Expression, purification, and biophysical characterization of the BRCT domain of human DNA ligase IIIalpha.

The C-terminal regions of several DNA repair and cell cycle checkpoint proteins are homologous to the breast-cancer-associated BRCA-1 protein C-terminal region. These regions, known as BRCT domains, have been found to mediate important protein-protein interactions. We produced the BRCT domain of DNA ligase IIIalpha (L3[86]) for biophysical and structural characterization. A glutathione S-transferase (GST) fusion with the L3[86] domain (residues 837-922 of ligase IIIalpha) was expressed in Escherichia coli and purified by glutathione affinity chromatography. The GST fusion protein was removed by thrombin digestion and further purification steps. Using this method, (15)N-labeled and (13)C/(15)N-double-labeled L3[86] proteins were prepared to enable a full determination of structure and dynamics using heteronuclear NMR spectroscopy. To obtain evidence of binding activity to the distal BRCT of the repair protein XRCC1 (X1BRCTb), as well as to provide insight into the interaction between these two BRCT binding partners, the corresponding BRCT heterocomplexes were also prepared and studied. Changes in the secondary structures (amount of helix and sheet components) of the two constituents were not observed upon complex formation. However, the melting temperature of the complex was significantly higher relative to the values obtained for the L3[86] or X1BRCTb proteins alone. This increased thermostability imparted by the interaction between the two BRCT domains may explain why cells require XRCC1 to maintain ligase IIIalpha activity.

Purification, characterization, and crystallization of the distal BRCT domain of the human XRCC1 DNA repair protein.

The XRCC1 DNA repair protein contains two regions of approximately 100 amino acids each that share homology with the BRCT (BRCA1 carboxyl terminus) domain superfamily. These two regions of XRCC1 have been shown to interact independently with DNA ligase III and poly(ADP-ribose)polymerase as part of a mechanism involved in the repair of DNA single-strand breaks. To understand how these BRCT regions specify protein-protein interactions and contribute to DNA repair function, we have overexpressed and purified the distal BRCT domain of XRCC1 with the goal of structure determination. The cDNA encoding this BRCT region (X1BRCTb) was inserted into the pET29 bacterial expression vector; the polypeptide was expressed in mostly soluble form and then purified by anion-exchange and gel filtration chromatography. Crystallization screening with the purified material resulted in the formation of large bipyramidal crystals. Crystals formed within several hours at room temperature from salt solutions of ammonium sulfate. Crystals diffract to approximately 2.85 A and were found to be in space group P4(1)2(1)2 (or its enantiomorph P4(3)2(1)2) with unit cell dimensions a = 100.43 A, c = 105.62 A. Crystals of similar character have also been obtained after incorporation of selenomethionine during expression of the protein. Efforts are now under way to determine the molecular structure of the X1BRCTb domain. These studies are likely to give insight into the interaction between XRCC1 and DNA ligase III and into general structural features of BRCT domains that exist in many other proteins.

Crystallization of the NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase from Saccharomyces cerevisiae.

Saccharomyces cerevisiae possesses three isozymes of 5,10-methylenetetrahydrofolate dehydrogenase (MTD). The NAD-dependent enzyme is the first monofunctional form found in eukaryotes. Here we report its crystallization in a form suitable for high-resolution structure. The space group is P4(2)2(1)2 with cell constants a = b = 75.9, c = 160.0 A, and there is one 36 kDa molecule in the asymmetric unit. Crystals diffract to 2.9 A resolution.

Metabolic role of cytoplasmic isozymes of 5,10-methylenetetrahydrofolate dehydrogenase in Saccharomyces cerevisiae.

Saccharomyces cerevisiae possesses two cytosolic 5,10-methylenetetrahydrofolate (CH2-THF) dehydrogenases that differ in their redox cofactor specificity: an NAD-dependent dehydrogenase encoded by the MTD1 gene and an NADP-dependent activity as part of the trifunctional C1-THF synthase encoded by the ADE3 gene. The experiments described here were designed to define the metabolic roles of the NAD- and NADP-dependent CH2-THF dehydrogenases in one-carbon interconversions and de novo purine biosynthesis. Growth studies showed that the NAD-dependent CH2-THF dehydrogenase is interchangeable with the NADP-dependent CH2-THF dehydrogenase when flow of one-carbon units is in the oxidative direction but that it does not participate significantly when flux is in the reductive direction. 13C NMR experiments with [2-13C]glycine and unlabeled formate confirmed the latter conclusion. Direct measurements of cellular folate coenzyme levels revealed substantial levels of 10-formyl-THF (CHO-THF), the one-carbon donor used in purine synthesis, in the purine-requiring ade3 deletion strain. Thus, CHO-THF is necessary but not sufficient for de novo purine synthesis in yeast. Disruption of the MTD1 gene in this strain resulted in undetectable CHO-THF, indicating that the NAD-dependent CH2-THF dehydrogenase was responsible for CHO-THF production in the ade3 deletion strain. Finally, we examined the ability of wild-type and catalytically-inactive domains of the cytoplasmic C1-THF synthase to complement the adenine auxotrophy of the ade3 deletion strain. Both the dehydrogenase/cyclohydrolase (D/C) domain and the synthetase domain could functionally replace the full-length protein, but, at least for the D/C domain, complementation was not dependent on catalytic activity. These results reveal a catalytic role for the NAD-dependent CH2-THF dehydrogenase in the oxidation of cytoplasmic one-carbon units and indicate that the cytoplasmic C1-THF synthase plays both catalytic and noncatalytic roles in de novo purine biosynthesis in yeast.

Cloning and characterization of the Saccharomyces cerevisiae gene encoding NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase.

Saccharomyces cerevisiae possess a monofunctional, cytoplasmic NAD-dependent 5,10-methylenetetrahydrofolate (THF) dehydrogenase that converts 5,10-methylene-THF to 5,10-methenyl-THF (Barlowe, C. K., and Appling, D.R. (1990) Biochemistry 29, 7089-7094). We have now isolated the gene encoding this enzyme from a yeast genomic library using oligonucleotide probes based on internal peptide sequences from the purified protein. Nucleotide sequence analysis reveals a 320-amino acid open reading frame that contains both of the internal peptide sequences. The predicted molecular weight (36,236) is consistent with the estimated size (33,000-38,000) of the purified protein. Disruption of the chromosomal copy of the gene resulted in loss of NAD-dependent 5,10-methylene-THF dehydrogenase activity and led to a purine requirement in certain genetic backgrounds, confirming a role for this enzyme in the oxidation of cytoplasmic one-carbon units. A single gene was mapped to chromosome XI by hybridization to a yeast chromosomal blot. We propose MTD1 as the name for this gene. Northern analysis of total yeast RNA revealed a single transcript of approximately 1,100 nucleotides. Multiple transcription initiation sites were identified between 58 and 83 base pairs upstream of the start of translation. The amino acid sequences derived from the nucleic acid sequences of seven other methylene-THF dehydrogenases cloned to date have been found to be highly homologous. Although the predicted amino acid sequence of the yeast NAD-dependent enzyme shows slight homology to the other sequences, it appears to be only distantly related to the other 5,10-methylene-THF dehydrogenases.

Patient participation in health care: an underused resource.

The CCM has been in development for more than 3 years and in operation for more than 2 years. According to Peters, "Developing a vision is a messy, artistic process. Living it convincingly is a passionate one beyond any doubt." This statement expresses our personal experience in development of the CCM in terms of time, effort, hurdles, growth, and satisfaction. An environment has been created that strengthens the nurse's role as clinical educator, advocate, and coordinator. Capable patients and families on pilot units express satisfaction because they have learned to participate actively in their care during hospitalization, to better understand their disease, and to better manage their care at home. The time saved for nurses allows them to be engaged in activities of health promotion and education, deliver selected aspects of care, and consult with other team members on issues of problematic patient management. In this environment, professional nursing practice has been enhanced and nurses are influencing positive patient outcomes. In addition, our nurse recruiter reports that it is easier to recruit nurses for CCM pilot units than for nonpilot units with similar patient populations. This project has tapped an often underused resource, the patient's self-care ability, and created an environment that benefits not only the care recipients but also the caregivers.

Rat C1-tetrahydrofolate synthase. cDNA isolation, tissue-specific levels of the mRNA, and expression of the protein in yeast.

We have isolated and characterized cDNA clones encoding rat cytoplasmic C1-tetrahydrofolate (H4folate) synthase. In eukaryotes, this enzyme is trifunctional and contains the activities of 10-formyl-H4folate synthetase, 5,10-methenyl-H4folate cyclohydrolase, and 5,10-methylene-H4folate dehydrogenase. The deduced sequence of the 935-amino acid open reading frame contained exact matches to NH2-terminal (15 residues) and internal (residues 436-450) peptide sequences obtained from the purified enzyme. The amino acid sequence derived from the rat cDNA shows extensive homology to analogous proteins from bacterial, yeast, and mammalian sources. We have used the cDNA to determine the steady-state levels of the mRNA in various rat tissues and have found that gene expression is regulated in a tissue-specific manner. Transcript levels are highest in kidney and liver with liver transcripts reduced about 30% relative to those found in kidney. Brain, heart, testis, lung and skeletal muscle display even lower transcript levels; reductions range from 70 to 80% of transcript levels found in kidney. Comparison to the levels of enzyme in these tissues allows us to conclude that pretranslational events predominate in the tissue-specific expression. The rat enzyme has been functionally expressed in Saccharomyces cerevisiae as evidenced by its capacity to complement a chromosomal deletion of ADE3, the yeast gene encoding cytoplasmic C1-H4folate synthase.