Mammalian Cell Extracts Research Articles

Identification of a Novel Protein, PDIP38, That Interacts with the p50 Subunit of DNA Polymerase δ and Proliferating Cell Nuclear Antigen

The yeast two-hybrid screening method was used to identify novel proteins that associate with human DNA polymerase delta (pol delta). Two baits were used in this study. These were the large (p125) and small (p50) subunits of the core pol delta heterodimer. p50 was the only positive isolated with p125 as the bait. Two novel protein partners, named PDIP38 and PDIP46, were identified from the p50 screen. In this study, the interaction of PDIP38 with pol delta was further characterized. PDIP38 encodes a protein of 368 amino acids whose C terminus is conserved with the bacterial APAG protein and with the F box A protein. It was found that PDIP38 also interacts with proliferating cell nuclear antigen (PCNA). The ability of PDIP38 to interact with both the p50 subunit of pol delta and with PCNA was confirmed by pull-down assays using glutathione S-transferase (GST)-PDIP38 fusion proteins. The PCNA-PDIP38 interaction was also demonstrated by PCNA overlay experiments. The association of PDIP38 with pol delta was shown to occur in calf thymus tissue and mammalian cell extracts by GST-PDIP38 pull-down and coimmunoprecipitation experiments. PDIP38 was associated with pol delta isolated by immunoaffinity chromatography. The association of PDIP38 with pol delta could also be demonstrated by native gel electrophoresis.

A rapid ELISA based method to determine peptidyl-arginine deiminase activity in biological samples

Peptidylarginine deiminases (PADs; EC 3.5.3.15) are a family of calcium-dependent enzymes that convert peptidylarginine into peptidylcitrulline. The recent finding that patients with rheumatoid arthritis (RA) produce autoantibodies against citrulline-containing epitopes greatly increased the interest in the PAD enzymes and their activities. It is not yet known whether there is a causative relationship between the generation of antibodies targeting citrullinated epitopes and the development of the disease. Characterisation of the structure and function of PADs may help to understand the production process of citrullinated antigens and possibly also the aetiology of RA. Several assays are known to monitor PAD activity in biological samples. However, these assays either have a low sensitivity or are laborious. Here, we describe the development of a simple, rapid method for the simultaneous analysis of many PAD-containing samples. This new method is based on the binding of an antibody specifically recognising a citrulline-containing epitope in a defined peptide. We show that this method is very sensitive and can be applied to monitor PAD activity in many types of biological samples, such as bacterial lysates, mammalian cell extracts and tissue extracts.

Borg/Septin Interactions and the Assembly of Mammalian Septin Heterodimers, Trimers, and Filaments

Septins constitute a family of guanine nucleotide-binding proteins that were first discovered in the yeast Saccharomyces cerevisiae but are also present in many other eukaryotes. In yeast they congregate at the bud neck and are required for cell division. Their function in metazoan cells is uncertain, but they have been implicated in exocytosis and cytokinesis. Septins have been purified from cells as hetero-oligomeric filaments, but their mechanism of assembly is unknown. Further studies have been limited by the difficulty in expressing functional septin proteins in bacteria. We now show that stable, soluble septin heterodimers can be produced by co-expression from bicistronic vectors in bacteria and that the co-expression of three septins results in their assembly into filaments. Pre-assembled dimers and trimers bind guanine nucleotide and show a slow GTPase activity. The assembly of a heterodimer from monomers in vitro is accompanied by GTP hydrolysis. Borg3, a downstream effector of the Cdc42 GTPase, binds specifically to a septin heterodimer composed of Sept6 and Sept7 and to the Sept2/6/7 trimer, but not to septin monomers or to other heterodimers. Septins associate through their C-terminal coiled-coil domains, and Borg3 appears to recognize the interface between these domains in Sept6 and Sept7.

The histone 3'-terminal stem-loop-binding protein enhances translation through a functional and physical interaction with eukaryotic initiation factor 4G (eIF4G) and eIF3.

Metazoan cell cycle-regulated histone mRNAs are unique cellular mRNAs in that they terminate in a highly conserved stem-loop structure instead of a poly(A) tail. Not only is the stem-loop structure necessary for 3'-end formation but it regulates the stability and translational efficiency of histone mRNAs. The histone stem-loop structure is recognized by the stem-loop-binding protein (SLBP), which is required for the regulation of mRNA processing and turnover. In this study, we show that SLBP is required for the translation of mRNAs containing the histone stem-loop structure. Moreover, we show that the translation of mRNAs ending in the histone stem-loop is stimulated in Saccharomyces cerevisiae cells expressing mammalian SLBP. The translational function of SLBP genetically required eukaryotic initiation factor 4E (eIF4E), eIF4G, and eIF3, and expressed SLBP coisolated with S. cerevisiae initiation factor complexes that bound the 5' cap in a manner dependent on eIF4G and eIF3. Furthermore, eIF4G coimmunoprecipitated with endogenous SLBP in mammalian cell extracts and recombinant SLBP and eIF4G coisolated. These data indicate that SLBP stimulates the translation of histone mRNAs through a functional interaction with both the mRNA stem-loop and the 5' cap that is mediated by eIF4G and eIF3.

HMutSβ Is Required for the Recognition and Uncoupling of Psoralen Interstrand Cross-Links In Vitro

The removal of interstrand cross-links (ICLs) from DNA in higher eucaryotes is not well understood. Here, we show that processing of psoralen ICLs in mammalian cell extracts is dependent upon the mismatch repair complex hMutSbeta but is not dependent upon the hMutSalpha complex or hMlh1. The processing of psoralen ICLs is also dependent upon the nucleotide excision repair proteins Ercc1 and Xpf but not upon other components of the excision stage of this pathway or upon Fanconi anemia proteins. Products formed during the in vitro reaction indicated that the ICL has been removed or uncoupled from the cross-linked substrate in the mammalian cell extracts. Finally, the hMutSbeta complex is shown to specifically bind to psoralen ICLs, and this binding is stimulated by the addition of PCNA. Thus, a novel pathway for processing ICLs has been identified in mammalian cells which involves components of the mismatch repair and nucleotide excision repair pathways.

Resistance of 3'-phosphoglycolate DNA ends to digestion by mammalian DNase III.

An essential step in the repair of free radical-mediated DNA strand breaks is the removal of sugar fragments such as phosphoglycolate from the 3' termini. While the abasic endonuclease Ape1 can remove phosphoglycolate from single-strand breaks in double-stranded DNA, an enzyme capable of removing it from 3' overhangs of double-strand breaks has yet to be identified. We therefore tested DNase III, the predominant 3' --> 5' exonuclease in mammalian cell extracts, for possible 3'-phosphoglycolate-removing activity. However, all 3'-phosphoglycolate substrates, as well as a 3'-phosphate substrate, were resistant to DNase III under conditions in which the analogous 3'-hydroxyl substrates were extensively degraded. The DNA end-binding protein Ku (an equimolar mixture of Ku70, now known as G22P1, and Ku86, now known as XRCC5) did not alter the resistance of the 3'-phosphoglycolate substrates, but the protein modulated the susceptibility of 3'-hydroxyl substrates, allowing DNase III to remove a 3' overhang but inhibiting digestion of the double-stranded portion of the substrate.

DNA polymerase beta is the major dRP lyase involved in repair of oxidative base lesions in DNA by mammalian cell extracts.

The repair of oxidative base lesions in DNA is a coordinated chain of reactions that includes removal of the damaged base, incision of the phosphodiester backbone at the abasic sugar residue, incorporation of an undamaged nucleotide and sealing of the DNA strand break. Although removal of a damaged base in mammalian cells is initiated primarily by a damage-specific DNA glycosylase, several lyases and DNA polymerases may contribute to the later stages of repair. DNA polymerase beta (Pol beta) was implicated recently as the major polymerase involved in repair of oxidative base lesions; however, the identity of the lyase participating in the repair of oxidative lesions is unclear. We studied the mechanism by which mammalian cell extracts process DNA substrates containing a single 8-oxoguanine or 5,6-dihydrouracil at a defined position. We find that, when repair synthesis proceeds through a Pol beta-dependent single nucleotide replacement mechanism, the 5'-deoxyribosephosphate lyase activity of Pol beta is essential for repair of both lesions.

A Nonradioactive High-Performance Liquid Chromatographic Microassay for Uridine 5′-Monophosphate Synthase, Orotate Phosphoribosyltransferase, and Orotidine 5′-Monophosphate Decarboxylase

A novel nonradioactive, microassay method has been developed to determine simultaneously the two enzymatic activities of orotate phosphoribosyltransferase (OPRTase) and orotidine 5′-monophosphate decarboxylase (ODCase), either as a bifunctional protein (uridine 5′-monophosphate synthase, UMPS) or as separate enzymes. Substrates (orotate for OPRTase or orotidine 5′-monophosphate for ODCase) and a product (UMP) of the enzymatic assay were separated by high-performance liquid chromatography (HPLC) using a reversed-phase column and an ion-pairing system; the amount of UMP was quantified by dual-wavelength uv detection at 260 and 278 nm. This HPLC assay can easily detect picomole levels of UMP in enzymatic reactions using low specific activity UMPS of mammalian cell extracts, which is difficult to do with the other nonradioactive assays that have been described. The HPLC assay is suitable for use in protein purification and for kinetic study of these enzymes.

MRNA decay during herpesvirus infections: interaction between a putative viral nuclease and a cellular translation factor.

During lytic infections, the virion host shutoff (Vhs) protein (UL41) of herpes simplex virus destabilizes both host and viral mRNAs. By accelerating mRNA decay, it helps determine the levels and kinetics of viral and cellular gene expression. In vivo, Vhs shows a strong preference for mRNAs, as opposed to non-mRNAs, and degrades the 5' end of mRNAs prior to the 3' end. In contrast, partially purified Vhs is not restricted to mRNAs and causes cleavage of target RNAs at various sites throughout the molecule. To explain this discrepancy, we searched for cellular proteins that interact with Vhs using the Saccharomyces cerevisiae two-hybrid system. Vhs was found to interact with the human translation initiation factor, eIF4H. This interaction was verified by glutathione S-transferase pull-down experiments and by coimmunoprecipitation of Vhs and epitope-tagged eIF4H from extracts of mammalian cells. The interaction was abolished by several point mutations in Vhs that abrogate its ability to degrade mRNAs in vivo. The results suggest that Vhs is a viral mRNA degradation factor that is targeted to mRNAs, and to regions of translation initiation, through an interaction with eIF4H.

Decreased sensitivity of tristetraprolin-deficient cells to p38 inhibitors suggests the involvement of tristetraprolin in the p38 signaling pathway.

Treatment of macrophages with pyridinyl imidazole inhibitors of p38 protein kinases can inhibit lipopolysaccharide-stimulated tumor necrosis factor alpha secretion. However, bone marrow-derived macrophages from tristetraprolin (TTP)-deficient mice were less sensitive than normal macrophages to this effect of p38 inhibitors, despite evidence for normal p38 activation in response to lipopolysaccharide. TTP is known to cause decreased stability of tumor necrosis factor alpha and granulocyte-macrophage colony-stimulating factor mRNAs after binding to an AU-rich element in their 3'-untranslated regions. A recombinant TTP fusion protein could be phosphorylated by a recombinant p38 kinase in cell-free assays and was phosphorylated to the same extent by immunoprecipitated p38 derived from normal and TTP-deficient cells stimulated with lipopolysaccharide; in both cases, the enzyme activity was inhibited by the p38 inhibitors. TTP phosphorylation also was increased in intact macrophages after lipopolysaccharide stimulation, an effect that was blocked by the p38 inhibitors. Finally, TTP in mammalian cell extracts bound less well to an AU-rich element RNA probe than did the same amount of TTP following dephosphorylation. These results suggest that TTP may be a component of the signaling cascade, initiated by inflammatory stimuli and mediated in part by activation of p38, that ultimately leads to enhanced secretion of tumor necrosis factor alpha.

In VitroRNASplicing in Mammalian Cell Extracts

Almost every eukaryotic pre-mRNA generated by RNA polymerase II transcription requires the removal of introns to create mRNA. The correct splicing of constitutive exons is thus critical for normal protein expression and function. Moreover, the removal of many introns by the spliceosome is controlled in a tissue-specific or developmentally specific manner. In order to study RNA splicing at a biochemical level, it is necessary to employ an in vitro, or cell-free, system. Cell-free splicing systems require two main components: (1) an extract made from mammalian cell nuclei and (2) the introns and exons of the eukaryotic gene of interest. This minigene construct allows the synthesis of sufficient quantities of pre-mRNA substrates in vitro, which are then incubated in the nuclear extract and analyzed for splicing. Nuclear extracts, first developed for studying transcription in vitro, are modified for splicing. This unit describes how to set up an in vitro splicing reaction using a mammalian nuclear extract derived from either cell line or tissue, and how to analyze the splicing reaction products.

Towards global analysis of mammalian proteomes using sample prefractionation prior to narrow pH range two-dimensional gels and using one-dimensional gels for insoluble and large proteins.

The number of unique protein species in proteomes from a single mammalian cell type is not well defined but is likely to be at least 10000-20000. Since standard-size two-dimensional gels typically resolve only about 1500 to 3000 spots, they merely analyze a small portion of these proteomes. In addition, all insoluble proteins and typically proteins > 100 kDa are seldom resolved on two-dimensional (2-D) gels. The current study demonstrates the feasibility of an overall strategy for more comprehensive quantitative comparisons of complex proteomes derived from physiological fluids or mammalian cell extracts. A key feature of this approach is to prefractionate samples into a few well-resolved fractions based on the proteins' isoelectric points (pIs) using microscale solution isoelectric focusing. These fractions are then separated on narrow pH range two-dimensional gels approximately +/- 0.1 pH unit wider than the prefractionated pool. When this prefractionation approach is applied to complex mammalian proteomes, it improves resolution and spot recovery at high protein loads compared with use of parallel narrow pH range gels without prefractionation. The minimal cross-contamination between fractions allows quantitative comparisons in contrast to most alternative prefractionation methods. In addition, complementary data can be obtained by parallel analysis of the solubilized fraction on high-resolution large-pore-gradient one-dimensional gels followed by mass spectrometric identification to analyze proteins between 100 and approximately 500 kDa. Similarly, insoluble proteins can be analyzed using large-pore gels for large proteins and 10-12% one-dimensional sodium dodecyl sulfate (SDS) gels for smaller proteins. Together, these strategies should permit more reliable quantitative comparisons of complex mammalian proteomes where detection of at least 10000 protein spots is needed in order to analyze the majority of the unique protein species.

Human DNA polymerase beta initiates DNA synthesis during long-patch repair of reduced AP sites in DNA.

Simple base damages are repaired through a short-patch base excision pathway where a single damaged nucleotide is removed and replaced. DNA polymerase beta (Pol beta) is responsible for the repair synthesis in this pathway and also removes a 5'-sugar phosphate residue by catalyzing a beta-elimination reaction. How ever, some DNA lesions that render deoxyribose resistant to beta-elimination are removed through a long-patch repair pathway that involves strand displacement synthesis and removal of the generated flap by specific endonuclease. Three human DNA polymerases (Pol beta, Pol delta and Pol epsilon) have been proposed to play a role in this pathway, however the identity of the polymerase involved and the polymerase selection mechanism are not clear. In repair reactions catalyzed by cell extracts we have used a substrate containing a reduced apurinic/apyrimidinic (AP) site resistant to beta-elimination and inhibitors that selectively affect different DNA polymerases. Using this approach we find that in human cell extracts Pol beta is the major DNA polymerase incorporating the first nucleotide during repair of reduced AP sites, thus initiating long-patch base excision repair synthesis.

DNA synthesis and dRPase activities of polymerase beta are both essential for single-nucleotide patch base excision repair in mammalian cell extracts.

In mammalian cells the majority of altered bases in DNA are processed through a single-nucleotide patch base excision repair mechanism. Base excision repair is initiated by a DNA glycosylase that removes a damaged base and generates an abasic site (AP site). This AP site is further processed by an AP endonuclease activity that incises the phosphodiester bond adjacent to the AP site and generates a strand break containing 3'-OH and 5'-sugar phosphate ends. In mammalian cells, the 5'-sugar phosphate is removed by the AP lyase activity of DNA polymerase beta (Pol beta). The same enzyme also fills the gap, and the DNA ends are finally rejoined by DNA ligase. We measured repair of oligonucleotide substrates containing a single AP site in cell extracts prepared from normal and Pol beta-null mouse cells and show that the reduced repair in Pol beta-null extracts can be complemented by addition of purified Pol beta. Using this complementation assay, we demonstrate that mutated Pol beta without dRPase activity is able to stimulate long patch BER. Mutant Pol beta deficient in DNA synthesis, but with normal dRPase activity, does not stimulate repair in Pol beta-null cells. However, under conditions where we measure base excision repair accomplished exclusively through a single-nucleotide patch BER, neither dRPase nor DNA synthesis mutants of Pol beta alone, or the two together, were able to complement the repair defect. These data suggest that the dRPase and DNA synthesis activities of Pol beta are coupled and that both of these Pol beta functions are essential during short patch BER and cannot be efficiently substituted by other cellular enzymes.

Reduced ligation during DNA base excision repair supported by BRCA2 mutant cells.

The breast cancer predisposing genes BRCA1 and BRCA2 appear to be involved in DNA repair. In particular, the sensitivity of BRCA2-deficient mouse embryonic fibroblasts to ionizing radiation and the demonstrated interaction of the BRCA2 protein with Rad51, a major factor in recombinational repair, indicate that BRCA2 is important for double strand break repair. The human BRCA2-deficient human cell line Capan-1, whilst being sensitive to ionizing radiation, is also sensitive to the alkylating agent methymethanesulfonate. The major lesions induced by this agent are methylated bases which are removed primarily by the base excision repair (BER) pathway. We have investigated the efficiency of BER in Capan-1 cells by an in vitro assay in which plasmid substrates containing a single lesion are repaired by mammalian cell extracts. In comparison to the control cell lines BxPC-3, T24 and MCF7, Capan-1 cells exhibited a reduced rate of DNA ligation during both the single-nucleotide insertion and PCNA-dependent pathways of BER. The reduced rate of DNA ligation exhibited by Capan-1 cell extracts was complemented by addition of bacteriophage T4 DNA ligase or human DNA ligase III. BRCA2-mutant Capan-1 cells may possess reduced DNA ligase activity during BER.

The Oxidative DNA Lesion 8,5′-(S)-Cyclo-2′-deoxyadenosine Is Repaired by the Nucleotide Excision Repair Pathway and Blocks Gene Expression in Mammalian Cells

Xeroderma pigmentosum (XP) patients with inherited defects in nucleotide excision repair (NER) are unable to excise from their DNA bulky photoproducts induced by UV radiation and therefore develop accelerated actinic damage, including cancer, on sun-exposed tissue. Some XP patients also develop a characteristic neurodegeneration believed to result from their inability to repair neuronal DNA damaged by endogenous metabolites since the harmful UV radiation in sunlight does not reach neurons. Free radicals, which are abundant in neurons, induce DNA lesions that, if unrepaired, might cause the XP neurodegeneration. Searching for such a lesion, we developed a synthesis for 8,5'-(S)-cyclo-2'-deoxyadenosine (cyclo-dA), a free radical-induced bulky lesion, and incorporated it into DNA to test its repair in mammalian cell extracts and living cells. Using extracts of normal and mutant Chinese hamster ovary (CHO) cells to test for NER and adult rat brain extracts to test for base excision repair, we found that cyclo-dA is repaired by NER and not by base excision repair. We measured host cell reactivation, which reflects a cell's capacity for NER, by transfecting CHO and XP cells with DNA constructs containing a single cyclo-dA or a cyclobutane thymine dimer at a specific site on the transcribed strand of a luciferase reporter gene. We found that, like the cyclobutane thymine dimer, cyclo-dA is a strong block to gene expression in CHO and human cells. Cyclo-dA was repaired extremely poorly in NER-deficient CHO cells and in cells from patients in XP complementation group A with neurodegeneration. Based on these findings, we propose that cyclo-dA is a candidate for an endogenous DNA lesion that might contribute to neurodegeneration in XP.

Comparative repair of the endogenous lesions 8-oxo-7,8-dihydroguanine (8-oxoG), uracil and abasic site by mammalian cell extracts: 8-oxoG is poorly repaired by human cell extracts

The repair of the endogenous lesions 8-oxo-7,8-dihydroguanine (8-oxoG), uracil (U) and natural abasic site (AP site) was investigated using an in vitro base excision repair assay in which a plasmid substrate containing a single lesion at a defined position was repaired by mammalian cell extracts. Repair replication of an 8-oxoG/cytosine base pair performed by normal human cell extracts was approximately 5-fold less efficient than repair of a U/adenine base pair and, in turn, the latter was repaired approximately 10-fold less efficiently than an AP site placed in front of an adenine. A similar pattern of repair capacity for the three lesions was observed in Chinese hamster extracts. Repair of 8-oxoG was performed by the one nucleotide insertion pathway only. The lower repair replication ability of 8-oxoG with respect to U was linked to a lower DNA glycosylase (base removal) activity rather than to inability to process the beta-elimination cleaved strand left by the AP lyase activity associated with human oxoguanine DNA glycosylase 1. The data show that DNA repair of 8-oxoG is poor in human cells in comparison with other frequent endogenous lesions.

Comparative repair of the endogenous lesions 8-oxo-7,8-dihydroguanine (8-oxoG), uracil and abasic site by mammalian cell extracts: 8-oxoG is poorly repaired by human cell extracts

The repair of the endogenous lesions 8-oxo-7,8-dihydroguanine (8-oxoG), uracil (U) and natural abasic site (AP site) was investigated using an in vitro base excision repair assay in which a plasmid substrate containing a single lesion at a defined position was repaired by mammalian cell extracts. Repair replication of an 8-oxoG/cytosine base pair performed by normal human cell extracts was ~5-fold less efficient than repair of a U/adenine base pair and, in turn, the latter was repaired ~10-fold less efficiently than an AP site placed in front of an adenine. A similar pattern of repair capacity for the three lesions was observed in Chinese hamster extracts. Repair of 8-oxoG was performed by the one nucleotide insertion pathway only. The lower repair replication ability of 8-oxoG with respect to U was linked to a lower DNA glycosylase (base removal) activity rather than to inability to process the β-elimination cleaved strand left by the AP lyase activity associated with human oxoguanine DNA glycosylase 1. The data show that DNA repair of 8-oxoG is poor in human cells in comparison with other frequent endogenous lesions.

Chromatin-bound Cdc6 persists in S and G2 phases in human cells, while soluble Cdc6 is destroyed in a cyclin A-cdk2 dependent process.

Cdc6 is essential for the initiation of DNA replication in all organisms in which it has been studied. In addition, recombinant Cdc6 can stimulate initiation in G(1) nuclei in vitro. We have analysed the behaviour of recombinant Cdc6 in mammalian cell extracts under in vitro replication conditions. We find that Cdc6 is imported into the nucleus in G(1 )phase, where it binds to chromatin and remains relatively stable. In S phase, exogenous Cdc6 is destroyed in a process that requires import into the nucleus and phosphorylation by a chromatin-bound protein kinase. Recombinant cyclin A-cdk2 can completely substitute for the nucleus in promoting destruction of soluble Xenopus and human Cdc6. Despite this regulated destruction, endogenous Cdc6 persists in the nucleus after initiation, although the amount falls. Cdc6 levels remain constant in G(2) then fall again before mitosis. We propose that cyclin A-cdk2 phosphorylation results in destruction of any Cdc6 not assembled into replication complexes, but that assembled proteins remain, in the phosphorylated state, in the nucleus. This process could contribute to the prevention of reinitiation in human cells by making free Cdc6 unavailable for re-assembly into replication complexes after G(1) phase.

DNA interstrand cross-links induce futile repair synthesis in mammalian cell extracts.

DNA interstrand cross-links are induced by many carcinogens and anticancer drugs. It was previously shown that mammalian DNA excision repair nuclease makes dual incisions 5' to the cross-linked base of a psoralen cross-link, generating a gap of 22 to 28 nucleotides adjacent to the cross-link. We wished to find the fates of the gap and the cross-link in this complex structure under conditions conducive to repair synthesis, using cell extracts from wild-type and cross-linker-sensitive mutant cell lines. We found that the extracts from both types of strains filled in the gap but were severely defective in ligating the resulting nick and incapable of removing the cross-link. The net result was a futile damage-induced DNA synthesis which converted a gap into a nick without removing the damage. In addition, in this study, we showed that the structure-specific endonuclease, the XPF-ERCC1 heterodimer, acted as a 3'-to-5' exonuclease on cross-linked DNA in the presence of RPA. Collectively, these observations shed some light on the cellular processing of DNA cross-links and reveal that cross-links induce a futile DNA synthesis cycle that may constitute a signal for specific cellular responses to cross-linked DNA.