Claire A. Adams

The Gts1 protein stabilizes the autonomous oscillator in yeast.

Continuous cultures of Saccharomyces cerevisiae show a robust autonomous temperature compensated oscillation in many metabolic functions. Respiratory activity, a convenient output to measure, oscillates with a period of 40 min. Deletion of GTS1, whose protein product has homology to the circadian per protein, has been implicated in temporal events within yeast, causes a reduction in periodicity to 18 min (wild‐type period 40–60 min). The dilution rate was steadily increased from 0.04/h to 0.085/h and the oscillation stabilized after four to six dilutions. However, Gts1ps involvement in the maintenance and generation of metabolic synchrony, and in the central oscillating loop, appear to be minimal, as the mutant oscillation was robust and autonomous. Deletion of GTS1 did cause decreased temperature compensation of the period of the oscillation from Q10 = 1.07 for the wild‐type to Q10 = 1.6 for the mutant. Also the degree of nutrient compensation observed for the wild‐type was not observed in the GTS1‐null mutant strain. It is postulated that Gts1p is involved in the mechanism that communicates external conditions, such as temperature, to the central oscillating loop. Copyright

EbfC (YbaB) Is a New Type of Bacterial Nucleoid-Associated Protein and a Global Regulator of Gene Expression in the Lyme Disease Spirochete

Nearly every known species of Eubacteria encodes a homolog of the Borrelia burgdorferi EbfC DNA-binding protein. We now demonstrate that fluorescently tagged EbfC associates with B. burgdorferi nucleoids in vivo and that chromatin immunoprecipitation (ChIP) of wild-type EbfC showed it to bind in vivo to sites throughout the genome, two hallmarks of nucleoid-associated proteins. Comparative RNA sequencing (RNA-Seq) of a mutant B. burgdorferi strain that overexpresses EbfC indicated that approximately 4.5% of borrelial genes are significantly impacted by EbfC. The ebfC gene was highly expressed in rapidly growing bacteria, but ebfC mRNA was undetectable in stationary phase. Combined with previous data showing that EbfC induces bends in DNA, these results demonstrate that EbfC is a nucleoid-associated protein and lead to the hypothesis that B. burgdorferi utilizes cellular fluctuations in EbfC levels to globally control transcription of numerous genes. The ubiquity of EbfC proteins in Eubacteria suggests that these results apply to a wide range of pathogens and other bacteria.

Topologies of Complexes Containing O6-Alkylguanine–DNA Alkyltransferase and DNA

The mutagenic and cytotoxic effects of many alkylating agents are reduced by O(6)-alkylguanine-DNA alkyltransferase (AGT). In humans, this protein not only protects the integrity of the genome, but also contributes to the resistance of tumors to DNA-alkylating chemotherapeutic agents. Here we describe and test models for cooperative multiprotein complexes of AGT with single-stranded and duplex DNAs that are based on in vitro binding data and the crystal structure of a 1:1 AGT-DNA complex. These models predict that cooperative assemblies contain a three-start helical array of proteins with dominant protein-protein interactions between the amino-terminal face of protein n and the carboxy-terminal face of protein n+3, and they predict that binding duplex DNA does not require large changes in B-form DNA geometry. Experimental tests using protein cross-linking analyzed by mass spectrometry, electrophoretic and analytical ultracentrifugation binding assays, and topological analyses with closed circular DNA show that the properties of multiprotein AGT-DNA complexes are consistent with these predictions.

BpaB and EbfC DNA-binding proteins regulate production of the Lyme disease spirochete's infection-associated Erp surface proteins

Vector-borne pathogens regulate their protein expression profiles, producing factors during host infection that differ from those produced during vector colonization. The Lyme disease agent, Borrelia burgdorferi, produces Erp surface proteins throughout mammalian infection and represses their synthesis during colonization of vector ticks. Known functions of Erp proteins include binding of host laminin, plasmin(ogen), and regulators of complement activation. A DNA region immediately 5 of erp operons, the erp operator, is required for transcriptional regulation. The B. burgdorferi BpaB and EbfC proteins exhibit high in vitro affinities for erp operator DNA. In the present studies, chromatin immunoprecipitation (ChIP) demonstrated that both proteins bind erp operator DNA in vivo. Additionally, a combination of in vivo and in vitro methods demonstrated that BpaB functions as a repressor of erp transcription, while EbfC functions as an antirepressor.

BpaB, a novel protein encoded by the Lyme disease spirochete's cp32 prophages, binds to erp Operator 2 DNA.

Borrelia burgdorferi produces Erp outer surface proteins throughout mammalian infection, but represses their synthesis during colonization of vector ticks. A DNA region 5′ of the start of erp transcription, Operator 2, was previously shown to be essential for regulation of expression. We now report identification and characterization of a novel erp Operator 2-binding protein, which we named BpaB. erp operons are located on episomal cp32 prophages, and a single bacterium may contain as many as 10 different cp32s. Each cp32 family member encodes a unique BpaB protein, yet the three tested cp32-encoded BpaB alleles all bound to the same DNA sequence. A 20-bp region of erp Operator 2 was determined to be essential for BpaB binding, and initial protein binding to that site was required for binding of additional BpaB molecules. A 36-residue region near the BpaB carboxy terminus was found to be essential for high-affinity DNA-binding. BpaB competed for binding to erp Operator 2 with a second B. burgdorferi DNA-binding protein, EbfC. Thus, cellular levels of free BpaB and EbfC could potentially control erp transcription levels.

Mutations That Probe the Cooperative Assembly of O6-Alkylguanine-DNA Alkyltransferase Complexes

O(6)-Alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O(6)-alkylguanine and O(4)-alkylthymine adducts present in DNA that has been exposed to alkylating agents. AGT binds DNA cooperatively, and models of cooperative complexes predict that residues 1-7 of one protein molecule and residues 163-169 of a neighboring protein are closely juxtaposed. To test these models, we used directed mutagenesis to substitute triplets of alanine for triplets of native residues across these two sequences. Six of eight designed mutants expressed AGT at detectable levels. All mutant AGTs that were expressed were folded compactly, bound DNA with stoichiometries equivalent to that of the wild-type protein, and were able to protect Escherichia coli to varying degrees from the potent alkylating agent N-methyl-N-nitro-N-nitrosoguanidine (MNNG). All mutations attenuated DNA binding cooperativity, but unexpectedly, they also reduced the affinity of AGT for DNA. This suggests that the protein-protein and protein-DNA interactions of AGT are strongly coupled. When normalized for differences in AGT expression, cells expressing mutants KDC(3-5)-AAA, DCE(4-6)-AAA, and KEW(165-167)-AAA were significantly more susceptible to MNNG than wild-type cells. This is the first evidence, to the best of our knowledge, of a role for residues at the protein-protein interface and, by implication, cooperative protein-protein interactions in the cell-protective mechanisms of AGT.

Borrelia burgdorferi cp32 BpaB Modulates Expression of the Prophage NucP Nuclease and SsbP Single-Stranded DNA-Binding Protein

The Borrelia burgdorferi BpaB proteins of the spirochetes ubiquitous cp32 prophages are DNA-binding proteins, required both for maintenance of the bacteriophage episomes and for transcriptional regulation of the cp32 erp operons. Through use of DNase I footprinting, we demonstrate that BpaB binds the erp operator initially at the sequence 5-TTATA-3. Electrophoretic mobility shift assays indicated that BpaB also binds with high affinity to sites located in the 5 noncoding regions of two additional cp32 genes. Characterization of the proteins encoded by those genes indicated that they are a single-stranded DNA-binding protein and a nuclease, which we named SsbP and NucP, respectively. Chromatin immunoprecipitation indicated that BpaB binds erp, ssbP, and nucP in live B. burgdorferi. A mutant bacterium that overexpressed BpaB produced significantly higher levels of ssbP and nucP transcript than did the wild-type parent.

Analysis of Protein-DNA Equilibria by Native Gel Electrophoresis

The electrophoretic mobility-shift assay (EMSA) is widely used to detect protein–nucleic acid interactions [for representative reviews, see Garner and Revzin (1986), Chodosh (1988), Fried (1989), Carey (1991), Lane et al. (1992), Fried and Garner (1998)]. A particular strength of this method is its ability to detect the simultaneous binding of several proteins to a single molecule of nucleic acid (Figure 14.1), or one (or more) protein(s) to several nucleic acids. Such interactions are of interest because nucleic acid complexes containing large number of proteins play central roles in important cellular transactions including (but not limited to) DNA replication, recombination and repair, and RNA transcription and processing. Although mobility-shift assays are often used for qualitative purposes, under appropriate conditions they can provide quantitative data for the determination of binding stoichiometries, affinities, and kinetics.

GLR1 Plays an Essential Role in the Homeodynamics of Glutathione and the Regulation of H2S Production during Respiratory Oscillation of Saccharomyces cerevisiae

The role of glutathione (GSH) and its homeodynamics during respiratory oscillation of Saccharomyces cerevisiae were investigated. Pulse injection of thiol redox modifying agents, such as diethylmaleate, N-ethylmaleimide, DL-butione-[S,R]-sulfoxamine, or 5-nitro-2-furaldehyde into the culture perturbed oscillation, although the degree of perturbation varied. Analysis of the expression profiles of GSH1 and GLR1, the activities of glutathione reductase, oscillations in cysteine and GSH concentrations, and the chemostat culture of the GLR1 disruptant indicated that GLR1 plays an essential role in the homeodynamics of GSH and the regulation of H2S production.