Exchanges across land-water-scape boundaries in urban systems: strategies for reducing nitrate pollution.

Conservation in urban areas typically focuses on biodiversity and large green spaces. However, opportunities exist throughout urban areas to enhance ecological functions. An important function of urban landscapes is retaining nitrogen thereby reducing nitrate pollution to streams and coastal waters. Control of nonpoint nitrate pollution in urban areas was originally based on the documented importance of riparian zones in agricultural and forested ecosystems. The watershed and boundary frameworks have been used to guide stream research and a riparian conservation strategy to reduce nitrate pollution in urban streams. But is stream restoration and riparian-zone conservation enough? Data from the Baltimore Ecosystem Study and other urban stream research indicate that urban riparian zones do not necessarily prevent nitrate from entering, nor remove nitrate from, streams. Based on this insight, policy makers in Baltimore extended the conservation strategy throughout larger watersheds, attempting to restore functions that no longer took place in riparian boundaries. Two urban revitalization projects are presented as examples aimed at reducing nitrate pollution to stormwater, streams, and the Chesapeake Bay. An adaptive cycle of ecological urban design synthesizes the insights from the watershed and boundary frameworks, from new data, and from the conservation concerns of agencies and local communities. This urban example of conservation based on ameliorating nitrate water pollution extends the initial watershed-boundary approach along three dimensions: 1) from riparian to urban land-water-scapes; 2) from discrete engineering solutions to ecological design approaches; and 3) from structural solutions to inclusion of individual, household, and institutional behavior.

Replication protein A is sequentially phosphorylated during meiosis.

Phosphorylation of the cellular single-stranded DNA-binding protein, replication protein A (RPA), occurs during normal mitotic cell cycle progression and also in response to genotoxic stress. In budding yeast, these reactions require the ATM homolog Mec1, a central regulator of the DNA replication and DNA damage checkpoint responses. We now demonstrate that the middle subunit of yeast RPA (Rfa2) becomes phosphorylated in two discrete steps during meiosis. Primary Rfa2 phosphorylation occurs early in meiotic progression and is independent of DNA replication, recombination and Mec1. In contrast, secondary Rfa2 phosphorylation is activated upon initiation of recombination and requires Mec1. While the primary Rfa2 phosphoisomer is detectable throughout most of meiosis, the secondary Rfa2 phosphoisomer is only transiently generated and begins to disappear soon after recombination is complete. Extensive secondary Rfa2 phosphorylation is observed in a recombination mutant defective for the pachytene checkpoint, indicating that Mec1-dependent Rfa2 phosphorylation does not function to maintain meiotic delay in response to DNA double-strand breaks. Our results suggest that Mec1-dependent RPA phosphorylation could be involved in regulating recombination rather than cell cycle or meiotic progression.

Phosphorylation of the replication protein A large subunit in the Saccharomyces cerevisiae checkpoint response.

The checkpoint mechanisms that delay cell cycle progression in response to DNA damage or inhibition of DNA replication are necessary for maintenance of genetic stability in eukaryotic cells. Potential targets of checkpoint-mediated regulation include proteins directly involved in DNA metabolism, such as the cellular single-stranded DNA (ssDNA) binding protein, replication protein A (RPA). Studies in Saccharomyces cerevisiae have revealed that the RPA large subunit (Rfa1p) is involved in the G1 and S phase DNA damage checkpoints. We now demonstrate that Rfa1p is phosphorylated in response to various forms of genotoxic stress, including radiation and hydroxyurea exposure, and further show that phosphorylation of Rfa1p is dependent on the central checkpoint regulator Mec1p. Analysis of the requirement for other checkpoint genes indicates that different mechanisms mediate radiation- and hydroxyurea-induced Rfa1p phosphorylation despite the common requirement for functional Mec1p. In addition, experiments with mutants defective in the Cdc13p telomere-binding protein indicate that ssDNA formation is an important signal for Rfa1p phosphorylation. Because Rfa1p contains the major ssDNA binding activity of the RPA heterotrimer and is required for DNA replication, repair and recombination, it is possible that phosphorylation of this subunit is directly involved in modulating RPA activity during the checkpoint response.

Identification and characterization of a single-stranded DNA-binding protein from the archaeon Methanococcus jannaschii.

Single-stranded DNA-binding proteins (SSBs) play essential roles in DNA replication, recombination, and repair in bacteria and eukarya. We report here the identification and characterization of the SSB of an archaeon, Methanococcus jannaschii. The M. jannaschii SSB (mjaSSB) has significant amino acid sequence similarity to the eukaryotic SSB, replication protein A (RPA), and contains four tandem repeats of the core single-stranded DNA (ssDNA) binding domain originally defined by structural studies of RPA. Homologous SSBs are encoded by the genomes of other archaeal species, including Methanobacterium thermoautotrophicum and Archaeoglobus fulgidus. The purified mjaSSB binds to ssDNA with high affinity and selectivity. The apparent association constant for binding to ssDNA is similar to that of RPA under comparable experimental conditions, and the affinity for ssDNA exceeds that for double-stranded DNA by at least two orders of magnitude. The binding site size for mjaSSB is approximately 20 nucleotides. Given that RPA is related to mjaSSB at the sequence level and to Escherichia coli SSB at the structural level, we conclude that the SSBs of archaea, eukarya, and bacteria share a common core ssDNA-binding domain. This ssDNA-binding domain was presumably present in the common ancestor to all three major branches of life.

The ATM homologue MEC1 is required for phosphorylation of replication protein A in yeast.

Replication protein A (RPA) is a highly conserved single-stranded DNA-binding protein, required for cellular DNA replication, repair, and recombination. In human cells, RPA is phosphorylated during the S and G2 phases of the cell cycle and also in response to ionizing or ultraviolet radiation. Saccharomyces cerevisiae exhibits a similar pattern of cell cycle-regulated RPA phosphorylation, and our studies indicate that the radiation-induced reactions occur in yeast as well. We have examined yeast RPA phosphorylation during the normal cell cycle and in response to environmental insult, and have demonstrated that the checkpoint gene MEC1 is required for the reaction under all conditions tested. Through examination of several checkpoint mutants, we have placed RPA phosphorylation in a novel pathway of the DNA damage response. MEC1 is similar in sequence to human ATM, the gene mutated in patients with ataxia-telangiectasia (A-T). A-T cells are deficient in multiple checkpoint pathways and are hypersensitive to killing by ionizing radiation. Because A-T cells exhibit a delay in ionizing radiation-induced RPA phosphorylation, our results indicate a functional similarity between MEC1 and ATM, and suggest that RPA phosphorylation is involved in a conserved eukaryotic DNA damage-response pathway defective in A-T.

Crystal structure of bacteriophage T4 deoxynucleotide kinase with its substrates dGMP and ATP.

NMP kinases catalyse the phosphorylation of the canonical nucleotides to the corresponding diphosphates using ATP as a phosphate donor. Bacteriophage T4 deoxynucleotide kinase (DNK) is the only member of this family of enzymes that recognizes three structurally dissimilar nucleotides: dGMP, dTMP and 5-hydroxymethyl-dCMP while excluding dCMP and dAMP. The crystal structure of DNK with its substrate dGMP has been determined at 2.0 A resolution by single isomorphous replacement. The structure of the ternary complex with dGMP and ATP has been determined at 2.2 A resolution. The polypeptide chain of DNK is folded into two domains of equal size, one of which resembles the mononucleotide binding motif with the glycine-rich P-loop. The second domain, consisting of five alpha-helices, forms the NMP binding pocket. A hinge connection between the domains allows for large movements upon substrate binding which are not restricted by dimerization of the enzyme. The mechanism of active centre formation via domain closure is described. Comparison with other P-loop-containing proteins indicates an induced-fit mode of NTP binding. Protein-substrate interactions observed at the NMP and NTP sites provide the basis for understanding the principles of nucleotide discrimination.

Crystallization and preliminary X-ray analysis of bacteriophage T4 deoxynucleotide kinase.

T4 deoxynucleotide kinase catalyzes the phosphorylation of 5-hydroxymethyldeoxycytidylate, dTMP and dGMP while excluding dCMP and dAMP. In order to understand the mechanism of this remarkable specificity, the enzyme was over-expressed in Escherichia coli, purified and crystallized for X-ray diffraction analysis. The crystals belong to the monoclinic space group C2 with cell dimensions a = 155.2, b = 58.5, c = 75.7 A, beta = 108.1 degrees. There are two protein monomers in the asymmetric unit related by a twofold axis. Diffraction data to 2.0 A resolution have been collected.

The DNA-activated protein kinase is required for the phosphorylation of replication protein A during simian virus 40 DNA replication.

The 32-kDa subunit of replication protein A (RPA) is phosphorylated during the S phase of the cell cycle in vivo and during simian virus 40 DNA replication in vitro. To explore the functional significance of this modification, we purified a HeLa cell protein kinase that phosphorylates RPA in the presence of single-stranded DNA. By several criteria we identified the purified enzyme as a form of the DNA-activated protein kinase (DNA-PK), a previously described high molecular weight protein kinase that is capable of phosphorylating a number of nuclear DNA binding proteins. Phosphorylation of RPA by DNA-PK is stimulated by natural single-stranded DNAs but not by homopolymers lacking secondary structure. Studies with the simian virus 40 model system indicate that DNA-PK is required for DNA-replication-dependent RPA phosphorylation. Depletion of the kinase activity, however, has no effect on the extent of DNA replication in vitro. Our data support a model in which phosphorylation of RPA by DNA-PK is activated by formation of replication intermediates containing single- and double-stranded regions. This event may be involved in a signaling mechanism that coordinates DNA replication with the cell cycle.

Chemical modification of bacteriophage T4 deoxynucleotide kinase. Evidence of a single catalytic region.

The mechanism underlying the unusual specificity of bacteriophage T4 deoxynucleotide kinase, which catalyzes the phosphorylation of 5-hydroxymethyldeoxycytidylate, dTMP, and dGMP, has been investigated by chemical modification of the protein. Pyridoxal 5'-phosphate inactivates deoxynucleotide kinase by modifying a single lysine out of the 17 per monomer. Lysine 10 has been tentatively identified as the site of modification, although the possibility of mutually exclusive reactive residues has not been eliminated. Diethylpyrocarbonate also inactivates the enzyme, suggesting that histidine plays a role in catalytic function. With either reagent, the three activities are lost at equal rates, supporting the contention that one active site is responsible for the exclusive phosphorylation of three dissimilar deoxynucleotides. These studies also identify two distant regions of the primary sequence that are likely to be closely associated in the active region of the folded protein.

Long-term history of chesapeake bay anoxia.

Stratigraphic records from four sediment cores collected along a transect across the Chesapeake Bay near the mouth of the Choptank River were used to reconstruct a 2000-year history of anoxia and eutrophication in the Chesapeake Bay. Variations in pollen, diatoms, concentration of organic carbon, nitrogen, sulfur, acid-soluble iron, and an estimate of the degree of pyritization of iron indicate that sedimentation rates, anoxic conditions and eutrophication have increased in the Chesapeake Bay since the time of European settlement.

Bacteriophage T4 deoxynucleotide kinase: gene cloning and enzyme purification.

Gene 1 of bacteriophage T4 has been cloned into a lambda pL expression vector, resulting in the overproduction of deoxynucleotide kinase. A procedure that includes affinity chromatography on Cibacron Blue F3GA-agarose has been used to purify milligram quantities of enzymes from a 2-liter culture. The enzyme has been partially characterized in vitro and in vivo, and it appears to be identical to the deoxynucleotide kinase isolated from T4-infected Escherichia coli. These results prove the earlier contention that the phosphorylation of three dissimilar deoxynucleotides (5-hydroxymethyldeoxycytidylate, dTMP, and dGMP), to the exclusion of most others, is catalyzed by a single protein.

Seizure activity and lesions after intrahippocampal quinolinic acid injection.

Electroencephalographic, behavioral, and neuropathologic changes were monitored after infusions of the endogenous excitatory amino acid, quinolinic acid (QUIN), into the dorsal hippocampus of unanesthetized, freely moving rats. A dose of 120 nmol QUIN was required to reliably precipitate seizures although EEG changes were observed with doses as small as 3 nmol. Seizure episodes were characterized by repetitive periods of high-voltage spiking typically lasting 20 s but occasional longer multicomponent episodes (60 s) were also observed. The latency of specific QUIN-induced seizures was similar for all doses tested (19 to 32 min); however, the total number of seizures and total time in seizures increased in a dose-dependent fashion from 30 to 300 nmol QUIN. Seizure episodes were often associated with a frozen appearance of the animal and intermittent "wet dog shakes". Ataxia was apparent in animals receiving 120 and 300 nmol QUIN. Using light microscopic analyses, pyramidal cell degeneration was observed in the QUIN-injected hippocampus (CA3 and CA1 cells more susceptible than CA2 cells); dentate granule cells showed signs of degeneration only at the largest QUIN dose. No neuropathologic changes were found outside the injected hippocampus. Seizures and neuropathologic changes induced by 120 nmol QUIN were completely blocked by pre- or cotreatment with 12 nmol (-)2-amino-7-phosphonoheptanoic acid. Experiments with [3H]QUIN indicated that only 3% of the injected radioactivity was present in the dorsal hippocampus at the average time of seizure onset (25 min), and consisted entirely of unmetabolized QUIN. The potent convulsant properties of QUIN, an endogenous metabolite, may prove to be of relevance for the etiology of human temporal lobe epilepsy.