Heinz-Peter Nasheuer

Eukaryotic DNA polymerases, a growing family

In eukaryotic cells, DNA polymerases are required to maintain the integrity of the genome during processes, such as DNA replication, various DNA repair events, translesion DNA synthesis, DNA recombination, and also in regulatory events, such as cell cycle control and DNA damage checkpoint function. In the last two years, the number of known DNA polymerases has increased to at least nine (called alpha, beta, gamma, delta, epsilon, zeta, eta, t and iota), and yeast Saccharomyces cerevisiae contains REV1 deoxycytidyl transferase.

Post-transcriptional Expression Regulation in the Yeast Saccharomyces cerevisiae on a Genomic Scale

Based on large-scale data for the yeast Saccharomyces cerevisiae (protein and mRNA abundance, translational status, transcript length), we investigate the relation of transcription, translation, and protein turnover on a genome-wide scale. We elucidate variations between different spatial cell compartments and functional modules by comparing protein-to-mRNA ratios, translational activity, and a novel descriptor for protein-specific degradation (protein half-life descriptor). This analysis helps to understand the cell’s strategy to use transcriptional and post-transcriptional regulation mechanisms for managing protein levels. For instance, it is possible to identify modules that are subject to suppressed translation under normal conditions (“translation on demand”). In order to reduce inconsistencies between the datasets, we compiled a new reference mRNA abundance dataset and we present a novel approach to correct large microarray signals for a saturation bias. Accounting for ribosome density based on transcript length rather than ORF length improves the correlation of observed protein levels to translational activity. We discuss potential causes for the deviations of these correlations. Finally, we introduce a quantitative descriptor for protein degradation (protein half-life descriptor) and compare it to measured half-lives. The study demonstrates significant post-transcriptional control of protein levels for a number of different compartments and functional modules, which is missed when exclusively focusing on transcript levels.

Modification of Histones by Sugar β-N-Acetylglucosamine (GlcNAc) Occurs on Multiple Residues, Including Histone H3 Serine 10, and Is Cell Cycle-regulated

The monosaccharide, β-N-acetylglucosamine (GlcNAc), can be added to the hydroxyl group of either serines or threonines to generate an O-linked β-N-acetylglucosamine (O-GlcNAc) residue (Love, D. C., and Hanover, J. A. (2005) Sci. STKE 2005 312, 1–14; Hart, G. W., Housley, M. P., and Slawson, C. (2007) Nature 446, 1017–1022). This post-translational protein modification, termed O-GlcNAcylation, is reversible, analogous to phosphorylation, and has been implicated in many cellular processes. Here, we present evidence that in human cells all four core histones of the nucleosome are substrates for this glycosylation in the relative abundance H3, H4/H2B, and H2A. Increasing the intracellular level of UDP-GlcNAc, the nucleotide sugar donor substrate for O-GlcNAcylation enhanced histone O-GlcNAcylation and partially suppressed phosphorylation of histone H3 at serine 10 (H3S10ph). Expression of recombinant H3.3 harboring an S10A mutation abrogated histone H3 O-GlcNAcylation relative to its wild-type version, consistent with H3S10 being a site of histone O-GlcNAcylation (H3S10glc). Moreover, O-GlcNAcylated histones were lost from H3S10ph immunoprecipitates, whereas immunoprecipitation of either H3K4me3 or H3K9me3 (active or inactive histone marks, respectively) resulted in co-immunoprecipitation of O-GlcNAcylated histones. We also examined histone O-GlcNAcylation during cell cycle progression. Histone O-GlcNAcylation is high in G1 cells, declines throughout the S phase, increases again during late S/early G2, and persists through late G2 and mitosis. Thus, O-GlcNAcylation is a novel histone post-translational modification regulating chromatin conformation during transcription and cell cycle progression.

The Chk1-mediated S-phase checkpoint targets initiation factor Cdc45 via a Cdc25A/Cdk2-independent mechanism.

DNA damage induced by the carcinogen benzo[a]pyrene dihydrodiol epoxide (BPDE) induces a Chk1-dependent S-phase checkpoint. Here, we have investigated the molecular basis of BPDE-induced S-phase arrest. Chk1-dependent inhibition of DNA synthesis in BPDE-treated cells occurred without detectable changes in Cdc25A levels, Cdk2 activity, or Cdc7/Dbf4 interaction. Overexpression studies showed that Cdc25A, cyclin A/Cdk2, and Cdc7/Dbf4 were not rate-limiting for DNA synthesis when the BPDE-induced S-phase checkpoint was active. To investigate other potential targets of the S-phase checkpoint, we tested the effects of BPDE on the chromatin association of DNA replication factors. The levels of chromatin-associated Cdc45 (but not soluble Cdc45) were reduced concomitantly with BPDE-induced Chk1 activation and inhibition of DNA synthesis. The chromatin association of Mcm7, Mcm10, and proliferating cell nuclear antigen was unaffected by BPDE treatment. However, the association between Mcm7 and Cdc45 in the chromatin fraction was inhibited in BPDE-treated cells. Chromatin immunoprecipitation analyses demonstrated reduced association of Cdc45 with the β-globin origin of replication in BPDE-treated cells. The inhibitory effects of BPDE on DNA synthesis, Cdc45/Mcm7 associations, and interactions between Cdc45 and the β-globin locus were abrogated by the Chk1 inhibitor UCN-01. Taken together, our results show that the association between Cdc45 and Mcm7 at origins of replication is negatively regulated by Chk1 in a Cdk2-independent manner. Therefore, Cdc45 is likely to be an important target of the Chk1-mediated S-phase checkpoint.

Phosphorylation of DNA polymerase α-primase by Cyclin A-dependent kinases regulates initiation of DNA replication in vitro

DNA polymerase α-primase is the only known eukaryotic enzyme that can start DNA replication de novo. In this study, we investigated the regulation of DNA replication by phosphorylation of DNA polymerase α-primase. The p180 and the p68 subunits of DNA polymerase α-primase were phosphorylated using Cyclin A-, B- and E- dependent kinases. This phosphorylation did not influence its DNA polymerase activity on activated DNA, but slightly stimulated primase activity using poly(dT) single-stranded DNA (ssDNA) without changing the product length of primers. In contrast, site-specific initiation of replication on plasmid DNA containing the SV40 origin is affected: Cyclin A-Cdk2 and Cyclin A-Cdc2 inhibited initiation of SV40 DNA replication in vitro, Cyclin B-Cdc2 had no effect and Cyclin E-Cdk2 stimulated the initation reaction. DNA polymerase α-primase that was pre-phosphorylated by Cyclin A-Cdk2 was completely unable to initiate the SV40 DNA replication in vitro; Cyclin B-Cdc2-phosphorylated enzyme was moderately inhibited, while Cyclin E-Cdk2-treated DNA polymerase α-primase remained fully active in the initiation reaction.

Quantitative assignment of reaction directionality in constraint-based models of metabolism: application to Escherichia coli.

Constraint-based modeling is an approach for quantitative prediction of net reaction flux in genome-scale biochemical networks. In vivo, the second law of thermodynamics requires that net macroscopic flux be forward, when the transformed reaction Gibbs energy is negative. We calculate the latter by using (i) group contribution estimates of metabolite species Gibbs energy, combined with (ii) experimentally measured equilibrium constants. In an application to a genome-scale stoichiometric model of Escherichia coli metabolism, iAF1260, we demonstrate that quantitative prediction of reaction directionality is increased in scope and accuracy by integration of both data sources, transformed appropriately to in vivo pH, temperature and ionic strength. Comparison of quantitative versus qualitative assignment of reaction directionality in iAF1260, assuming an accommodating reactant concentration range of 0.02-20mM, revealed that quantitative assignment leads to a low false positive, but high false negative, prediction of effectively irreversible reactions. The latter is partly due to the uncertainty associated with group contribution estimates. We also uncovered evidence that the high intracellular concentration of glutamate in E. coli may be essential to direct otherwise thermodynamically unfavorable essential reactions, such as the leucine transaminase reaction, in an anabolic direction.

Interactions of human Cdc45 with the Mcm2-7 complex, the GINS complex, and DNA polymerases ! and " during S phase

Cdc45 is an essential cellular protein that functions in both the initiation and elongation of DNA replication. Here, we analyzed the localization of human Cdc45 and its interactions with other proteins during the cell cycle. Human Cdc45 showed a diffuse distribution in G1 phase, a spot‐like pattern in S and G2, and again a diffuse distribution in M phase of the cell cycle. The co‐localization of Cdc45 with active replication sites during S phase suggested that the human Cdc45 protein was part of the elongation complex. This view was corroborated by findings that Cdc45 interacted with the elongating DNA polymerases δ and ɛ, with Psf2, which is a component of the GINS complex as well as with Mcm5 and 7, subunits of the putative replicative DNA helicase complex. Hence, Cdc45 may play an important role in elongation of DNA replication by bridging the processive DNA polymerases δ and ɛ with the replicative helicase in the elongating machinery.

Integrated stoichiometric, thermodynamic and kinetic modelling of steady state metabolism ☆

The quantitative analysis of biochemical reactions and metabolites is at frontier of biological sciences. The recent availability of high-throughput technology data sets in biology has paved the way for new modelling approaches at various levels of complexity including the metabolome of a cell or an organism. Understanding the metabolism of a single cell and multi-cell organism will provide the knowledge for the rational design of growth conditions to produce commercially valuable reagents in biotechnology. Here, we demonstrate how equations representing steady state mass conservation, energy conservation, the second law of thermodynamics, and reversible enzyme kinetics can be formulated as a single system of linear equalities and inequalities, in addition to linear equalities on exponential variables. Even though the feasible set is non-convex, the reformulation is exact and amenable to large-scale numerical analysis, a prerequisite for computationally feasible genome scale modelling. Integrating flux, concentration and kinetic variables in a unified constraint-based formulation is aimed at increasing the quantitative predictive capacity of flux balance analysis. Incorporation of experimental and theoretical bounds on thermodynamic and kinetic variables ensures that the predicted steady state fluxes are both thermodynamically and biochemically feasible. The resulting in silico predictions are tested against fluxomic data for central metabolism in Escherichia coli and compare favourably with in silico prediction by flux balance analysis.

Protein-protein interactions of the primase subunits p58 and p48 with simian virus 40 T antigen are required for efficient primer synthesis in a cell-free system.

DNA polymerase α-primase (pol-prim, consisting of p180-p68-p58-p48), and primase p58-p48 (prim2) synthesize short RNA primers on single-stranded DNA. In the SV40 DNA replication system, only pol-prim is able to start leading strand DNA replication that needs unwinding of double-stranded (ds) DNA prior to primer synthesis. At high concentrations, pol-prim and prim2 indistinguishably reduce the unwinding of dsDNA by SV40 T antigen (Tag). RNA primer synthesis on ssDNA in the presence of replication protein A (RPA) and Tag has served as a model system to study the initiation of Okazaki fragments on the lagging strandin vitro. On ssDNA, Tag stimulates whereas RPA inhibits the initiation reaction of both enzymes. Tag reverses and even overcompensates the inhibition of primase by RPA. Physical binding of Tag to the primase subunits and RPA, respectively, is required for these activities. Each subunit of the primase complex, p58 and p48, performs physical contacts with Tag and RPA independently of p180 and p68. Using surface plasmon resonance, the dissociation constants of the Tag/pol-prim and Tag/primase interactions were 1.2·10−8 m and 1.3·10−8 m, respectively.

Purification and functional characterization of bovine RP-A in anin vitro SV40 DNA replication system

The single-stranded DNA binding protein RP-A is required in SV40 DNAin vitro replication. The RP-A purified from calf thymus contains 4 polypeptides with molecular weights 70kDa, 53kDa, 32kDa, and 14kDa. The p70 subunit and its proteolysed form p53 are recognized by the monoclonal antibody 70C (Kenny et al. (1990)) and bind to ssDNA. The p70 and p32 subunits of bovine RP-A are phosphorylated by CDC2-cyclin B kinase. Bovine RP-A supports the origin dependent unwinding of SV40 DNA by T antigen. Furthermore, bovine RP-A can efficiently substitute for human RP-A in SV40 DNA replicationin vitro. A modified blotting technique revealed that RP-A interacts specifically and directly with the p48 subunit of DNA polymerase α-primase complex.