The mouse DNA polymerase alpha-primase subunit p48 mediates species-specific replication of polyomavirus DNA in vitro.

Mouse cell extracts support vigorous replication of polyomavirus (Py) DNA in vitro, while human cell extracts do not. However, the addition of purified mouse DNA polymerase alpha-primase to human cell extracts renders them permissive for Py DNA replication, suggesting that mouse polymerase alpha-primase determines the species specificity of Py DNA replication. We set out to identify the subunit of mouse polymerase alpha-primase that mediates this species specificity. To this end, we cloned and expressed cDNAs encoding all four subunits of mouse and human polymerase alpha-primase. Purified recombinant mouse polymerase alpha-primase and a hybrid DNA polymerase alpha-primase complex composed of human subunits p180 and p68 and mouse subunits p58 and p48 supported Py DNA replication in human cell extracts depleted of polymerase alpha-primase, suggesting that the primase heterodimer or one of its subunits controls host specificity. To determine whether both mouse primase subunits were required, recombinant hybrid polymerase alpha-primases containing only one mouse primase subunit, p48 or p58, together with three human subunits, were assayed for Py replication activity. Only the hybrid containing mouse p48 efficiently replicated Py DNA in depleted human cell extracts. Moreover, in a purified initiation assay containing Py T antigen, replication protein A (RP-A) and topoisomerase I, only the hybrid polymerase alpha-primase containing the mouse p48 subunit initiated primer synthesis on Py origin DNA. Together, these results indicate that the p48 subunit is primarily responsible for the species specificity of Py DNA replication in vitro. Specific physical association of Py T antigen with purified recombinant DNA polymerase alpha-primase, mouse DNA primase heterodimer, and mouse p48 suggested that direct interactions between Py T antigen and primase could play a role in species-specific initiation of Py replication.

A single essential gene, PRI2, encodes the large subunit of DNA primase in Saccharomyces cerevisiae.

DNA primase activity of the yeast DNA polymerase-primase complex is related to two polypeptides, p58 and p48. The reciprocal role of these protein species has not yet been clarified, although both participate in formation of the active center of the enzyme. The gene encoding the p58 subunit has been cloned by screening of a lambda gt11 yeast genomic DNA library, using specific anti-p58 antiserum. Antibodies that inhibited DNA primase activity could be purified by lysates of Escherichia coli cells infected with a recombinant bacteriophage containing the entire gene, which we designate PR12. The gene was found to be transcribed in a 1.7-kilobase mRNA whose level appeared to fluctuate during the mitotic cell cycle. Nucleotide sequence determination indicated that PR12 encodes a 528-amino-acid polypeptide with a calculated molecular weight of 62,262. The gene is unique in the haploid yeast genome, and its product is essential for cell viability, as has been shown for other components of the yeast DNA polymerase-primase complex.

The B subunit of the DNA polymerase alpha-primase complex in Saccharomyces cerevisiae executes an essential function at the initial stage of DNA replication.

The four-subunit DNA polymerase alpha-primase complex is unique in its ability to synthesize DNA chains de novo, and some in vitro data suggest its involvement in initiation and elongation of chromosomal DNA replication, although direct in vivo evidence for a role in the initiation reaction is still lacking. The function of the B subunit of the complex is unknown, but the Saccharomyces cerevisiae POL12 gene, which encodes this protein, is essential for cell viability. We have produced different pol12 alleles by in vitro mutagenesis of the cloned gene. The in vivo analysis of our 18 pol12 alleles indicates that the conserved carboxy-terminal two-thirds of the protein contains regions that are essential for cell viability, while the more divergent NH2-terminal portion is partially dispensable. The characterization of the temperature-sensitive pol12-T9 mutant allele demonstrates that the B subunit is required for in vivo DNA synthesis and correct progression through S phase. Moreover, reciprocal shift experiments indicate that the POL12 gene product plays an essential role at the early stage of chromosomal DNA replication, before the hydroxyurea-sensitive step. A model for the role of the B subunit in initiation of DNA replication at an origin is presented.

Human DBR1 modulates the recycling of snRNPs to affect alternative RNA splicing and contributes to the suppression of cancer development.

The contribution of RNA processing to tumorigenesis is understudied. Here, we report that the human RNA debranching enzyme (hDBR1), when inappropriately regulated, induces oncogenesis by causing RNA processing defects, for example, splicing defects. We found that wild-type p53 and hypoxia-inducible factor 1 co-regulate hDBR1 expression, and insufficient hDBR1 leads to a higher rate of exon skipping. Transcriptomic sequencing confirmed the effect of hDBR1 on RNA splicing, and metabolite profiling supported the observation that neoplasm is triggered by a decrease in hDBR1 expression both in vitro and in vivo. Most importantly, when modulating the expression of hDBR1, which was found to be generally low in malignant human tissues, higher expression of hDBR1 only affected exon-skipping activity in malignant cells. Together, our findings demonstrate previously unrecognized regulation and functions of hDBR1, with immediate clinical implications regarding the regulation of hDBR1 as an effective strategy for combating human cancer.

Expression of the DEAD-box/RNA helicase rck/p54 in mouse tissues: implications for heterogeneous protein expression.

rck/p54, a DEAD-box RNA helicase, is closely associated with the basic modification of RNA molecules in the process of mRNA transport, RNA decay, and translation initiation. In the current study, Western blot analysis revealed that rck/p54 protein was ubiquitously expressed in mouse tissues. Interestingly, three different-sized rck/p54 proteins were detected by antibodies against mouse rck/p54, and these products were differentially expressed in the tissues. An immunohistochemical study revealed that rck/p54 was strongly expressed in basal cells of the crypt in the gastrointestinal tract and in neuronal bodies of the cerebral cortex, and was localized in epithelial cells of the convoluted tubules of the kidneys, suggesting that the heterogeneous rck/p54 may play pivotal roles in cells committed to become specialized in these tissues.

Inhibition of primer RNA formation in CCRF-CEM leukemia cells by fludarabine triphosphate.

The effects of fludarabine triphosphate (Fara-ATP), 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP), and aphidicolin on primer RNA and DNA synthesis in human CCRF-CEM leukemia cells were investigated. RNA-primed Okazaki fragment synthesis was monitored by first incubating whole cell lysates for 10 min in the presence or absence of the compound and then following the incorporation of [alpha-32P]ATP and [3H]dTTP into the primer RNA and DNA portions, respectively, of the Okazaki fragments. In whole cell lysates the degree of DNA synthesis inhibition induced by Fara-ATP was directly related to the extent of primer RNA synthesis inhibition over the entire range of Fara-ATP concentrations tested (10-50 microM). In contrast, primer RNA formation was stimulated by concentrations of ara-CTP (25-200 microM) and aphidicolin (0.5-5 micrograms/ml) that inhibited DNA synthesis. The primer RNA recovered from cell lysates incubated with either Fara-ATP, ara-CTP, or aphidicolin was of normal length, predominately 11 nucleotides. Fara-ATP was a more potent inhibitor of the polydeoxythymidylate primase activity than of the DNA polymerase alpha/delta activities present in the 100,000 x g supernatants of CCRF-CEM cells. Fara-ATP was a noncompetitive inhibitor of DNA primase with respect to ATP [50% inhibitory concentration, 2.3 +/- 0.3 (SD) microM, Ki = 6.1 +/- 0.3 (SE) microM] and the Km(ATP)/Ki (Fara-ATP) was 25. The 50% inhibitory concentration values of Fara-ATP for DNA polymerases alpha/delta activities on calf thymus DNA were 43 +/- 1.6 (SD) microM and greater than 100 microM with respect to dATP and dTTP. The effects of ara-CTP and aphidicolin on these enzymes were opposite those seen with Fara-ATP, since 50% inhibitory concentrations of either ara-CTP or aphidicolin for DNA polymerases alpha/delta did not inhibit polydeoxythymidylate primase activity. The results provide evidence that fludarabine phosphate blocks DNA synthesis in CCRF-CEM cells through inhibition of primer RNA formation. In contrast, the accumulation of primer RNA and RNA-primed Okazaki fragments that is induced by ara-CTP and aphidicolin could lead to the rereplication and amplification of chromosomal DNA segments.

Molecular cloning and functional analysis of a novel cadmium-responsive proto-oncogene.

The molecular mechanisms potentially responsible for cell transformation and tumorigenesis induced by cadmium, a human carcinogen, were investigated by differential gene expression analysis of BALB/c-3T3 cells transformed with cadmium chloride (CdCl(2)). Differential display analysis of gene expression revealed consistent overexpression of mouse translation initiation factor 3 (TIF3; GenBank accession number AF271072) in the cells transformed with CdCl(2) when compared with nontransformed cells. The predicted protein encoded by TIF3 cDNA exhibited 99% similarity to human eukaryotic initiation factor 3 p36 protein. A M(r) 36,000 protein was detected in cells transfected with an expression vector containing TIF3 cDNA. Transfection of NIH3T3 cells with an expression vector containing TIF3 cDNA resulted in overexpression of the encoded protein, and this was associated with cell transformation, as evidenced by the appearance of transformed foci exhibiting anchorage-independent growth on soft agar and tumorigenic potential in nude mice. Expression of the antisense RNA against TIF3 mRNA resulted in significant reversal of oncogenic potential of the CdCl(2)-transformed BALB/c-3T3 cells. Taken together, these findings demonstrate for the first time that the cell transformation and tumorigenesis induced by CdCl(2) are due, at least in part, to the overexpression of TIF3, a novel cadmium-responsive proto-oncogene.

Overproduction and functional analysis of DNA primase subunits from yeast and mouse.

Eukaryotic DNA primases are composed of two distinct subunits of 48-50 and 58-60 kDa. The amino acid sequences derived from the nucleotide sequences of the cloned genes are known only for the yeast and mouse polypeptides, and the extensive homology between the corresponding mouse and yeast subunits suggests conservation of functional domains. We were able to express in Saccharomyces cerevisiae the homologous and mouse primase-encoding genes under the control of both the constitutive ADH1 and the inducible GAL1 strong promoters, thus obtaining strains producing relevant amounts of the different polypeptides. In vivo complementation studies showed that neither one of the wild-type mouse primase-encoding genes was able to rescue the lethal or temperature-sensitive phenotype caused by mutations in the yeast PRI1 or PRI2 genes, indicating that these proteins, even if structurally and functionally very similar, might be involved in critical species-specific interactions during DNA replication.

An over-expression plasmid for Escherichia coli primase.

To facilitate the overexpression of Escherichia coli primase, the dnaG gene has been reconstructed using polymerase chain reaction to remove the 5' transcription terminator and the 3' RNA processing site. This construct was cloned into the T7 polymerase-transcribed expression vector, pET-3d. Cells containing the resulting plasmid (pGNG1) express up to 30% of the cellular protein as primase. The pGNG1-encoded primase has normal activity in synthesizing primer RNA on a single-stranded DNA template in vitro. Plasmid pGNG1 can also be used to synthesize [35S]methionine-labelled primase in in vitro transcription-translation systems. In addition, the small amount of transcription in the absence of T7 polymerase is sufficient to complement temperature-sensitive and amber dnaG chromosomal mutations in vivo. Plasmid pGNG1 can therefore be used not only to overproduce wild-type primase, but to change and manipulate the primase structure in vivo and in vitro. These mutant proteins can be overproduced and used for structural and functional studies.

Growth inhibition by overexpression of human DEAD box protein rck/p54 in cells of a guinea pig cell line.

We transfected cells of a guinea pig cell line with RCK cDNA inserted in a pIRES1neo expression vector. The overexpression of rck/p54 was confirmed by Western blot and RT-PCR analysis. In two clones expressing rck/p54, the cell growth was highly inhibited; and their anchorage-independent growth, which is an important character of malignant transformation, was not found. These findings are the first evidence that the overexpression of a DEAD box protein/RNA helicase could inhibit substantially cell growth at the translational level.

Identification of differentially transcribed RNA and DNA helicase-related genes of Plasmodium falciparum.

Chloroquine antimalarial action was assessed by the analysis of changes in gene expression. With this aim, Plasmodium falciparum cultures were submitted to chloroquine and to other stresses to determine which transcripts were specifically induced. P. falciparum in vitro control culture was compared to cultures where chloroquine was added and to cultures where serum was omitted, or where higher partial oxygen pressure was used, and, finally, at a temperature of 40 degrees C instead of 37 degrees C. Poly (A)+RNAs were reverse-transcribed and detected by the differential display technique. Two specific cDNAs were obtained and cloned, and a part of the genes was sequenced. The deduced protein, referred to as Pfhel-1, was related to a RNA helicase and was thought to be involved in protein translation control. The second deduced protein, called Pfhel-2, possessed consensus sequences of ATP-dependent helicase domains. Pfhel-2 may be involved either in mitotic control or in DNA repair. The possible roles of both helicase-related genes in chloroquine therapeutic activity are discussed.

High-level expression in male germ cells of murine P68 RNA helicase mRNA.

The complete cDNA coding for mouse P68 RNA helicase was cloned and its nucleotide sequence was determined. The sequence is about 95% identical to the human equivalent. Whereas the 5'-untranslated region is less conserved (71%), the 3'-ends of mouse and human mRNAs are nearly identical. Between stop codon and poly(A)-tail both sequences are 97% conserved. At the level of amino acid sequence, the similarity of both, mouse and human, DEAD box family proteins is as high as 98%. In situ hybridizations using cDNA subfragments as probes revealed a testis-selective expression of P68 RNA helicase mRNA. The signal was restricted to late pachytene spermatocytes and haploid spermatids. Northern blot analyses corroborated these results but suggested that expression of related mRNA species occurs in a variety of other tissues.