Mutations in yeast proliferating cell nuclear antigen define distinct sites for interaction with DNA polymerase delta and DNA polymerase epsilon.

The importance of the interdomain connector loop and of the carboxy-terminal domain of Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) for functional interaction with DNA polymerases delta (Poldelta) and epsilon (Pol epsilon) was investigated by site-directed mutagenesis. Two alleles, pol30-79 (IL126,128AA) in the interdomain connector loop and pol30-90 (PK252,253AA) near the carboxy terminus, caused growth defects and elevated sensitivity to DNA-damaging agents. These two mutants also had elevated rates of spontaneous mutations. The mutator phenotype of pol30-90 was due to partially defective mismatch repair in the mutant. In vitro, the mutant PCNAs showed defects in DNA synthesis. Interestingly, the pol30-79 mutant PCNA (pcna-79) was most defective in replication with Poldelta, whereas pcna-90 was defective in replication with Pol epsilon. Protein-protein interaction studies showed that pcna-79 and pcna-90 failed to interact with Pol delta and Pol epsilon, respectively. In addition, pcna-90 was defective in interaction with the FEN-1 endo-exonuclease (RTH1 product). A loss of interaction between pcna-79 and the smallest subunit of Poldelta, the POL32 gene product, implicates this interaction in the observed defect with the polymerase. Neither PCNA mutant showed a defect in the interaction with replication factor C or in loading by this complex. Processivity of DNA synthesis by the mutant holoenzyme containing pcna-79 was unaffected on poly(dA) x oligo(dT) but was dramatically reduced on a natural template with secondary structure. A stem-loop structure with a 20-bp stem formed a virtually complete block for the holoenzyme containing pcna-79 but posed only a minor pause site for wild-type holoenzyme, indicating a function of the POL32 gene product in allowing replication past structural blocks.     

Exonuclease V from Saccharomyces cerevisiae. A 5'----3'-deoxyribonuclease that produces dinucleotides in a sequential fashion

A novel deoxyribonuclease, exonuclease V, has been purified approximately 30,000-fold from Saccharomyces cerevisiae. Exonuclease V is localized in the nucleus. The nuclease degrades single-stranded, but not double-stranded, DNA from the 5'-end. The products of exonuclease action are dinucleotides, except the 3'-terminal tri- and tetranucleotides which are not degraded. Studies with synthetic oligo- and polynucleotides with specified sequence elements showed that exonuclease V cleaves off dinucleotides as primary digestion products. Thus, the polymers (pT)9pA(pT)n and (pT)10pA(pT)n yielded pTpA and pApT as digestion products, respectively. Removal of the 5'-terminal phosphate from the DNA substrate results in reduced binding of the enzyme to the substrate. In addition, the initial hydrolytic cut by exonuclease V on the dephosphorylated substrate produces a mixture of dinucleoside monophosphates and trinucleoside diphosphates. The enzyme is processive in action.     

Contributions of the two accessory subunits,RNASEH2B and RNASEH2C,to the activity and properties of the human RNase H2 complex

Eukaryotic RNase H2 is a heterotrimeric enzyme. Here, we show that the biochemical composition and stoichiometry of the human RNase H2 complex is consistent with the properties previously deduced from genetic studies. The catalytic subunit of eukaryotic RNase H2, RNASEH2A, is well conserved and similar to the monomeric prokaryotic RNase HII. In contrast, the RNASEH2B and RNASEH2C subunits from human and Saccharomyces cerevisiae share very little homology, although they both form soluble B/C complexes that may serve as a nucleation site for the addition of RNASEH2A to form an active RNase H2, or for interactions with other proteins to support different functions. The RNASEH2B subunit has a PIP-box and confers PCNA binding to human RNase H2. Unlike Escherichia coli RNase HII, eukaryotic RNase H2 acts processively and hydrolyzes a variety of RNA/DNA hybrids with similar efficiencies, suggesting multiple cellular substrates. Moreover, of five analyzed mutations in human RNASEH2B and RNASEH2C linked to Aicardi-Goutières Syndrome (AGS), only one, R69W in the RNASEH2C protein, exhibits a significant reduction in specific activity, revealing a role for the C subunit in enzymatic activity. Near-normal activity of four AGS-related mutant enzymes was unexpected in light of their predicted impairment causing the AGS phenotype.     

Proteomics Comparison of Cerebrospinal Fluid of Relapsing Remitting and Primary Progressive Multiple Sclerosis

Background

Based on clinical representation of disease symptoms multiple sclerosis (MScl) patients can be divided into two major subtypes; relapsing remitting (RR) MScl (85–90%) and primary progressive (PP) MScl (10–15%). Proteomics analysis of cerebrospinal fluid (CSF) has detected a number of proteins that were elevated in MScl patients. Here we specifically aimed to differentiate between the PP and RR subtypes of MScl by comparing CSF proteins.

Methodology/Principal Findings

CSF samples (n = 31) were handled according to the same protocol for quantitative mass spectrometry measurements we reported previously. In the comparison of PP MScl versus RR MScl we observed a number of differentially abundant proteins, such as protein jagged-1 and vitamin D-binding protein. Protein jagged-1 was over three times less abundant in PP MScl compared to RR MScl. Vitamin D-binding protein was only detected in the RR MScl samples. These two proteins were validated by independent techniques (western blot and ELISA) as differentially abundant in the comparison between both MScl types.

Conclusions/Significance

The main finding of this comparative study is the observation that the proteome profiles of CSF in PP and RR MScl patients overlap to a large extent. Still, a number of differences could be observed. Protein jagged-1 is a ligand for multiple Notch receptors and involved in the mediation of Notch signaling. It is suggested in literature that the Notch pathway is involved in the remyelination of MScl lesions. Aberration of normal homeostasis of Vitamin D, of which approximately 90% is bound to vitamin D-binding protein, has been widely implicated in MScl for some years now. Vitamin D directly and indirectly regulates the differentiation, activation of CD4+ T-lymphocytes and can prevent the development of autoimmune processes, and so it may be involved in neuroprotective elements in MScl.     

Identifying Ca2+-Binding Sites in Proteins by Liquid Chromatography-Mass Spectrometry Using Ca2+-Directed Dissociations

Here we describe a new method to identify calcium-binding sites in proteins using high-resolution liquid chromatography-mass spectrometry in concert with calcium-directed collision-induced dissociations. Our method does not require any modifications to the liquid chromatography-mass spectrometry apparatus, uses standard digestion protocols, and can be applied to existing high-resolution MS data files. In contrast to NMR, our method is applicable to very small amounts of complex protein mixtures (femtomole level). Calcium-bound peptides can be identified using three criteria: (1) the calculated exact mass of the calcium containing peptide; (2) specific dissociations of the calcium-containing peptide from threonine and serine residues; and (3) the very similar retention times of the calcium-containing peptide and the free peptide.Calcium-dependent protein interactions mostly organized in protein networks are responsible for the regulation of cell cycle progression, cell growth, differentiation, secretion, and cytoskeletal organization (1–3). As many of these proteins are linked to various pathological conditions, they are clinically important. The speed at which calcium can have an interplay between various cellular components is impressive and comes notably detectable in neurological processes and in muscle contraction. Calcium binding sites in proteins can be determined by NMR spectroscopy (4, 5). For example, by such NMR measurements, the Ca²⁺-binding sites of the tellurite-resistance protein TerD from Klebsiella pneumoniae were found to be formed in part by a highly conserved motif of 13 residues specified by the sequence GDN(R/L)TG(E/A)GDGDDE (4).Although NMR is the gold standard to study calcium binding in proteins, this approach has several drawbacks. For instance, protein size is limited (< 30 kDa) and proteins should be pure and isotopically labeled. In addition, although the information content is high, NMR is relatively insensitive compared with other techniques such as MS and fluorescence spectroscopy, and relatively large quantities of material (typically 0.5 ml at 0.5–1.0 mm in biological samples) are needed, although efforts are devoted to improve sensitivity in NMR, such as stripline NMR (6).In bottom-up proteomics, proteolytic peptides, generated by enzymatic digestion of complex protein mixtures, are sequenced by MS-based methods (MS/MS (7, 8)) using collision-induced dissociations. Because of the even higher complexity of these peptide mixtures, liquid chromatography (LC)¹ is used to separate the peptides prior to sequencing. In such an LC-MS/MS procedure, many peptides can be identified belonging to the same protein. It has been stated (9) that by this procedure more peptides are analyzed than strictly necessary for identification purposes, but it can equally well be argued that such large coverages enable more reliable protein identifications; moreover, these larger coverages allow the detection of post-translational modifications, including specific calcium complexation as described here.Considering the need of identifying calcium-bound proteins in complex biological samples at low concentrations, we set out to develop a novel method for detecting Ca²⁺-binding sites in proteins based on LC-MS.     

Probing the Mec1ATR Checkpoint Activation Mechanism with Small Peptides

Yeast Mec1, the ortholog of human ATR, is the apical protein kinase that initiates the cell cycle checkpoint in response to DNA damage and replication stress. The basal activity of Mec1 kinase is activated by cell cycle phase-specific activators. Three distinct activators stimulate Mec1 kinase using an intrinsically disordered domain of the protein. These are the Ddc1 subunit of the 9-1-1 checkpoint clamp (ortholog of human and Schizosaccharomyces pombe Rad9), the replication initiator Dpb11 (ortholog of human TopBP1 and S. pombe Cut5), and the multifunctional nuclease/helicase Dna2. Here, we use small peptides to determine the requirements for Mec1 activation. For Ddc1, we identify two essential aromatic amino acids in a hydrophobic environment that when fused together are proficient activators. Using this increased insight, we have been able to identify homologous motifs in S. pombe Rad9 that can activate Mec1. Furthermore, we show that a 9-amino acid Dna2-based peptide is sufficient for Mec1 activation. Studies with mutant activators suggest that binding of an activator to Mec1 is a two-step process, the first step involving the obligatory binding of essential aromatic amino acids to Mec1, followed by an enhancement in binding energy through interactions with neighboring sequences.     

Three new DNA helicases from<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

At least six DNA helicases have been identified during fractionation of extracts from the yeastSaccharomyces cerevisiae. Three of those, DNA helicases B, C, and D, have been further purified and characterized. DNA helicases B and C co-purified with DNA polymerse δ through several chromatographic steps, but were separated from the polymerase by hydrophobic chromatography. DNA helicase D co-purified with Replication Factor C over seven chromatographic steps, and was only separated from it by glycerol gradient centrifugation in the presence of 0.2 M NaCl. All three helicases are DNA dependent ATPases with K_m values for ATP of 190 μM, 325 μM, and 60 μM for DNA helicases B, C, and D, respectively. Their DNA helicase activities are comparable. They are 5′–3′ helicases and have pH optima of 6.5–7 and Mg²⁺ optima of 1–2 mM. However, they differ in the nucleotide requirement for helicase action. Whereas all three helicases preferred ATP, dATP, UTP, CTP, and dCTP as cofactors, DNA helicase C also used GTP, but not dTTP. On the other hand, DNA helicase D used dTTP, but not GTP, and DNA helicase B used neither nucleotide as cofactor. These studies allowed us to conclude that DNA helicases B, C, and D are not only distinct enzymes, but also different from two previously identified yeast DNA helicases, the RAD3 protein and ATPase III.     

Synthesis of DNA by DNA polymerase epsilon in vitro.

The isolation of DNA polymerase (Pol) epsilon from extracts of HeLa cells is described. The final fractions contained two major subunits of 210 and 50 kDa which cosedimented with Pol epsilon activity, similar to those described previously (Syvaoja, J., and Linn, S. (1989) J. Biol. Chem. 264, 2489-2497). The properties of the human Pol epsilon and the yeast Pol epsilon were compared. Both enzymes elongated singly primed single-stranded circular DNA templates. Yeast Pol epsilon required the presence of a DNA binding protein (SSB) whereas human Pol epsilon required the addition of SSB, Activator 1 and proliferating cell nuclear antigen (PCNA) for maximal activity. Both enzymes were totally unable to elongate primed DNA templates in the presence of salt; however, activity could be restored by the addition of Activator 1 and PCNA. Like Pol delta, Pol epsilon formed complexes with SSB-coated primed DNA templates in the presence of Activator 1 and PCNA which could be isolated by filtration through Bio-Gel A-5m columns. Unlike Pol delta, Pol epsilon bound to SSB-coated primed DNA in the absence of the auxiliary factors. In the presence of salt, Pol epsilon complexes were less stable than they were in the absence of salt. In the in vitro simian virus 40 (SV40) T antigen-dependent synthesis of DNA containing the SV40 origin of replication, yeast Pol epsilon but not human Pol epsilon could substitute for yeast or human Pol delta in the generation of long DNA products. However, human Pol epsilon did increase slightly the length of DNA chains formed by the DNA polymerase alpha-primase complex in SV40 DNA synthesis. The bearing of this observation on the requirement for a PCNA-dependent DNA polymerase in the synthesis and maturation of Okazaki fragments is discussed. However, no unique role for human Pol epsilon in the in vitro SV40 DNA replication system was detected.