Lysines in the lyase active site of DNA polymerase ß destabilize nonspecific DNA binding, facilitating searching and DNA gap recognition.

DNA polymerase (pol) ß catalyzes two reactions at DNA gaps generated during base excision repair, gap-filling DNA synthesis and lyase-dependent 5´-end deoxyribose phosphate removal. The lyase domain of pol ß has been proposed to function in DNA gap recognition and to facilitate DNA scanning during substrate search. However, the mechanisms and molecular interactions used by pol ß for substrate search and recognition are not clear. To provide insight into this process, a comparison was made of the DNA binding affinities of WT pol ß, pol λ, and pol µ, and several variants of pol ß, for 1-nt-gap-containing and undamaged DNA. Surprisingly, this analysis revealed that mutation of three lysine residues in the lyase active site of pol ß, 35, 68, and 72, to alanine (pol ß KΔ3A) increased the binding affinity for nonspecific DNA ∼11-fold compared with that of the WT. WT pol µ, lacking homologous lysines, displayed nonspecific DNA binding behavior similar to that of pol ß KΔ3A, in line with previous data demonstrating both enzymes were deficient in processive searching. In fluorescent microscopy experiments using mouse fibroblasts deficient in PARP-1, the ability of pol ß KΔ3A to localize to sites of laser-induced DNA damage was strongly decreased compared with that of WT pol ß. These data suggest that the three lysines in the lyase active site destabilize pol ß when bound to DNA nonspecifically, promoting DNA scanning and providing binding specificity for gapped DNA.

DNA polymerase ß nucleotide-stabilized template misalignment fidelity depends on local sequence context.

DNA polymerase ß has two DNA-binding domains that interact with the opposite sides of short DNA gaps. These domains contribute two activities that modify the 5' and 3' margins of gapped DNA during base excision repair. DNA gaps greater than 1 nucleotide (nt) pose an architectural and logistical problem for the two domains to interact with their respective DNA termini. Here, crystallographic and kinetic analyses of 2-nt gap-filling DNA synthesis revealed that the fidelity of DNA synthesis depends on local sequence context. This was due to template dynamics that altered which of the two template nucleotides in the gap served as the coding nucleotide. We observed that, when a purine nucleotide was in the first coding position, DNA synthesis fidelity was similar to that observed with a 1-nt gap. However, when the initial templating nucleotide was a pyrimidine, fidelity was decreased. If the first templating nucleotide was a cytidine, there was a significantly higher probability that the downstream template nucleotide coded for the incoming nucleotide. This dNTP-stabilized misalignment reduced base substitution and frameshift deletion fidelities. A crystal structure of a binary DNA product complex revealed that the cytidine in the first templating site was in an extrahelical position, permitting the downstream template nucleotide to occupy the coding position. These results indicate that DNA polymerase ß can induce a strain in the DNA that modulates the position of the coding nucleotide and thereby impacts the identity of the incoming nucleotide. Our findings demonstrate that "correct" DNA synthesis can result in errors when template dynamics induce coding ambiguity.

Molecular basis for the faithful replication of 5-methylcytosine and its oxidized forms by DNA polymerase ß.

DNA methylation is an epigenetic mark that regulates gene expression in mammals. One method of methylation removal is through ten-eleven translocation-catalyzed oxidation and the base excision repair pathway. The iterative oxidation of 5-methylcytosine catalyzed by ten-eleven translocation enzymes produces three oxidized forms of cytosine: 5-hydroxmethylcytosine, 5-formylcytosine, and 5-carboxycytosine. The effect these modifications have on the efficiency and fidelity of the base excision repair pathway during the repair of opposing base damage, and in particular DNA polymerization, remains to be elucidated. Using kinetic assays, we show that the catalytic efficiency for the incorporation of dGTP catalyzed by human DNA polymerase ß is not affected when 5-methylcytosine, 5-hydroxmethylcytosine, and 5-formylcytosine are in the DNA template. In contrast, the catalytic efficiency of dGTP insertion decreases ∼20-fold when 5-carboxycytosine is in the templating position, as compared with unmodified cytosine. However, DNA polymerase fidelity is unaltered when these modifications are in the templating position. Structural analysis reveals that the methyl, hydroxymethyl, and formyl modifications are easily accommodated within the polymerase active site. However, to accommodate the carboxyl modification, the phosphate backbone on the templating nucleotide shifts ∼2.5 Å to avoid a potential steric/repulsive clash. This altered conformation is stabilized by lysine 280, which makes a direct interaction with the carboxyl modification and the phosphate backbone of the templating strand. This work provides the molecular basis for the accommodation of epigenetic base modifications in a polymerase active site and suggests that these modifications are not mutagenically copied during base excision repair.

DNA polymerase ß uses its lyase domain in a processive search for DNA damage.

DNA polymerase (Pol) ß maintains genome fidelity by catalyzing DNA synthesis and removal of a reactive DNA repair intermediate during base excision repair (BER). Situated within the middle of the BER pathway, Pol ß must efficiently locate its substrates before damage is exacerbated. The mechanisms of damage search and location by Pol ß are largely unknown, but are critical for understanding the fundamental features of the BER pathway. We developed a processive search assay to determine if Pol ß has evolved a mechanism for efficient DNA damage location. These assays revealed that Pol ß scans DNA using a processive hopping mechanism and has a mean search footprint of ∼24 bp at predicted physiological ionic strength. Lysines within the lyase domain are required for processive searching, revealing a novel function for the lyase domain of Pol ß. Application of our processive search assay into nucleosome core particles revealed that Pol ß is not processive in the context of a nucleosome, and its single-turnover activity is reduced ∼500-fold, as compared to free DNA. These data suggest that the repair footprint of Pol ß mainly resides within accessible regions of the genome and that these regions can be scanned for damage by Pol ß.

Unencumbered Pol ß lyase activity in nucleosome core particles.

Packaging of DNA into the nucleosome core particle (NCP) is considered to exert constraints to all DNA-templated processes, including base excision repair where Pol ß catalyzes two key enzymatic steps: 5'-dRP lyase gap trimming and template-directed DNA synthesis. Despite its biological significance, knowledge of Pol ß activities on NCPs is still limited. Here, we show that removal of the 5'-dRP block by Pol ß is unaffected by NCP constraints at all sites tested and is even enhanced near the DNA ends. In contrast, strong inhibition of DNA synthesis is observed. These results indicate 5'-dRP gap trimming proceeds unperturbed within the NCP; whereas, gap filling is strongly limited. In the absence of additional factors, base excision repair in NCPs will stall at the gap-filling step.

Processive searching ability varies among members of the gap-filling DNA polymerase X family.

DNA repair proteins must locate rare damaged sites within the genome. DNA polymerase ß (Pol ß), a member of the DNA polymerase X family that is involved in base excision repair, uses a processive hopping search mechanism to locate substrates. This effectively enhances its search footprint on DNA, increasing the probability of locating damaged sites. Processive searching has been reported or proposed for many DNA-binding proteins, raising the question of how widespread or specific to certain enzymes the ability to perform this function is. To provide insight into this question, we compared the ability of three homologous DNA Pol X family members to perform a processive search for 1-nucleotide gaps in DNA using a previously developed biochemical assay. We found that at near-predicted physiological ionic strengths, the intramolecular searching ability of Pol ß is at least 4-fold higher than that of Pol µ and ∼2-fold higher than that of Pol λ. Pol ß also was able to perform intersegmental transfer with the intersegmental searching ability of Pol ß being at least 6- and ∼2-fold higher than that of Pols µ and λ, respectively. Mutational analysis suggested that differences in the N-terminal domains of these polymerases are responsible for the varying degrees of searching competence. Of note, the differences in processive searching ability observed among the DNA Pol X family members correlated with their proposed biological functions in base excision repair and nonhomologous end joining.

Differential substrate recognition by isozymes of plant protein-only Ribonuclease P.

Ribonuclease P (RNase P) catalyzes the cleavage of leader sequences from precursor tRNA (pre-tRNA). Typically, these enzymes are ribonucleic protein complexes that are found in all domains of life. However, a new class of RNase P has been discovered that is composed entirely of protein, termed protein-only RNase P (PRORP). To investigate the molecular determinants of PRORP substrate recognition, we measured the binding affinities and cleavage kinetics of Arabidopsis PRORP1 for varied pre-tRNA substrates. This analysis revealed that PRORP1 does not make significant contacts within the trailer or beyond N-1of the leader, indicating that this enzyme recognizes primarily the tRNA body. To determine the extent to which sequence variation within the tRNA body modulates substrate selectivity and to provide insight into the evolution and function of PRORP enzymes, we measured the reactivity of the three Arabidopsis PRORP isozymes (PRORP1-3) with four pre-tRNA substrates. A 13-fold range in catalytic efficiencies (10(4)-10(5)M(-1)s(-1)) was observed, demonstrating moderate selectivity for pre-tRNA substrates. Although PRORPs bind the different pre-tRNA species with affinities varying by as much as 100-fold, the three isozymes have similar affinities for a given pre-tRNA, suggesting similar binding modes. However, PRORP isozymes have varying degrees of cleavage fidelity, which is dependent on the pre-tRNA species and the presence of a 3'-discriminator base. This work defines molecular determinants of PRORP substrate recognition that provides insight into this new class of RNA processing enzymes.

Mechanistic Studies Reveal Similar Catalytic Strategies for Phosphodiester Bond Hydrolysis by Protein-only and RNA-dependent Ribonuclease P.

Ribonuclease P (RNase P) is an endonuclease that catalyzes the essential removal of the 5' end of tRNA precursors. Until recently, all identified RNase P enzymes were a ribonucleoprotein with a conserved catalytic RNA component. However, the discovery of protein-only RNase P (PRORP) shifted this paradigm, affording a unique opportunity to compare mechanistic strategies used by naturally evolved protein and RNA-based enzymes that catalyze the same reaction. Here we investigate the enzymatic mechanism of pre-tRNA hydrolysis catalyzed by the NYN (Nedd4-BP1, YacP nuclease) metallonuclease of Arabidopsis thaliana, PRORP1. Multiple and single turnover kinetic data support a mechanism where a step at or before chemistry is rate-limiting and provide a kinetic framework to interpret the results of metal alteration, mutations, and pH dependence. Catalytic activity has a cooperative dependence on the magnesium concentration (nH = 2) under kcat/Km conditions, suggesting that PRORP1 catalysis is optimal with at least two active site metal ions, consistent with the crystal structure. Metal rescue of Asp-to-Ala mutations identified two aspartates important for enhancing metal ion affinity. The single turnover pH dependence of pre-tRNA cleavage revealed a single ionization (pKa ∼ 8.7) important for catalysis, consistent with deprotonation of a metal-bound water nucleophile. The pH and metal dependence mirrors that observed for the RNA-based RNase P, suggesting similar catalytic mechanisms. Thus, despite different macromolecular composition, the RNA and protein-based RNase P act as dynamic scaffolds for the binding and positioning of magnesium ions to catalyze phosphodiester bond hydrolysis.

Mitochondrial ribonuclease P structure provides insight into the evolution of catalytic strategies for precursor-tRNA 5' processing.

Ribonuclease P (RNase P) catalyzes the maturation of the 5' end of tRNA precursors. Typically these enzymes are ribonucleoproteins with a conserved RNA component responsible for catalysis. However, protein-only RNase P (PRORP) enzymes process precursor tRNAs in human mitochondria and in all tRNA-using compartments of Arabidopsis thaliana. PRORP enzymes are nuclear encoded and conserved among many eukaryotes, having evolved recently as yeast mitochondrial genomes encode an RNase P RNA. Here we report the crystal structure of PRORP1 from A. thaliana at 1.75 Å resolution, revealing a prototypical metallonuclease domain tethered to a pentatricopeptide repeat (PPR) domain by a structural zinc-binding domain. The metallonuclease domain is a unique high-resolution structure of a Nedd4-BP1, YacP Nucleases (NYN) domain that is a member of the PIN domain-like fold superfamily, including the FLAP nuclease family. The structural similarity between PRORP1 and the FLAP nuclease family suggests that they evolved from a common ancestor. Biochemical data reveal that conserved aspartate residues in PRORP1 are important for catalytic activity and metal binding and that the PPR domain also enhances activity, likely through an interaction with pre-tRNA. These results provide a foundation for understanding tRNA maturation in organelles. Furthermore, these studies allow for a molecular-level comparison of the catalytic strategies used by the only known naturally evolved protein and RNA-based catalysts that perform the same biological function, pre-tRNA maturation, thereby providing insight into the differences between the prebiotic RNA world and the present protein-dominated world.

RNase P enzymes: divergent scaffolds for a conserved biological reaction.

Ribonuclease P (RNase P) catalyzes the maturation of the 5' end of precursor-tRNAs (pre-tRNA) and is conserved in all domains of life. However, the composition of RNase P varies from bacteria to archaea and eukarya, making RNase P one of the most diverse enzymes characterized. Most known RNase P enzymes contain a large catalytic RNA subunit that associates with one to 10 proteins. Recently, a protein-only form of RNase P was discovered in mitochondria and chloroplasts of many higher eukaryotes. This proteinaceous RNase P (PRORP) represents a new class of metallonucleases. Here we discuss our recent crystal structure of PRORP1 from Arabidopsis thaliana and speculate on the reasons for the replacement of catalytic RNA by a protein catalyst. We conclude, based on an analysis of the catalytic efficiencies of ribonucleoprotein (RNP) and PRORP enzymes, that the need for greater catalytic efficiency is most likely not the driving force behind the replacement of the RNA with a protein catalyst. The emergence of a protein-based RNase P more likely reflects the increasing complexity of the biological system, including difficulties in importation into organelles and vulnerability of organellar RNAs to cleavage.

An afferent vagal nerve pathway links hepatic PPARalpha activation to glucocorticoid-induced insulin resistance and hypertension.

Glucocorticoid excess causes insulin resistance and hypertension. Hepatic expression of PPARalpha (Ppara) is required for glucocorticoid-induced insulin resistance. Here we demonstrate that afferent fibers of the vagus nerve interface with hepatic Ppara expression to disrupt blood pressure and glucose homeostasis in response to glucocorticoids. Selective hepatic vagotomy decreased hyperglycemia, hyperinsulinemia, hepatic insulin resistance, Ppara expression, and phosphoenolpyruvate carboxykinase (PEPCK) enzyme activity in dexamethasone-treated Ppara(+/+) mice. Selective vagotomy also decreased blood pressure, adrenergic tone, renin activity, and urinary sodium retention in these mice. Hepatic reconstitution of Ppara in nondiabetic, normotensive dexamethasone-treated PPARalpha null mice increased glucose, insulin, hepatic PEPCK enzyme activity, blood pressure, and renin activity in sham-operated animals but not hepatic-vagotomized animals. Disruption of vagal afferent fibers by chemical or surgical means prevented glucocorticoid-induced metabolic derangements. We conclude that a dynamic interaction between hepatic Ppara expression and a vagal afferent pathway is essential for glucocorticoid induction of diabetes and hypertension.

The Escherichia coli alkylation response protein AidB is a redox partner of flavodoxin and binds RNA and acyl carrier protein.

Microorganisms are exposed to a wide variety of exogenous and endogenous chemical agents that alkylate DNA. Escherichia coli cells exhibit an adaptive response that recognizes and repairs alkylated DNA lesions using Ada, AlkA, and AlkB enzymes. Another alkylation response protein, the DNA-binding flavoprotein AidB, was proposed to repair DNA or protect it from chemical alkylating agents, but direct evidence for its role is lacking. Here, AidB was shown to form tight complexes with both flavodoxin and acyl carrier protein. In addition, electron transfer between 1-electron and 2-electron reduced flavodoxin to oxidized AidB was observed, although with very small rate constants. AidB was found to bind to RNA, raising the prospect that the protein may have a role in protection of RNA from chemical alkylation. Finally, the reagent N-methyl-N'-nitro-N-nitrosoguanidine was eliminated as a direct substrate of the enzyme.

Unit-level voluntary turnover rates and customer service quality: implications of group cohesiveness, newcomer concentration, and size.

Despite substantial growth in the service industry and emerging work on turnover consequences, little research examines how unit-level turnover rates affect essential customer-related outcomes. The authors propose an operational disruption framework to explain why voluntary turnover impairs customers' service quality perceptions. On the basis of a sample of 75 work units and data from 5,631 employee surveys, 59,602 customer surveys, and organizational records, results indicate that unit-level voluntary turnover rates are negatively related to service quality perceptions. The authors also examine potential boundary conditions related to the disruption framework. Of 3 moderators studied (group cohesiveness, group size, and newcomer concentration), results show that turnover's negative effects on service quality are more pronounced in larger units and in those with a greater concentration of newcomers.

DNA scanning by base excision repair enzymes and implications for pathway coordination.

Site-specific DNA binding proteins must search the genome to locate their target sites, and many DNA modifying enzymes have the ability to scan along DNA in search of their substrates. This process is termed processive searching, and it serves to decrease the search time by effectively increasing the DNA binding footprint of a protein. The repertoire of proteins capable of processive searching is expanding, highlighting the need to understand the governing principles behind this fundamental process. Many of the enzymes in the base excision DNA repair pathway are capable of processive searching. Here, we briefly summarize methodology for determining if a protein can scan DNA and highlight the discovery that the base excision repair DNA polymerase ß performs a processive search. Elucidation of physical models for DNA searching has also provided a plausible mechanism for pathway coordination during repair. The ability of BER enzymes to transiently sample adjacent DNA sites while bound to their product confers accessibility to downstream enzymes and does not require protein-protein interactions for coordination.

Nuclear Protein-Only Ribonuclease P2 Structure and Biochemical Characterization Provide Insight into the Conserved Properties of tRNA 5' End Processing Enzymes.

Protein-only RNase Ps (PRORPs) are a recently discovered class of RNA processing enzymes that catalyze maturation of the 5' end of precursor tRNAs in Eukaryotes. PRORPs are found in the nucleus and/or organelles of most eukaryotic organisms. Arabidopsis thaliana is a representative organism that contains PRORP enzymes (PRORP1, PRORP2 and PRORP3) in both its nucleus and its organelles; PRORP2 and PRORP3 localize to the nucleus and PRORP1 localizes to the chloroplast and the mitochondria. Apart from their identification, almost nothing is known about the structure and function of PRORPs that act in the nucleus. Here, we use a combination of biochemical assays and X-ray crystallography to characterize A. thaliana PRORP2. We solved the crystal structure of PRORP2 (3.2Å) revealing an overall V-shaped protein and conserved metallonuclease active-site structure. Our biochemical studies indicate that PRORP2 requires Mg(2+) for catalysis and catalyzes the maturation of nuclear encoded substrates up to 10-fold faster than mitochondrial encoded precursor nad6 t-element under single-turnover conditions. We also demonstrate that PRORP2 preferentially binds precursor tRNAs containing short 5' leaders and 3' trailers; however, leader and trailer lengths do not significantly alter the observed rate constants of PRORP2 in single-turnover cleavage assays. Our data provide a biochemical and structural framework to begin understanding how nuclear localized PRORPs recognize and cleave their substrates.

The Diversity of Ribonuclease P: Protein and RNA Catalysts with Analogous Biological Functions.

Ribonuclease P (RNase P) is an essential endonuclease responsible for catalyzing 5' end maturation in precursor transfer RNAs. Since its discovery in the 1970s, RNase P enzymes have been identified and studied throughout the three domains of life. Interestingly, RNase P is either RNA-based, with a catalytic RNA subunit, or a protein-only (PRORP) enzyme with differential evolutionary distribution. The available structural data, including the active site data, provides insight into catalysis and substrate recognition. The hydrolytic and kinetic mechanisms of the two forms of RNase P enzymes are similar, yet features unique to the RNA-based and PRORP enzymes are consistent with different evolutionary origins. The various RNase P enzymes, in addition to their primary role in tRNA 5' maturation, catalyze cleavage of a variety of alternative substrates, indicating a diversification of RNase P function in vivo. The review concludes with a discussion of recent advances and interesting research directions in the field.

Repairing the damaged spinal cord: a summary of our early success with embryonic stem cell transplantation and remyelination.

Demyelination contributes to the loss of function consequent to central nervous system (CNS) injury. Optimizing remyelination through transplantation of myelin-producing cells may offer a pragmatic approach to restoring meaningful neurological function. An unlimited source of cell suitable for such transplantation therapy can be derived from embryonic stem (ES) cells, which are both pluripotent and genetically flexible. Here we review work from our group showing that neural precursor cells can be derived from ES cells and that transplantation of these cells into the injured spinal cord leads to some recovery of function. We have further examined and optimized methods for enriching oligodendrocyte differentiation from ES cells. ES cell-derived oligodendrocytes are capable of rapid differentiation and myelination in mixed neuron/glia cultures. When transplanted into the injured spinal cord of adult rodents, the neural-induced precursor cells are capable of differentiating into oligodendrocytes and myelinating host axons. The role of myelination and remyelination will be discussed in the context of regeneration strategies.

Transplantation of apoptosis-resistant embryonic stem cells into the injured rat spinal cord.

Murine embryonic stem cells were induced to differentiate into neural lineage cells by exposure to retinoic acid. Approximately one million cells were transplanted into the lesion site in the spinal cords of adult rats which had received moderate contusion injuries 9 days previously. One group received transplants of cells genetically modified to over-express bcl-2, which codes for an anti-apoptotic protein. A second group received transplants of the wild-type ES cells from which the bcl-2 line was developed. In the untransplanted control group, only medium was injected. Locomotor abilities were assessed using the Basso, Beattie and Bresnahan (BBB) rating scale for 6 weeks. There was no incremental locomotor improvement in either transplant group when compared to control over the survival period. Morbidity and mortality were significantly more prevalent in the transplant groups than in controls. At the conclusion of the 6-week survival period, the spinal cords were examined. Two of six cords from the bcl-2 group and one of 12 cords from the wild-type group showed gross evidence of abnormal growths at the site of transplantation. No similar growth was seen in the control. Pathological examination of the abnormal cords showed very large numbers of undifferentiated cells proliferating at the injection site and extending up to 1.5 cm rostrally and caudally. These results suggest that transplanting KD3 ES cells, or apoptosis-resistant cells derived from the KD3 line, into the injured spinal cord does not improve locomotor recovery and can lead to tumor-like growth of cells, accompanied by increased debilitation, morbidity and mortality.