Biomedical subjects

Reid C Johnson

Publications and source records attributed to Reid C Johnson.

At least 19 recordsLinked to original sources

PubMed2006

Mechanism of chromosome compaction and looping by the Escherichia coli nucleoid protein Fis.

Fis, the most abundant DNA-binding protein in Escherichia coli during rapid growth, has been suspected to play an important role in defining nucleoid structure. Using bulk-phase and single-DNA molecule experiments, we analyze the structural consequences of non-specific binding by Fis to DNA. Fis binds DNA in a largely sequence-neutral fashion at nanomolar concentrations, resulting in mild compaction under applied force due to DNA bending. With increasing concentration, Fis first coats DNA to form an ordered array with one Fis dimer bound per 21 bp and then abruptly shifts to forming a higher-order Fis-DNA filament, referred to as a low-mobility complex (LMC). The LMC initially contains two Fis dimers per 21 bp of DNA, but additional Fis dimers assemble into the LMC as the concentration is increased further. These complexes, formed at or above 1 microM Fis, are able to collapse large DNA molecules via stabilization of DNA loops. The opening and closing of loops on single DNA molecules can be followed in real time as abrupt jumps in DNA extension. Formation of loop-stabilizing complexes is sensitive to high ionic strength, even under conditions where DNA bending-compaction is unaltered. Analyses of mutants indicate that Fis-mediated DNA looping does not involve tertiary or quaternary changes in the Fis dimer structure but that a number of surface-exposed residues located both within and outside the helix-turn-helix DNA-binding region are critical. These results suggest that Fis may play a role in vivo as a domain barrier element by organizing DNA loops within the E. coli chromosome.

Chromosomes, Bacterial↗

PubMed2006

Crystallization, dehydration and preliminary X-ray analysis of excisionase (Xis) proteins cooperatively bound to DNA.

This paper describes the crystallization, dehydration and preliminary X-ray data analysis of a complex containing several bacteriophage lambda excisionase (Xis) [Bushman et al. (1984). Cell, 39, 699-706] proteins cooperatively bound to a 33-mer DNA duplex (Xis-DNA(X1-X2)). Xis is expected to recognize this regulatory element in a novel manner by cooperatively binding and distorting multiple head-to-tail orientated DNA-binding sites. Crystals of this complex belonged to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 107.7, c = 73.5 angstroms, alpha = beta = 90, gamma = 120 degrees. Based on the unit-cell parameters for the asymmetric unit, V(M) is 3.0 A(3) Da(-1), which corresponds to a solvent content of approximately 59%. The approaches used to crystallize the unusually long DNA fragment in the complex and the dehydration technique applied that dramatically improved the diffraction of the crystals from 10 to 2.6 angstroms are discussed.

Bacteriophage lambda↗

PubMed2005

Low-force DNA condensation and discontinuous high-force decondensation reveal a loop-stabilizing function of the protein Fis.

We report single-DNA-stretching experiments showing that the protein Fis, an abundant bacterial chromosome protein of E. coli, mediates a dramatic DNA condensation to zero length. This condensation occurs abruptly when DNA tension is reduced below a protein-concentration-dependent threshold f* < 1 pN. Following condensation, reopening under larger forces proceeds via a series of discrete jumps, indicating that Fis is able to stabilize DNA crossings. Our experiments suggest that Fis may play a role in vivo stabilizing the "loop-domain" structure of the bacterial chromosome.

DNA, Bacterial↗

PubMed2005

Determinants of HMGB proteins required to promote RAG1/2-recombination signal sequence complex assembly and catalysis during V(D)J recombination.

Efficient assembly of RAG1/2-recombination signal sequence (RSS) DNA complexes that are competent for V(D)J cleavage requires the presence of the nonspecific DNA binding and bending protein HMGB1 or HMGB2. We find that either of the two minimal DNA binding domains of HMGB1 is effective in assembling RAG1/2-RSS complexes on naked DNA and stimulating V(D)J cleavage but that both domains are required for efficient activity when the RSS is incorporated into a nucleosome. The single-domain HMGB protein from Saccharomyces cerevisiae, Nhp6A, efficiently assembles RAG1/2 complexes on naked DNA; however, these complexes are minimally competent for V(D)J cleavage. Nhp6A forms much more stable DNA complexes than HMGB1, and a variety of mutations that destabilize Nhp6A binding to bent microcircular DNA promote increased V(D)J cleavage. One of the two DNA bending wedges on Nhp6A and the analogous phenylalanine wedge at the DNA exit site of HMGB1 domain A were found to be essential for promoting RAG1/2-RSS complex formation. Because the phenylalanine wedge is required for specific recognition of DNA kinks, we propose that HMGB proteins facilitate RAG1/2-RSS interactions by recognizing a distorted DNA structure induced by RAG1/2 binding. The resulting complex must be sufficiently dynamic to enable the series of RAG1/2-mediated chemical reactions on the DNA.

Amino Acid Sequence↗

PubMed2004

Micromechanical analysis of the binding of DNA-bending proteins HMGB1, NHP6A, and HU reveals their ability to form highly stable DNA-protein complexes.

The mechanical response generated by binding of the nonspecific DNA-bending proteins HMGB1, NHP6A, and HU to single tethered 48.5 kb lambda-DNA molecules is investigated using DNA micromanipulation. As protein concentration is increased, the force needed to extend the DNA molecule increases, due to its compaction by protein-generated bending. Most significantly, we find that for each of HMGB1, NHP6A, and HU there is a well-defined protein concentration, not far above the binding threshold, above which the proteins do not spontaneously dissociate. In this regime, the amount of protein bound to the DNA, as assayed by the degree to which the DNA is compacted, is unperturbed either by replacing the surrounding protein solution with protein-free buffer or by straightening of the molecule by applied force. Thus, the stability of the protein-DNA complexes formed is dependent on the protein concentration during the binding. HU is distinguished by a switch to a DNA-stiffening function at the protein concentration where the formation of highly stable complexes occurs. Finally, introduction of competitor DNA fragments into the surrounding solution disassembles the stable DNA complexes with HMGB1, NHP6A, and HU within seconds. Since spontaneous dissociation of protein does not occur on a time scale of hours, we conclude that this rapid protein exchange in the presence of competitor DNA must occur only via "direct" DNA-DNA contact. We therefore observe that protein transport along DNA by direct transfers occurs even for proteins such as NHP6A and HU that have only one DNA-binding domain.

Animals↗

PubMed2004

Architecture of the hin synaptic complex during recombination: the recombinase subunits translocate with the DNA strands.

Most site-specific recombinases can be grouped into two mechanistically distinct families. Whereas tyrosine recombinases exchange DNA strands through a Holliday intermediate, serine recombinases such as Hin generate double-strand breaks in each recombining partner. Here, site-directed protein crosslinking is used to elucidate the configuration of protein subunits and DNA within the Hin synaptic complex and to follow the movement of protein subunits during DNA strand exchange. Our results show that the protein interface mediating synapsis is localized to a region within the catalytic domains, thereby positioning the DNA strands on the outside of the Hin tetrameric complex. Unexpected crosslinks between residues within the dimerization helices provide evidence for a conformational change that accompanies DNA cleavage. We demonstrate that the Hin subunits, which are linked to the cleaved DNA ends by serine-phosphodiester bonds, translocate between synapsed dimers to exchange the DNA strands.

Binding Sites↗

PubMed2004

Stepwise dissection of the Hin-catalyzed recombination reaction from synapsis to resolution.

The Hin DNA invertase promotes a site-specific DNA recombination reaction in the Salmonella chromosome. The native Hin reaction exhibits overwhelming selectivity for promoting inversions between appropriately oriented recombination sites and requires the Fis regulatory protein, a recombinational enhancer, and a supercoiled DNA substrate. Here, we report a robust recombination reaction employing oligonucleotide substrates and a hyperactive mutant form of Hin. Synaptic complex intermediates purified by gel electrophoresis were found to contain four Hin protomers bound to two recombination sites. Each Hin protomer is associated covalently with a cleaved DNA end. The cleaved complexes can be ligated into both parental and recombinant orientations at equivalent frequencies, provided the core residues can base-pair, and are readily disassembled into separated DNA fragments bound by Hin dimers. Kinetic analyses reveal that synapsis occurs rapidly, followed by comparatively slow Hin-catalyzed DNA cleavage. Subsequent steps of the reaction, including DNA exchange and ligation, are fast. Thus, post-synaptic step(s) required for DNA cleavage limit the overall rate of the recombination reaction.

Amino Acid Substitution↗

PubMed2004

Crystal structure of the excisionase-DNA complex from bacteriophage lambda.

The excisionase (Xis) protein from bacteriophage lambda is the best characterized member of a large family of recombination directionality factors that control integrase-mediated DNA rearrangements. It triggers phage excision by cooperatively binding to sites X1 and X2 within the phage, bending DNA significantly and recruiting the phage-encoded integrase (Int) protein to site P2. We have determined the co-crystal structure of Xis with its X2 DNA-binding site at 1.7A resolution. Xis forms a unique winged-helix motif that interacts with the major and minor grooves of its binding site using an alpha-helix and an ordered beta-hairpin (wing), respectively. Recognition is achieved through an elaborate water-mediated hydrogen-bonding network at the major groove interface, while the preformed hairpin forms largely non-specific interactions with the minor groove. The structure of the complex provides insights into how Xis recruits Int cooperatively, and suggests a plausible mechanism by which it may distort longer DNA fragments significantly. It reveals a surface on the protein that is likely to mediate Xis-Xis interactions required for its cooperative binding to DNA.

Amino Acid Sequence↗

PubMed2004

Topological analysis of Hin-catalysed DNA recombination in vivo and in vitro.

In vitro studies have demonstrated that Hin-catalysed site-specific DNA inversion occurs within a tripartite invertasome complex assembled at a branch on a supercoiled DNA molecule. Multiple DNA exchanges within a recombination complex (processive recombination) have been found to occur with particular substrates or reaction conditions. To investigate the mechanistic properties of the Hin recombination reaction in vivo, we have analysed the topology of recombination products generated by Hin catalysis in growing cells. Recombination between wild-type recombination sites in vivo is primarily limited to one exchange. However, processive recombination leading to knotted DNA products is efficient on substrates containing recombination sites with non-identical core nucleotides. Multiple exchanges are limited by a short DNA segment between the Fis-bound enhancer and closest recombination site and by the strength of Fis-Hin interactions, implying that the enhancer normally remains associated with the recombining complex throughout a single exchange reaction, but that release of the enhancer leads to multiple exchanges. This work confirms salient mechanistic aspects of the reaction in vivo and provides strong evidence for the propensity of plectonemically branched DNA in prokaryotic cells. We also demonstrated that a single DNA exchange resulting in inversion in vitro is accompanied by a loss of four negative supercoils.

Bacterial Proteins↗

PubMed2004

Activation of transcription initiation from a stable RNA promoter by a Fis protein-mediated DNA structural transmission mechanism.

The leuV operon of Escherichia coli encodes three of the four genes for the tRNA1Leu isoacceptors. Transcription from this and other stable RNA promoters is known to be affected by a cis-acting UP element and by Fis protein interactions with the carboxyl-terminal domain of the alpha-subunits of RNA polymerase. In this report, we suggest that transcription from the leuV promoter also is activated by a Fis-mediated, DNA supercoiling-dependent mechanism similar to the IHF-mediated mechanism described previously for the ilvP(G) promoter (S. D. Sheridan et al., 1998, J Biol Chem 273: 21298-21308). We present evidence that Fis binding results in the translocation of superhelical energy from the promoter-distal portion of a supercoiling-induced DNA duplex destabilized (SIDD) region to the promoter-proximal portion of the leuV promoter that is unwound within the open complex. A mutant Fis protein, which is defective in contacting the carboxyl-terminal domain of the alpha-subunits of RNA polymerase, remains competent for stimulating open complex formation, suggesting that this DNA supercoiling-dependent component of Fis-mediated activation occurs in the absence of specific protein interactions between Fis and RNA polymerase. Fis-mediated translocation of superhelical energy from upstream binding sites to the promoter region may be a general feature of Fis-mediated activation of transcription at stable RNA promoters, which often contain A+T-rich upstream sequences.

Base Sequence↗

PubMed2004

The DNA architectural protein HMGB1 facilitates RTA-mediated viral gene expression in gamma-2 herpesviruses.

Replication and transcription activator (RTA), an immediate-early gene product of gamma-2 herpesviruses including Kaposi's sarcoma-associated herpesvirus (KSHV) and murine gamma herpesvirus 68 (MHV-68), plays a critical role in controlling the viral life cycle. RTA acts as a strong transcription activator for several downstream genes of KSHV and MHV-68 through direct DNA binding, as well as via indirect mechanisms. HMGB1 (also called HMG-1) protein is a highly conserved nonhistone chromatin protein with the ability to bind and bend DNA. HMGB1 protein promoted RTA binding to different RTA target sites in vitro, with greater enhancement to low-affinity sites than to high-affinity sites. Box A or box B and homologues of HMGB1 also enhanced RTA binding to DNA. Transient transfection of HMGB1 stimulated RTA transactivation of RTA-responsive promoters from KSHV and MHV-68. Furthermore, MHV-68 viral gene expression, as well as viral replication, was significantly reduced in HMGB1-deficient cells than in the wild type. This abated viral gene expression was partially restored by HMGB1 transfection into HMGB1(-/-) cells. These results suggest an important function of the DNA architectural protein, HMGB1, in RTA-mediated gene expression, as well as viral replication in gamma-2 herpesviruses.

Animals↗

PubMed2003

Fis stabilizes the interaction between RNA polymerase and the ribosomal promoter rrnB P1, leading to transcriptional activation.

It has been shown that Fis activates transcription of the ribosomal promoter rrnB P1; however, the mechanism by which Fis activates rrnB P1 transcription is not fully understood. Paradoxically, although Fis activates transcription of rrnB P1 in vitro, transcription from the promoter containing Fis sites (as measured from rrnB P1-lacZ fusions) is not reduced in a fis null mutant strain. In this study, we further investigated the mechanism by which Fis activates transcription of the rrnB P1 promoter and the role of Fis in rRNA synthesis and cell growth in Escherichia coli. Like all other stringent promoters investigated so far, open complex of rrnB P1 has been shown to be intrinsically unstable, making open complex stability a potential regulatory step in transcription of this class of promoters. Our results show that Fis acts at this regulatory step by stabilizing the interaction between RNA polymerase and rrnB P1 in the absence of NTPs. Mutational analysis of the Fis protein demonstrates that there is a complete correlation between Fis-mediated transcriptional activation of rrnB P1 and Fis-mediated stabilization of preinitiation complexes of the promoter. Thus, our study indicates that Fis-mediated stabilization of RNA polymerase-rrnB P1 preinitiation complexes, presumably at the open complex step, contributes prominently to transcriptional activation. Furthermore, our in vivo results show that rRNA synthesis from the P1 promoters of several rRNA operons are reduced 2-fold in a fis null mutant compared with the wild type strain, indicating that Fis plays an important role in the establishment of robust rRNA synthesis when E. coli cells are emerging from a growth-arrested phase to a rapid growth phase. Thus, our results resolve an apparent paradox of the role of Fis in vitro and in vivo in the field.

Cell Division↗

PubMed2002

Regulation of directionality in bacteriophage lambda site-specific recombination: structure of the Xis protein.

Upon induction of a bacteriophage lambda lysogen, a site-specific recombination reaction excises the phage genome from the chromosome of its bacterial host. A critical regulator of this process is the phage-encoded excisionase (Xis) protein, which functions both as a DNA architectural factor and by cooperatively recruiting integrase to an adjacent binding site specifically required for excision. Here we present the three-dimensional structure of Xis and the results of a structure-based mutagenesis study to define the molecular basis of its function. Xis adopts an unusual "winged"-helix motif that is modeled to interact with the major- and minor-grooves of its binding site through a single alpha-helix and loop structure ("wing"), respectively. The C-terminal tail of Xis, which is required for cooperative binding with integrase, is unstructured in the absence of DNA. We propose that asymmetric bending of DNA by Xis positions its unstructured C-terminal tail for direct contacts with the N-terminal DNA-binding domain of integrase and that an ensuing disordered to ordered transition of the tail may act to stabilize the formation of the tripartite integrase-Xis-DNA complex required for phage excision.

Amino Acid Motifs↗

PubMed2002

Differential affinity and cooperativity functions of the amino-terminal 70 residues of lambda integrase.

The site-specific recombinase (Int) of bacteriophage lambda is a heterobivalent DNA-binding protein that binds two different classes of DNA-binding sites within its recombination target sites. The several functions of Int are apportioned between a large carboxy-terminal domain that cleaves and ligates DNA at each of its four "core-type" DNA-binding sites and a small amino-terminal domain, whose primary function is binding to each of its five "arm-type" DNA sites, which are distant from the core region. Int bridges between the two classes of binding sites are facilitated by accessory DNA-bending proteins that along with Int comprise higher-order recombinogenic complexes. We show here that although the 64 amino-terminal residues of Int bind efficiently to a single arm site, this protein cannot form doubly bound complexes on adjacent arm sites. However, 1-70 Int does show the same cooperative binding to adjacent arm sites as the full length protein. We also found that 1-70 Int specifies cooperative interactions with the accessory protein Xis when the two are bound to their adjacent cognate sites P2 and X1, respectively. To complement the finding that these two amino-terminal domain functions (along with arm DNA binding) are all specified by residues 1-70, we determined that Thr75 is the first residue of the minimal carboxy-terminal domain, thereby identifying a specific interdomain linker region. We have measured the affinity constants for Int binding to each of the five arm sites and the cooperativity factors for Int binding to the two pairs of adjacent arm sites, and we have identified several DNA structural features that contribute to the observed patterns of Int binding to arm sites. Taken together, the results highlight several interesting features of arm DNA binding that invite speculation about additional levels of complexity in the regulation of lambda site-specific recombination.

Amino Acid Sequence↗

PubMed2002

The S. cerevisiae architectural HMGB protein NHP6A complexed with DNA: DNA and protein conformational changes upon binding.

NHP6A is a non-sequence-specific DNA-binding protein from Saccharomyces cerevisiae which belongs to the HMGB protein family. Previously, we have solved the structure of NHP6A in the absence of DNA and modeled its interaction with DNA. Here, we present the refined solution structures of the NHP6A-DNA complex as well as the free 15bp DNA. Both the free and bound forms of the protein adopt the typical L-shaped HMGB domain fold. The DNA in the complex undergoes significant structural rearrangement from its free form while the protein shows smaller but significant conformational changes in the complex. Structural and mutational analysis as well as comparison of the complex with the free DNA provides insight into the factors that contribute to binding site selection and DNA deformations in the complex. Further insight into the amino acid determinants of DNA binding by HMGB domain proteins is given by a correlation study of NHP6A and 32 other HMGB domains belonging to both the DNA-sequence-specific and non-sequence-specific families of HMGB proteins. The resulting correlations can be rationalized by comparison of solved structures of HMGB proteins.

Amino Acid Sequence↗

PubMed2002

Subunit exchange and the role of dimer flexibility in DNA binding by the Fis protein.

Fis is an abundant bacterial DNA binding protein that functions in many different reactions. We show here that Fis subunits rapidly exchange between dimers in solution by disulfide cross-linking mixtures of Fis mutants with different electrophoretic mobilities and by monitoring energy transfer between fluorescently labeled Fis subunits upon heterodimer formation. The effects of detergents and salt concentrations on subunit exchange imply that the dimer is predominantly stabilized by hydrophobic forces, consistent with the X-ray crystal structures. Specific and nonspecific DNA strongly inhibit Fis subunit exchange. In all crystal forms of Fis, the separation between the DNA recognition helices within the Fis dimer is too short to insert into adjacent major grooves on canonical B-DNA, implying that conformational changes within the Fis dimer and/or the DNA must occur upon binding. We therefore investigated the functional importance of dimer interface flexibility for Fis-DNA binding by studying the DNA binding properties of Fis mutants that were cross-linked at different positions in the dimer. Flexibility within the core dimer interface does not appear to be required for efficient DNA binding, Fis-DNA complex dissociation, or Fis-induced DNA bending. Moreover, FRET-based experiments provided no evidence for a change in the spatial relationship between the two helix-turn-helix motifs in the Fis dimer upon DNA binding. These results support a model in which the unusually short distance between DNA recognition helices on Fis is accommodated primarily through bending of the DNA.

Blotting, Western↗

PubMed2002

Binding to cisplatin-modified DNA by the Saccharomyces cerevisiae HMGB protein Nhp6A.

Nhp6A is an abundant non-histone chromatin-associated protein in Saccharomyces cerevisiae that contains a minor groove DNA binding motif called the HMG box. In this report, we show that Nhp6Ap binds to cisplatin intrastrand cross-links on duplex DNA with a 40-fold greater affinity than to unmodified DNA with the same sequence. Nevertheless, Nhp6Ap bound to cisplatinated DNA readily exchanges onto unmodified DNA. Phenanthroline-copper footprinting and two-dimensional NMR on complexes of wild-type and mutant Nhp6Ap with DNA were employed to probe the mode of binding to the cisplatin lesion. Recognition of the cisplatin adduct requires a surface-exposed phenylalanine on Nhp6Ap that promotes bending of DNA by inserting into the helix from the minor groove. We propose that Nhp6Ap targets the cisplatin adduct by means of intercalation by the phenylalanine and that it can bind in either orientation with respect to the DNA lesion. A methionine, which also inserts between base pairs and functions in target selection on unmodified DNA, plays no apparent role in recognition of the cisplatin lesion. Basic amino acids within the N-terminal arm of Nhp6Ap are required for high-affinity binding to the cisplatin adduct as well as to unmodified DNA. Cisplatin mediates its cytotoxicity by forming covalent adducts on DNA, and we find that Deltanhp6a/b mutants are hypersensitive to cisplatin in comparison with the wild-type strain. In contrast, Deltanhp6a/b mutants are slightly more resistant to hydrogen peroxide and ultraviolet irradiation. Therefore, Nhp6A/Bp appears to directly or indirectly function in yeast to enhance cellular resistance to cisplatin.

Amino Acid Motifs↗

PubMed2002

Identification of the tRNA-dihydrouridine synthase family.

5,6-Dihydrouridine (D) is a modified base found abundantly in the D-loops of tRNA from Archaea, Bacteria, and Eukarya. D is thought to be formed post-transcriptionally by the reduction of uridines in tRNA transcripts. Despite its abundance, no enzymes that catalyze D-formation have been identified. Using comparative genomics and computational methods we have identified members of the cluster of orthologous genes, COG0042, as putative dihydrouridine synthase encoding genes. Escherichia coli contains three COG0042 family members (yjbN, yhdG, and yohI). Strains were created where one, two, or all three of the COG0042 genes were deleted. Purified tRNA samples were investigated from the three single and the three double knockout strains, as well as from the triple deletion strain. The results showed that the COG0042 gene family is responsible for tRNA-dihydrouridine synthase activity in E. coli. They also suggest that the COG0042-encoded family members act site-specifically on the tRNA D-loop and contain non-redundant catalytic functions in vivo.

Escherichia coli↗