Based on large-scale data for the yeast Saccharomyces cerevisiae (protein and mRNA abundance, translational status, transcript length), we investigate relation of transcription, translation, protein turnover a genome-wide scale. We elucidate variations between different spatial cell compartments functional modules by comparing protein-to-mRNA ratios, activity, novel descriptor protein-specific degradation half-life descriptor). This analysis helps to understand cell's strategy use...

We have investigated the DNA polymerase alpha promoter sequence requirements for expression of a heterologous gene in actively cycling cells and following serum addition to serum-deprived cells. An 11.4-kb genomic clone that spans 5' end this includes 1.62 kb upstream from translation start site was isolated. The transcription mapped at 46 +/- 1 nucleotides site. is GC rich lacks TATA but has CCAAT on opposite strand. Analysis set deletion constructs transient transfection assays...

The monosaccharide, β-N-acetylglucosamine (GlcNAc), can be added to the hydroxyl group of either serines or threonines generate an O-linked (O-GlcNAc) residue (Love, D. C., and Hanover, J. A. (2005) Sci. STKE 2005 312, 1–14; Hart, G. W., Housley, M. P., Slawson, C. (2007) Nature 446, 1017–1022). This post-translational protein modification, termed O-GlcNAcylation, is reversible, analogous phosphorylation, has been implicated in many cellular processes. Here, we present evidence that human...

The expression of DNA polymerase alpha, a principal chromosome replication enzyme, is constitutive during the cell cycle. We show in this report that alpha catalytic polypeptide p180 phosphorylated throughout cycle and hyperphosphorylated G2/M phase. p70 subunit only This cycle-dependent phosphorylation due to kinase(s) not phosphatase(s). In vitro evidence indicates involvement p34cdc2 kinase mitotic alpha. Tryptic phosphopeptide maps demonstrate peptides are identical those vivo. from...

Affinity chromatography on double-stranded (ds) and single-stranded (ss) DNA-cellulose columns was employed to find analogs of the Escherichia coli T4 DNA binding proteins (SSB proteins) in calf thymus. The interaction several purified SSB with pure DNA-polymerase-alpha--primase complex synthesis activated primase-initiated M13 served as a criterion for possible involvement one these process replication. Two were essential homogeneity. These most abundant exhibited apparent relative...

Replication protein A (RPA) is a heterotrimeric complex and the main single-stranded DNA (ssDNA)-binding in eukaryotes. RPA has key functions most of DNA-associated metabolic pathways damage signalling. Its high affinity for ssDNA helps to stabilise structures protect sequence from nuclease attacks. consists multiple DNA-binding domains which are oligonucleotide/oligosaccharide-binding (OB)-folds that responsible binding interactions with proteins. These RPA-ssDNA RPA-protein crucial...

DNA damage induced by the carcinogen benzo[a]pyrene dihydrodiol epoxide (BPDE) induces a Chk1-dependent S-phase checkpoint. Here, we have investigated molecular basis of BPDE-induced arrest. inhibition synthesis in BPDE-treated cells occurred without detectable changes Cdc25A levels, Cdk2 activity, or Cdc7/Dbf4 interaction. Overexpression studies showed that Cdc25A, cyclin A/Cdk2, and were not rate-limiting for when checkpoint was active. To investigate other potential targets checkpoint,...

Cell division cycle protein 45 (Cdc45) plays a critical role in DNA replication to ensure that chromosomal is replicated only once per cell cycle. We analysed the expression of human Cdc45 proliferating and nonproliferating cells. Our findings show absent from long-term quiescent, terminally differentiated senescent cells, although it present throughout Moreover, much less abundant than minichromosome maintenance (Mcm) proteins supporting concept origin binding rate limiting for initiation....

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTImmunoaffinity-purified DNA polymerase .alpha. displays novel propertiesHeinz Peter Nasheuer and Frank GrosseCite this: Biochemistry 1987, 26, 25, 8458–8466Publication Date (Print):December 1, 1987Publication History Published online1 May 2002Published inissue 1 December 1987https://pubs.acs.org/doi/10.1021/bi00399a064https://doi.org/10.1021/bi00399a064research-articleACS PublicationsRequest reuse permissionsArticle...

Poly(ADP-ribosyl)ation is involved in numerous bio-logical processes including DNA repair, transcription and cell death. Cellular levels of poly(ADP-ribose) (PAR) are regulated by PAR polymerases (PARPs) the degrading enzyme glycohydrolase (PARG), controlling fate decision between life death response to damage. Replication stress a source damage, leading transient stalling replication forks or their collapse followed generation double-strand breaks (DSB). The involvement PARP-1 replicative...

DNA polymerase α-primase is known to be phosphorylated in human and yeast cells a cell cycle-dependent manner on the p180 p68 subunits. Here we show that phosphorylation of purified by cyclin A/cdk2 vitro reduced its ability initiate simian virus 40 (SV40) replication vitro, while E/cdk2 stimulated initiation activity. Tryptic phosphopeptide mapping revealed family peptides was modified well poorly E/cdk2. The phosphopeptides were identical with both kinases. By mass spectrometry, peptide...

DNA‐polymerase‐α‐primase complex contains four subunits, p180, p68, p58, and p48, comprises a minimum of two enzymic functions. We have cloned cDNAs encoding subunits from human mouse. Sequence comparisons showed high amino acid conservation among the mammalian proteins. over‐expressed single polypeptides co‐expressed various subunit complexes using baculovirus vectors, purified proteins investigated their biochemical properties. The mouse p48 (Mp48) alone had primase activity. Purification...

Immunoaffinity-purified DNA polymerase alpha-primase complex from calf thymus consists of subunits with molecular weights 148,000-180,000, 73,000, 59,000, and 48,000 (Nasheuer, H.-P., Grosse, F. (1987) Biochemistry 26, 8458-8466). Primase activity was separated the immobilized by washing extensively 2 M KCl or, alternatively, shifting to pH 11.5 in presence 1 KCl. From both elution procedures, primase found be associated polypeptides 59,000 48,000. The specific activity, using either...

Cdc45 is an essential cellular protein that functions in both the initiation and elongation of DNA replication. Here, we analyzed localization human its interactions with other proteins during cell cycle. Human showed a diffuse distribution G1 phase, spot-like pattern S G2, again M phase The co-localization active replication sites suggested was part complex. This view corroborated by findings interacted elongating polymerases delta epsilon, Psf2, which component GINS complex as well Mcm5 7,...

We have investigated the DNA polymerase alpha promoter sequence requirements for expression of a heterologous gene in actively cycling cells and following serum addition to serum-deprived cells. An 11.4-kb genomic clone that spans 5' end this includes 1.62 kb upstream from translation start site was isolated. The transcription mapped at 46 +/- 1 nucleotides site. is GC rich lacks TATA but has CCAAT on opposite strand. Analysis set deletion constructs transient transfection assays...

To analyze the interaction of human replication protein A (RPA) and its subunits with DNA template-primer junction in fork, we designed several systems differing size single-stranded template tail (4, 9, 13, 14,19 31 nt). Base substituted photoreactive dNTP analogs—5-[ N -(2-nitro-5-azidobenzoyl)- frans -3-amino-propenyl-1]-2′-deoxyuridine-5′-triphosphate (NAB-4-dUTP) 5-[ -[ -(2-nitro-5-azidobenzoyl)glycyl]- trans -3-aminopropenyl-1]-2′-deoxyuridine-5′-triphos-phate (NAB-7-dUTP)—were used as...

The human single-stranded DNA-binding protein, replication protein A (RPA), is regulated by the N-terminal phosphorylation of its 32-kDa subunit, RPA2. RPA2 hyperphosphorylated in response to various DNA-damaging agents and also phosphorylated a cell-cycle-dependent manner during S- M-phase, primarily at two CDK consensus sites, S23 S29. Here we generated monoclonal phospho-specific antibodies directed against these sites. These RPA2-(P)-S23 RPA2-(P)-S29 recognized mitotically with high...

DNA polymerase alpha-primase (pol-prim, consisting of p180-p68-p58-p48), and primase p58-p48 (prim(2)) synthesize short RNA primers on single-stranded DNA. In the SV40 replication system, only pol-prim is able to start leading strand that needs unwinding double-stranded (ds) prior primer synthesis. At high concentrations, prim(2) indistinguishably reduce dsDNA by T antigen (Tag). synthesis ssDNA in presence protein A (RPA) Tag has served as a model system study initiation Okazaki fragments...