Binding kinetics and activity of human poly(ADP‐ribose) polymerase‐1 on oligo‐deoxyribonucleotide substrates

Poly(ADP‐ribose) polymerase‐1 (PARP‐1) is a mammalian enzyme that attaches long branching chains of ADP‐ribose to specific nuclear proteins, including itself. Because its activity in vitro is dependent upon interaction with broken DNA, it has been postulated that PARP‐1 plays an important role in DNA strand‐break repair in vivo. The exact mechanism of binding to DNA and the structural determinants of binding remain to be defined, but regions of transition from single‐stranded to double‐strandedness may be important recognition sites. Here we employ surface plasmon resonance (SPR) to investigate this hypothesis. Oligodeoxynucleotide (ODN) substrates that mimic DNA with different degrees of single‐strandedness were used for measurements of both PARP‐1/DNA binding kinetics and PARP‐1's enzyme activities. We found that binding correlated with activity, but was unrelated to single‐strandedness of the ODN. Instead, PARP‐1 binding and activity were highest on ODNs that modeled a DNA double‐strand break (DSB). These results provide support for PARP‐1 recognizing and binding DSBs in a manner that is independent of single‐stranded features, and demonstrate the usefulness of SPR for simultaneously investigating both PARP‐1 binding and PARP‐1 auto‐poly(ADP‐ribosyl)ation activities within the same in vitro system. Copyright © 2009 John Wiley & Sons, Ltd.     

Phosphorylation of the cell division protein GpsB regulates PrkC kinase activity through a negative feedback loop in Bacillus subtilis

Although many membrane Ser/Thr‐kinases with PASTA motifs have been shown to control bacterial cell division and morphogenesis, inactivation of the Ser/Thr‐kinase PrkC does not impact Bacillus subtilis cell division. In this study, we show that PrkC localizes at the division septum. In addition, three proteins involved in cell division/elongation, GpsB, DivIVA and EzrA are required for stimulating PrkC activity in vivo. We show that GpsB interacts with the catalytic subunit of PrkC that, in turn, phosphorylates GpsB. These observations are not made with DivIVA and EzrA. Consistent with the phosphorylated residue previously detected for GpsB in a high‐throughput phosphoproteomic analysis of B. subtilis, we show that threonine 75 is the single PrkC‐mediated phosphorylation site in GpsB. Importantly, the substitution of this threonine by a phospho‐mimetic residue induces a loss of PrkC kinase activity in vivo and a reduced growth under high salt conditions as observed for gpsB and prkC null mutants. Conversely, substitution of threonine 75 by a phospho‐ablative residue does not induce such growth and PrkC kinase activity defects. Altogether, these data show that proteins of the divisome control PrkC activity and thereby phosphorylation of PrkC substrates through a negative feedback loop in B. subtilis.     

Bacillus subtilis SalA is a phosphorylation‐dependent transcription regulator that represses scoC and activates the production of the exoprotease AprE

Bacillus subtilis Mrp family protein SalA has been shown to indirectly promote the production of the exoprotease AprE by inhibiting the expression of scoC, which codes for a repressor of aprE. The exact mechanism by which SalA influences scoC expression has not been clarified previously. We demonstrate that SalA possesses a DNA‐binding domain (residues 1–60), which binds to the promoter region of scoC. The binding of SalA to its target DNA depends on the presence of ATP and is stimulated by phosphorylation of SalA at tyrosine 327. The B. subtilis protein‐tyrosine kinase PtkA interacts specifically with the C‐terminal domain of SalA in vivo and in vitro and is responsible for activating its DNA binding via phosphorylation of tyrosine 327. In vivo, a mutant mimicking phosphorylation of SalA (SalA Y327E) exhibited a strong repression of scoC and consequently overproduction of AprE. By contrast, the non‐phosphorylatable SalA Y327F and the ΔptkA exhibited the opposite effect, stronger expression of scoC and lower production of the exoprotease. Interestingly, both SalA and PtkA contain the same ATP‐binding Walker domain and have thus presumably arisen from the common ancestral protein. Their regulatory interplay seems to be conserved in other bacteria.     

Structural and biochemical analyses of YvgN and YtbE from Bacillus subtilis

Bacillus subtilis is one of the most studied gram‐positive bacteria. In this work, YvgN and YtbE from B. subtilis, assigned as AKR5G1 and AKR5G2 of aldo‐keto reductase (AKR) superfamily. AKR catalyzes the NADPH‐dependent reduction of aldehyde or aldose substrates to alcohols. YvgN and YtbE were studied by crystallographic and enzymatic analyses. The apo structures of these proteins were determined by molecular replacement, and the structure of holoenzyme YvgN with NADPH was also solved, revealing the conformational changes upon cofactor binding. Our biochemical data suggest both YvgN and YtbE have preferential specificity for derivatives of benzaldehyde, such as nitryl or halogen group substitution at the 2 or 4 positions. These proteins also showed broad catalytic activity on many standard substrates of AKR, such as glyoxal, dihydroxyacetone, and DL‐glyceraldehyde, suggesting a possible role in bacterial detoxification.     

The recognition of mismatched base pairs in DNA by DNase I from Ustilago maydis.

Summary The activity of Ustilago maydis DNAse I, an enzyme implicated in genetic recombination, on DNA substrates containing unpaired or mismatched bases, was examined. The enzyme nicked supercoiled PM-2 molecules, converting these to relaxed circular and linear molecules. Discrete double stranded linear fragments smaller than unit length were also observed after digestion at high enzyme concentration. Heteroduplex molecules were constructed using 80 bacteriophage derivatives which contained single base substitutions within the E. coli tRNA ₁ ^tyr gene. Single and double stranded nicking at or near the single mismatched site was observed with three out of the five pairs of heteroduplexes.     

A RAD52‐like single‐stranded DNA binding protein affects mitochondrial DNA repair by recombination

The plant mitochondrial DNA‐binding protein ODB1 was identified from a mitochondrial extract after DNA‐affinity purification. ODB1 (organellar DNA‐binding protein 1) co‐purified with WHY2, a mitochondrial member of the WHIRLY family of plant‐specific proteins involved in the repair of organellar DNA. The Arabidopsis thaliana ODB1 gene is identical to RAD52‐1, which encodes a protein functioning in homologous recombination in the nucleus but additionally localizing to mitochondria. We confirmed the mitochondrial localization of ODB1 by in vitro and in vivo import assays, as well as by immunodetection on Arabidopsis subcellular fractions. In mitochondria, WHY2 and ODB1 were found in large nucleo‐protein complexes. Both proteins co‐immunoprecipitated in a DNA‐dependent manner. In vitro assays confirmed DNA binding by ODB1 and showed that the protein has higher affinity for single‐stranded than for double‐stranded DNA. ODB1 showed no sequence specificity in vitro. In vivo, DNA co‐immunoprecipitation indicated that ODB1 binds sequences throughout the mitochondrial genome. ODB1 promoted annealing of complementary DNA sequences, suggesting a RAD52‐like function as a recombination mediator. Arabidopsis odb1 mutants were morphologically indistinguishable from the wild‐type, but following DNA damage by genotoxic stress, they showed reduced mitochondrial homologous recombination activity. Under the same conditions, the odb1 mutants showed an increase in illegitimate repair bypasses generated by microhomology‐mediated recombination. These observations identify ODB1 as a further component of homologous recombination‐dependent DNA repair in plant mitochondria.     

Simple,fast and high‐efficiency transformation system for directed evolution of cellulase in Bacillus subtilis

Bacillus subtilis can serve as a powerful platform for directed evolution, especially for secretory enzymes. However, cloning and transformation of a DNA mutant library in B. subtilis are not as easy as they are in Escherichia coli. For direct transformation of B. subtilis, here we developed a new protocol based on supercompetent cells prepared from the recombinant B. subtilis strain SCK6 and multimeric plasmids. This new protocol is simple (restriction enzyme‐, phosphatase‐ and ligase‐free), fast (i.e. 1 day) and of high efficiency (i.e. ～10⁷ or ～10⁴ transformants per µg of multimeric plasmid or ligated plasmid DNA respectively). Supercompetent B. subtilis SCK6 cells were prepared by overexpression of the competence master regulator ComK that was induced by adding xylose. The DNA mutant library was generated through a two‐round PCR: (i) the mutagenized DNA fragments were generated by error‐prone PCR and linearized plasmids were made using high‐fidelity PCR, and (ii) the multimeric plasmids were generated based on these two DNA templates by using overlap PCR. Both protein expression level and specific activity of glycoside hydrolase family 5 endoglucanse on regenerated amorphous cellulose were improved through this new system. To our limited knowledge, this study is the first report for enhancing secretory cellulase performance on insoluble cellulose.     

Structure and function of a novel endonuclease acting on branched DNA substrates

We show that Pyrococcus abyssi PAB2263 (dubbed NucS (nuc lease for s s DNA) is a novel archaeal endonuclease that interacts with the replication clamp PCNA. Structural determination of P. abyssi NucS revealed a two‐domain dumbbell‐like structure that in overall does not resemble any known protein structure. Biochemical and structural studies indicate that NucS orthologues use a non‐catalytic ssDNA‐binding domain to regulate the cleavage activity at another site, thus resulting into the specific cleavage at double‐stranded DNA (dsDNA)/ssDNA junctions on branched DNA substrates. Both 3′ and 5′ extremities of the ssDNA can be cleaved at the nuclease channel that is too narrow to accommodate duplex DNA. Altogether, our data suggest that NucS proteins constitute a new family of structure‐specific DNA endonucleases that are widely distributed in archaea and in bacteria, including Mycobacterium tuberculosis.     

Deinococcus radiodurans DR1088 is a novel RecF‐interacting protein that stimulates single‐stranded DNA annealing

RecF, together with the recombination mediators RecO and RecR, is required in the RecFOR homologous recombination repair pathway in bacteria. In this study, a recF‐dr1088 operon, which is highly conserved in the Deinococcus‐Thermus phylum, was identified in Deinococcus radiodurans. Interaction between DRRecF and DR1088 was confirmed by yeast two‐hybrid and pull‐down assays. DR1088 exhibited some RecO‐like biochemical properties including single/double‐stranded DNA binding activity, ssDNA binding protein (SSB) replacement ability and ssDNA (with or without SSB) annealing activity. However, unlike other recombination proteins, dr1088 is essential for cell viability. These results indicate that DR1088 might play a role in DNA replication and DNA repair processes.     

YocM a small heat shock protein can protect Bacillus subtilis cells during salt stress

Small heat shock proteins (sHsp) occur in all domains of life. By interacting with misfolded or aggregated proteins these chaperones fulfill a protective role in cellular protein homeostasis. Here, we demonstrate that the sHsp YocM of the Gram‐positive model organism Bacillus subtilis is part of the cellular protein quality control system with a specific role in salt stress response. In the absence of YocM the survival of salt shocked cells is impaired, and increased levels of YocM protect B. subtilis exposed to heat or salt. We observed a salt and heat stress‐induced localization of YocM to intracellular protein aggregates. Interestingly, purified YocM appears to accelerate protein aggregation of different model substrates in vitro. In addition, the combined presence of YocM and chemical chaperones, which accumulate in salt stressed cells, can facilitate in vitro a synergistic protective effect on protein misfolding. Therefore, the beneficial role of YocM during salt stress could be related to a mutual functional relationship with chemical chaperones and adds a new possible functional aspect to sHsp chaperone activities.     

Bacterial transformation: ComFA is a DNA‐dependent ATPase that forms complexes with ComFC and DprA

Pneumococcal natural transformation contributes to genomic plasticity, antibiotic resistance development and vaccine escape. Streptococcus pneumoniae, like many other naturally transformable species, has evolved sophisticated protein machinery for the binding and uptake of DNA. Two proteins encoded by the comF operon, ComFA and ComFC, are involved in transformation but their exact molecular roles remain unknown. In this study, we provide experimental evidence that ComFA binds to single stranded DNA (ssDNA) and has ssDNA‐dependent ATPase activity. We show that both ComFA and ComFC are essential for the transformation process in pneumococci. Moreover, we show that these proteins interact with each other and with other proteins involved in homologous recombination, such as DprA, thus placing the ComFA‐ComFC duo at the interface between DNA uptake and DNA recombination during transformation.     

New insights in the ϕ29 terminal protein DNA‐binding and host nucleoid localization functions

Protein‐primed DNA replication constitutes a strategy to initiate viral DNA synthesis in a variety of prokaryotic and eukaryotic organisms. Although the main function of viral terminal proteins (TPs) is to provide a free hydroxyl group to start initiation of DNA replication, there are compelling evidences that TPs can also play other biological roles. In the case of Bacillus subtilis bacteriophage ?29, the N‐terminal domain of the TP organizes viral DNA replication at the bacterial nucleoid being essential for an efficient phage DNA replication, and it contains a nuclear localization signal (NLS) that is functional in eukaryotes. Here we provide information about the structural properties of the ?29 TP N‐terminal domain, which possesses sequence‐independent DNA‐binding capacity, and dissect the amino acid residues important for its biological function. By mutating all the basic residues of the TP N‐terminal domain we identify the amino acids responsible for its interaction with the B. subtilis genome, establishing a correlation between the capacity of DNA‐binding and nucleoid localization of the protein. Significantly, these residues are important to recruit the DNA polymerase at the bacterial nucleoid and, subsequently, for an efficient phage DNA replication.     

Purification and Characterization of DNA Polymerase Obtained from the Membrane Fraction of E. coli

Purification studies were conducted on DNA polymerase bound to the membrane fraction of E. coli HF 4704. Purified enzyme (Fraction V) required Mg²⁺ and showed an optimun pH of 7.2. Various kinds of salt indicated a stimulative effect at concentrations lower than 0.1 m. Fraction V was unstable at an acidic condition (pH 5.0) but was rather stable at an alkaline condition (pH 9.0). The enzyme activity was lost by incubation at 45°C for 30min but was stabilized by the addition of DNA. The enzyme contained exonuclease activity but no endonuclease activity. The enzyme produced only light density DNA of various sizes. The function of this enzyme as considered to fill single stranded region of the double stranded primer DNA.     

Multi-tier regulation of the streptomycete morphogenetic peptide SapB

Streptomyces coelicolor is a morphologically complex bacterium requiring the secretion of surface‐active proteins to progress through its life cycle. SapB represents an important class of these biosurfactants, as illustrated by its ability to restore aerial hyphae formation when applied exogenously to developmental mutants. However, such aerial hyphae fail to sporulate, exemplifying the need to co‐ordinate the timing of SapB production with other developmental events. SapB has an unusual lantibiotic structure. Its structural gene, ramS, is only 38 nucleotides downstream of the gene encoding its putative modification enzyme, RamC. Transient, co‐ordinated expression of the operon was thought to be controlled by the response regulator RamR. However, we show that ramS is transcribed throughout the cell cycle with a dual expression profile dissimilar to the tightly controlled ramC expression. Surprisingly, post‐translational modification relies on prior membrane localization of the precursor peptide, RamS, as demonstrated by the absence of RamS modification in S. coelicolor hyphae treated with the Bacillus subtilis lipoprotein surfactin. Our results demonstrate that interspecies interaction can also be mediated by interference of post‐translational events. Further, temporal and spatial regulation of irreversible post‐translational modification of a surface‐active morphogenetic peptide suggests a new model for the control of key developmental events.     

Rhodobacter capsulatus DprA is essential for RecA‐mediated gene transfer agent (RcGTA) recipient capability regulated by quorum‐sensing and the CtrA response regulator

Gene transfer agents (GTAs) are genetic exchange elements that resemble small DNA bacteriophages that transfer random pieces of the producing cell's genome to recipient cells. The best‐studied GTA is that of Rhodobacter capsulatus, termed RcGTA. We discovered that the putative response regulator CtrA, which is essential for RcGTA production, is required for RcGTA‐mediated gene acquisition, and confirmed that a RecA homologue is required. It was also discovered that a DprA (DNA‐protecting protein A) homologue is essential for RcGTA‐mediated gene acquisition, and that dprA expression is induced by gtaI‐dependent quorum‐sensing and non‐phosphorylated CtrA. Modelling of the R. capsulatus DprA structure indicated the presence of a C‐terminal region that resembles a dsDNA‐binding protein domain. Purified His‐tagged R. capsulatus DprA protein bound to both single‐stranded (ss)DNA and double‐stranded (ds)DNA, but with a greater affinity for ssDNA. Additionally, DprA protected dsDNA from endonuclease digestion, and increased the rate of nucleation of Escherichia coli RecA onto ssDNA. Single‐cell expression analyses revealed that dprA is expressed in the majority of cells throughout a population. Overall, the results suggest that incorporation of RcGTA DNA into the recipient cell genome proceeds through a homologous recombination pathway resembling DNA recombination in natural transformation.