NMR in the SPINE Structural Proteomics project.

This paper describes the developments, role and contributions of the NMR spectroscopy groups in the Structural Proteomics In Europe (SPINE) consortium. Focusing on the development of high-throughput (HTP) pipelines for NMR structure determinations of proteins, all aspects from sample preparation, data acquisition, data processing, data analysis to structure determination have been improved with respect to sensitivity, automation, speed, robustness and validation. Specific highlights are protonless (13)C-direct detection methods and inferential structure determinations (ISD). In addition to technological improvements, these methods have been applied to deliver over 60 NMR structures of proteins, among which are five that failed to crystallize. The inclusion of NMR spectroscopy in structural proteomics pipelines improves the success rate for protein structure determinations.

First steps towards effective methods in exploiting high-throughput technologies for the determination of human protein structures of high biomedical value.

The EC 'Structural Proteomics In Europe' contract is aimed specifically at the atomic resolution structure determination of human protein targets closely linked to health, with a focus on cancer (kinesins, kinases, proteins from the ubiquitin pathway), neurological development and neurodegenerative diseases and immune recognition. Despite the challenging nature of the analysis of such targets, approximately 170 structures have been determined to date. Here, the impact of high-throughput technologies, such as parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens or the use of mass spectrometry to assist sample preparation, on the structural biology of mammalian protein targets is illustrated through selected examples.

DNA binding properties of the adenovirus DNA replication priming protein pTP.

The precursor terminal protein pTP is the primer for the initiation of adenovirus (Ad) DNA replication and forms a heterodimer with Ad DNA polymerase (pol). Pol can couple dCTP to pTP directed by the fourth nucleotide of the viral genome template strand in the absence of other replication proteins, which suggests that pTP/pol binding destabilizes the origin or stabilizes an unwound state. We analyzed the contribution of pTP to pTP/pol origin binding using various DNA oligonucleotides. We show that two pTP molecules bind cooperatively to short DNA duplexes, while longer DNA fragments are bound by single pTP molecules as well. Cooperative binding to short duplexes is DNA sequence independent and most likely mediated by protein/protein contacts. Furthermore, we observed that pTP binds single-stranded (ss)DNA with a minimal length of approximately 35 nt and that random ssDNA competed 25-fold more efficiently than random duplex DNA for origin binding by pTP. Remarkably, short DNA fragments with two opposing single strands supported monomeric pTP binding. pTP did not stimulate, but inhibited strand displacement by the Ad DNA binding and unwinding protein DBP. These observations suggest a mechanism in which the ssDNA affinity of pTP stabilizes Ad pol on partially unwound origin DNA.

Adenovirus DNA replication: protein priming, jumping back and the role of the DNA binding protein DBP.

The adenovirus (Ad) genome is a linear double-stranded (ds) molecule containing about 36 kilobase pairs. At each end of the genome an approximately 100 base pair (bp) inverted terminal repeat (ITR) is found, the exact length depending on the serotype. To the 5'-end of each ITR, a 55-kDa terminal protein (TP) is covalently coupled. The Ad DNA replication system was one of the first replication systems that could be reconstituted in vitro (Challberg and Kelly 1979). The system requires three virally encoded proteins: precursor TP (pTP), DNA polymerase (Pol) and the DNA binding protein (DBP). In addition, three stimulating human cellular proteins have been identified. These are the transcription factors NFI (Nagata et al. 1982) and Oct-1 (Pruijn et al. 1986) and the type I topoisomerase NFII (Nagata et al. 1983). Ad DNA replication uses a protein primer for replication initiation. The transition from initiation to elongation is marked by a jumping back mechanism (King and van der Vliet 1994), followed by elongation. In order to elongate DBP is required. In this review we discuss the roles of DBP during initiation and elongation and we relate biochemical data on the jumping back mechanism used by Ad Pol to the recently solved crystal structure of a Pol alpha-like replication complex (Franklin et al. 2001). We comment on the conditions and possible functions of jumping back and propose a model to describe the jumping back mechanism.

Recruitment of the priming protein pTP and DNA binding occur by overlapping Oct-1 POU homeodomain surfaces.

The human transcription factor Oct-1 can stimulate transcription from a variety of promoters by interacting with the coactivators OBF-1/OCA-B/BOB-1, SNAP190 and VP16. These proteins contact Oct-1 regions different from the DNA binding surface. Oct-1 also stimulates the DNA replication of adenovirus through its DNA binding site in the origin. The Oct-1 POU homeodomain (POUhd) binds the adenovirus precursor terminal protein pTP, which serves as the protein primer of DNA replication and recruits pTP to the origin. To map the interaction with pTP at the POUhd surface, we screened a library of randomly mutated POU domains and identified mutations that interfered with pTP interaction and DNA replication stimulation. These mutants clustered at a surface different from those recognized by OBF-1, SNAP190 and VP16. Unexpectedly, the pTP binding region largely overlapped with the DNA binding surface of POUhd. In agreement with this, pTP binding and DNA binding were mutually exclusive. We propose a model to reconcile pTP recruitment and DNA binding by Oct-1.

A direct interaction between proliferating cell nuclear antigen (PCNA) and Cdk2 targets PCNA-interacting proteins for phosphorylation.

Proliferating cell nuclear antigen is best known as a DNA polymerase accessory protein but has more recently also been shown to have different functions in important cellular processes such as DNA replication, DNA repair, and cell cycle control. PCNA has been found in quaternary complexes with the cyclin kinase inhibitor p21 and several pairs of cyclin-dependent protein kinases and their regulatory partner, the cyclins. Here we show a direct interaction between PCNA and Cdk2. This interaction involves the regions of the PCNA trimer close to the C termini. We found that PCNA and Cdk2 form a complex together with cyclin A. This ternary PCNA-Cdk2-cyclin A complex was able to phosphorylate the PCNA binding region of the large subunit of replication factor C as well as DNA ligase I. Furthermore, PCNA appears to be a connector between Cdk2 and DNA ligase I and to stimulate phosphorylation of DNA ligase I. Based on our results, we propose the model that PCNA brings Cdk2 to proteins involved in DNA replication and possibly might act as an "adaptor" for Cdk2-cyclin A to PCNA-binding DNA replication proteins.

Mechanism of DNA replication in eukaryotic cells: cellular host factors stimulating adenovirus DNA replication.

Replication of adenovirus (Ad) DNA depends on interactions between three viral and three cellular proteins. Human transcription factors NFI and Oct-1 recruit the Ad DNA polymerase to the origin of DNA replication as a complex with the Ad protein primer pTP. High affinity and specificity DNA binding to recognition sites in this origin by the transcription factors stimulate and stabilize pre-initiation complex formation to compensate for the low binding specificity of the pTP/pol complex. In this review, we discuss the properties of NFI and Oct-1 and the mechanism by which they enhance initiation of DNA replication. We propose a model that describes the dynamics of initiation and elongation as well as the assembly and disassembly of the pre-initiation complex.

Use of Women's Health Care Specialists in a women's clinic.

Women's Health Care Specialists (WHCS), with physician supervision, provide most of the ambulatory obstetric and gynecologic care at a major urban teaching hospital. WHCS see 75% of all patients without physician interaction. The performance of WHCS was compared to that of the previous clinic staff, which was composed of residents in training. WHCS identified surgical candidates at rates equal to those of previous resident staff. Visual and palpable gynecologic malignancies were identified at rates significantly greater than expected from national population-based data (P less than 0.06). Patient return for follow-up of Papanicolaou smear abnormalities increased significantly, from 43% to 58% (P less than 0.01). In this model, physician utilization is enhanced and residents in training benefit. WHCS, with physician backup, are able to provide ambulatory obstetric and gynecologic care comparable and in some ways superior to the previous resident service.