theory, control the termination of chromosome replication by restricting replication fork fusion to the terminus region. In contrast, a recent hypothesis suggested that termination occurs at the dif locus instead. Using two-dimensional agarose gel electrophoresis, we examined DNA replication intermediates at the Ter sites and at dif in wild-type cells. Two definitive signatures of site-specific termination—specific replication fork arrest and converging replication forks—were clearly detected at Ter sites, but not at dif. We also detected a significant pause during the latter stages of replication fork convergence at Ter sites. Quantification of fork pausing at the Ter sites in both their native chromosomal context and the plasmid context further supported the fork trap model.     

Identification of operator sites within the upstream region of the putative mce2R gene from mycobacteria

Mycobacterium tuberculosis harbors four mce operons. Among them, mce2 operon is preceded by a FadR-like regulator mce2R (Rv0586). Here, we report the operator sites of the mce2R and its orthologs in other sequenced mycobacteria and non-mycobacterial species Nocardia farciana. All the identified DNA motifs illustrate the FadR subfamily specific nucleotide preference. Moreover, these motifs from the upstream region share sequence conservation, which is in agreement with the similarity of their DNA binding domain. Using electrophoretic mobility shift assay, we demonstrate that the predicted DNA motifs specifically interact with the recombinant Mce2R-Rv0586. Our present study has implications in the understanding of cis-regulatory elements and the auto-regulatory nature of the FadR subfamily of regulators.     

Nucleosome positioning signals in the DNA sequence of the human and mouse H19 imprinting control regions

We have investigated the sequences of the mouse and human H19 imprinting control regions (ICRs) to see whether they contain nucleosome positioning information pertinent to their function as a methylation-regulated chromatin boundary. Positioning signals were identified by an in vitro approach that employs reconstituted chromatin to comprehensively describe the contribution of the DNA to the most basic, underlying level of chromatin structure. Signals in the DNA sequence of both ICRs directed nucleosomes to flank and encompass the short conserved sequences that constitute the binding sites for the zinc finger protein CTCF, an essential mediator of insulator activity. The repeat structure of the human ICR presented a conserved array of strong positioning signals that would preferentially flank these CTCF binding sites with positioned nucleosomes, a chromatin structure that would tend to maintain their accessibility. Conversely, all four CTCF binding sites in the mouse sequence were located close to the centre of positioning signals that were stronger than those in their flanks; these binding sites might therefore be expected to be more readily incorporated into positioned nucleosomes. We found that CpG methylation did not effect widespread repositioning of nucleosomes on either ICR, indicating that allelic methylation patterns were unlikely to establish allele-specific chromatin structures for H19 by operating directly upon the underlying DNA-histone interactions; instead, epigenetic modulation of ICR chromatin structure is likely to be mediated principally at higher levels of control. DNA methylation did, however, both promote and inhibit nucleosome positioning at several sites in both ICRs and substantially negated one of the strongest nucleosome positioning signals in the human sequence, observations that underline the fact that this epigenetic modification can, nevertheless, directly and decisively modulate core histone-DNA interactions within the nucleosome.     

GATC flanking sequences regulate Dam activity: evidence for how Dam specificity may influence pap expression

Escherichia coli DNA adenine methyltransferase (Dam) plays essential roles in DNA replication, mismatch repair and gene regulation. The differential methylation by Dam of the two GATC sequences in the pap promoter regulates the expression of pili genes necessary for uropathogenic E.coli cellular adhesion. Dam processively methylates GATC sites in various DNA substrates, yet the two pap GATC sites are not processively methylated. We previously proposed that the flanking sequences surrounding the two pap GATC sites contribute to the enzyme's distributive methylation. We show here that replacement of the poorly methylated pap GATC sites with sites predicted to be processively methylated indeed results in an increase in Dam processivity. The increased processivity is due to a change in the methyltransfer kinetics and not the binding efficiency of Dam. A competition experiment in which the flanking sequences of only one pap GATC site were altered demonstrates that the GATC flanking sequences directly regulate the enzyme's catalytic efficiency. The GATC flanking sequences in Dam-regulated promoters in E.coli and other bacteria are similar to those in the pap promoter. Gene regulation from some of these promoters involves mechanisms and proteins that are quite different from those in the pap operon. Further, GATC sequences previously identified to remain unmethylated within the E.coli genome, but whose function remains largely unassigned, are flanked by sequences predicted to be poorly methylated. We conclude that the GATC flanking sequences may be critical for expression of pap and other Dam-regulated genes by affecting the activity of Dam at such sites and, thus, its processivity. A model is proposed, illustrating how the sequences flanking the GATC sites in Dam-regulated promoters may contribute to this epigenetic mechanism of gene expression, and how flanking sequences contribute to the diverse biological roles of Dam.     

The spacing of Escherichia coli DNA gyrase sites cleaved in vivo by treatment with oxolinic acid and sodium dodecyl sulfate

In an attempt to locate gyrase binding sites in a specific region of the chromosome of E. coli, we have reinvestigated gyrase-promoted cleavage of chromosomal DNA by oxolinic acid and sodium dodecyl sulfate. Contrary to a previous report suggesting the presence of one site every 100 kb of DNA (Snyder and Drlica, J. Mol. Biol. 131, 287-302), we found frequencies of one cleavage every 25 or 12 kb depending on the growth medium. A search for cleavage sites by Southern blot hybridization failed to reveal any binding site cleaved at a high frequency. These results suggest that the actual spacing of sites is much closer than that determined from the frequency of cleavage. Measurement of the average size of fragments containing defined DNA sequences indicated that the frequency of sites varies along the chromosome. The region located opposite to oriC carries relatively few sites.     

Evidence for a replication function of FFA-1, the Xenopus orthologue of Werner syndrome protein

DNA replication in higher eukaryotic cells occurs at a large number of discrete sites called replication foci. We have previously purified a protein, focus-forming activity 1 (FFA-1), which is involved in the assembly of putative prereplication foci in Xenopus egg extracts. FFA-1 is the orthologue of the Werner syndrome gene product (WRN), a member of the RecQ helicase family. In this paper we show that FFA-1 colocalizes with sites of DNA synthesis and the single-stranded DNA binding protein, replication protein A (RPA), in nuclei reconstituted in the egg extract. In addition, we show that two glutathione S-transferase FFA-1 fusion proteins can inhibit DNA replication in a dominant negative manner. The dominant negative effect correlates with the incorporation of the fusion proteins into replication foci to form "hybrid foci," which are unable to engage in DNA replication. At the biochemical level, RPA can interact with FFA-1 and specifically stimulates its DNA helicase activity. However, in the presence of the dominant negative mutant proteins, the stimulation is prevented. These results provide the first direct biochemical evidence of an important role for FFA-1 in DNA replication.     

Crystal structure of a SeqA-N filament: implications for DNA replication and chromosome organization

Escherichia coli SeqA binds clusters of transiently hemimethylated GATC sequences and sequesters the origin of replication, oriC, from methylation and premature reinitiation. Besides oriC, SeqA binds and organizes newly synthesized DNA at replication forks. Binding to multiple GATC sites is crucial for the formation of stable SeqA-DNA complexes. Here we report the crystal structure of the oligomerization domain of SeqA (SeqA-N). The structural unit of SeqA-N is a dimer, which oligomerizes to form a filament. Mutations that disrupt filament formation lead to asynchronous DNA replication, but the resulting SeqA dimer can still bind two GATC sites separated from 5 to 34 base pairs. Truncation of the linker between the oligomerization and DNA-binding domains restricts SeqA to bind two GATC sites separated by one or two full turns. We propose a model of a SeqA filament interacting with multiple GATC sites that accounts for both origin sequestration and chromosome organization.     

Competitive Lrp and Dam assembly at the pap regulatory region: implications for mechanisms of epigenetic regulation

Escherichia coli DNA adenine methyltransferase (Dam) and Leucine-responsive regulatory protein (Lrp) are key regulators of the pap operon, which codes for the pilus proteins necessary for uropathogenic E. coli cellular adhesion. The pap operon is regulated by a phase variation mechanism in which the methylation states of two GATC sites in the pap regulatory region and the binding position of Lrp determine whether the pilus genes are expressed. The post-replicative reassembly of Dam, Lrp, and the local regulator PapI onto a hemimethylated pap intermediate is a critical step of the phase variation switching mechanism and is not well understood. We show that Lrp, in the presence and in the absence of PapI and nonspecific DNA, specifically protects pap regulatory GATC sites from Dam methylation when allowed to compete with Dam for assembly on unmethylated and hemimethylated pap DNA. The methylation protection is dependent upon the concentration of Lrp and does not occur with non-regulatory GATC sites. Our data suggest that only at low Lrp concentrations will Dam compete effectively for binding and methylation of the proximal GATC site, leading to a phase switch resulting in the expression of pili.     

A novel plant gene essential for meiosis is related to the human CtIP and the yeast COM1/SAE2 gene

Obligatory homologous recombination (HR) is required for chiasma formation and chromosome segregation in meiosis I. Meiotic HR is initiated by DNA double-strand breaks (DSBs), generated by Spo11, a homologue of the archaebacterial topoisomerase subunit Top6A. In Saccharomyces cerevisiae, Rad50, Mre11 and Com1/Sae2 are essential to process an intermediate of the cleavage reaction consisting of Spo11 covalently linked to the 5' termini of DNA. While Rad50 and Mre11 also confer genome stability to vegetative cells and are well conserved in evolution, Com1/Sae2 was believed to be fungal-specific. Here, we identify COM1/SAE2 homologues in all eukaryotic kingdoms. Arabidopsis thaliana Com1/Sae2 mutants are sterile, accumulate AtSPO11-1 during meiotic prophase and fail to form AtRAd51 foci despite the presence of unrepaired DSBs. Furthermore, DNA fragmentation in AtCom1 is suppressed by eliminating AtSPO11-1. In addition, AtCOM1 is specifically required for mitomycin C resistance. Interestingly, we identified CtIP, an essential protein interacting with the DNA repair machinery, as the mammalian homologue of Com1/Sae2, with important implications for the molecular role of CtIP.     

Preferential Breakpoints in the Recovery of Broken Dicentric Chromosomes in Drosophila melanogaster

We designed a system to determine whether dicentric chromosomes in Drosophila melanogaster break at random or at preferred sites. Sister chromatid exchange in a Ring-X chromosome produced dicentric chromosomes with two bridging arms connecting segregating centromeres as cells divide. This double bridge can break in mitosis. A genetic screen recovered chromosomes that were linearized by breakage in the male germline. Because the screen required viability of males with this X chromosome, the breakpoints in each arm of the double bridge must be closely matched to produce a nearly euploid chromosome. We expected that most linear chromosomes would be broken in heterochromatin because there are no vital genes in heterochromatin, and breakpoint distribution would be relatively unconstrained. Surprisingly, approximately half the breakpoints are found in euchromatin, and the breakpoints are clustered in just a few regions of the chromosome that closely match regions identified as intercalary heterochromatin. The results support the Laird hypothesis that intercalary heterochromatin can explain fragile sites in mitotic chromosomes, including fragile X. Opened rings also were recovered after male larvae were exposed to X-rays. This method was much less efficient and produced chromosomes with a strikingly different array of breakpoints, with almost all located in heterochromatin. A series of circularly permuted linear X chromosomes was generated that may be useful for investigating aspects of chromosome behavior, such as crossover distribution and interference in meiosis, or questions of nuclear organization and function.     

The initiator protein DnaA contributes to keeping new origins inactivated by promoting the presence of hemimethylated DNA

The Escherichia coli replication origin oriC and other regions with high numbers of GATC sites remain hemimethylated after replication much longer than regions with average numbers of GATC sites. The prolonged period of hemimethylation has been attributed to the presence of bound SeqA protein. Here, it was found that a GATC cluster inserted at the datA site, which binds large amounts of DnaA in vivo, did not become remethylated at all, unless the availability of the DnaA protein was severely reduced. Sequestration of oriC was also found to be affected by the availability of DnaA. The period of origin hemimethylation was reduced by approximately 30% upon a reduction in the availability of DnaA. The result shows that not only SeqA binding but also DnaA binding to newly replicated origins contributes to keeping them hemimethylated. It was also found that the number of SeqA foci increased in cells with a combination of DnaA-mediated protection and sequestration at the GATC::datA cluster.