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Classical DNA and RNA polymerase (pol) enzymes have defined roles with their respective substrates, but several pols have been found to have multiple functions. We reported previously that purified human DNA pol η (hpol η) can incorporate both deoxyribonucleoside triphosphates (dNTPs) and ribonucleoside triphosphates (rNTPs) and can use both DNA and RNA as substrates. X-ray crystal structures revealed that two pol η residues, Phe-18 and Tyr-92, behave as steric gates to influence sugar selectivity. However, the physiological relevance of these phenomena has not been established. Here, we show that purified hpol η adds rNTPs to DNA primers at physiological rNTP concentrations and in the presence of competing dNTPs. When two rATPs were inserted opposite a cyclobutane pyrimidine dimer, the substrate was less efficiently cleaved by human RNase H2. Human XP-V fibroblast extracts, devoid of hpol η, could not add rNTPs to a DNA primer, but the expression of transfected hpol η in the cells restored this ability. XP-V cell extracts did not add dNTPs to DNA primers hybridized to RNA, but could when hpol η was expressed in the cells. HEK293T cell extracts could add dNTPs to DNA primers hybridized to RNA, but lost this ability if hpol η was deleted. Interestingly, a similar phenomenon was not observed when other translesion synthesis (TLS) DNA polymerases-hpol ι, κ, or ζ-were individually deleted. These results suggest that hpol η is one of the major reverse transcriptases involved in physiological processes in human cells.
© 2019 Su et al.
Yeast retrotransposons form intracellular particles within which replication occurs. Because fungal nuclear membranes do not break down during mitosis, similar to retroviruses infecting nondividing cells, the cDNA produced must be translocated through nuclear pore complexes. The Saccharomyces cerevisiae long terminal repeat retrotransposon Ty3 assembles its Gag3 and Gag3-Pol3 precursor polyproteins into viruslike particles in association with perinuclear P-body foci. These perinuclear clusters of Ty3 viruslike particles localized to sites of clustered nuclear pore complexes (NPCs) in a nup120Delta mutant, indicating that Ty3 particles and NPCs interact physically. The NPC channels are lined with nucleoporins (Nups) with extended FG (Phe-Gly) motif repeat domains, further classified as FG, FxFG, or GLFG repeat types. These domains mediate partitioning of proteins between the cytoplasm and the nucleus. Here we have systematically examined the requirements for FG repeat domains in Ty3 nuclear transport. The GLFG domains interacted in vitro with virus-like particle Gag3, and this interaction was disrupted by mutations in the amino-terminal domain of Gag3, which is predicted to lie on the external surface of the particles. Accordingly, Ty3 transposition was decreased in strains with the GLFG repeats deleted. The spacer-nucleocapsid domain of Gag3, which is predicted to be internal to the particle, interacted with GLFG repeats and nucleocapsid localized to the nucleus. We conclude that Ty3 particle docking on nuclear pores is facilitated by interactions between Gag3 and GLFG Nups and that nuclear entry of the preintegration complex is further promoted by nuclear localization signals within the nucleocapsid and integrase.
The telomerase reverse transcriptase component (TERT) is not expressed in most primary somatic human cells and tissues, but is upregulated in the majority of immortalized cell lines and tumors. Here, we identify the c-Myc transcription factor as a direct mediator of telomerase activation in primary human fibroblasts through its ability to specifically induce TERT gene expression. Through the use of a hormone inducible form of c-Myc (c-Myc-ER), we demonstrate that Myc-induced activation of the hTERT promoter requires an evolutionarily conserved E-box and that c-Myc-ER-induced accumulation of hTERT mRNA takes place in the absence of de novo protein synthesis. These findings demonstrate that the TERT gene is a direct transcriptional target of c-Myc. Since telomerase activation frequently correlates with immortalization and telomerase functions to stabilize telomers in cycling cells, we tested whether Myc-induced activation of TERT gene expression represents an important mechanism through which c-Myc acts to immortalize cells. Employing the rat embryo fibroblast cooperation assay, we show that TERT is unable to substitute for c-Myc in the transformation of primary rodent fibroblasts, suggesting that the transforming activities of Myc extend beyond its ability to activate TERT gene expression and hence telomerase activity.
To learn about the V lambda gene segments that are expressed in lambda 3 light chains, the most recently identified lambda-chain subtype in inbred mice, we determined partial amino acid sequences of the V regions of two of these chains, L5-8 and Lc49 . The partial sequences were extended by establishing the complete V region sequence of cDNA for the lambda-chain mRNA from the hybridoma (RZ 5-8) and the myeloma ( CBPC -49) that produce these chains. The primer extension method used to sequence the cDNA is described in detail, because the same primer (a synthetic heptadecadeoxynucleotide ) can be used for sequencing cDNA for lambda-chains of all three subtypes of inbred mice and probably for lambda-chains from some other vertebrate species as well. The results confirm earlier preliminary findings that for both chains the V region is encoded by the V lambda 1 and J lambda 3 gene segments. The unmutated germ-line sequences of these gene segments are present in both chains, but the two chains, nevertheless, differ at codon 97, the V lambda-J lambda boundary. A T/G difference in the third position of this codon resulted in a codon for histidine (CAT) in one chain (L5-8) and for glutamine (CAG) in the other chain ( Lc49 ). This difference can be accounted for by variation in the site of V lambda 1-J lambda 3 recombination. Though the V region amino acid sequences of L5-8 and Lc49 differ only by the His/Gln substitution at position 97, the two chains have been shown (manuscript in preparation) to differ in their ability to form an effective combining site for the 2,4-dinitrophenyl group.
Avian erythroblastosis virus (AEV) induces both erythroblastosis and fibrosarcomas in susceptible birds. Two domains within its replication-defective genome, erb-A and erb-B, have been implicated in AEV-mediated oncogenesis. An efficient transfection system for generating infectious, transforming virus from molecular clones of AEV and RAV-1 (helper virus) was combined with the techniques of site-specific mutagenesis to investigate the contribution of erb-B to the two forms of oncogenesis induced by AEV. Deletion and frameshift mutations were constructed in the erb-B locus of cloned AEV DNA in vitro. Infectious retroviruses harboring these mutations were recovered and their ability to transform fibroblasts in vitro or induce erythroleukemia in vivo was assessed. The presence of mutant viral genomes in chick embryo fibroblasts or erythroblasts of infected birds was confirmed by suitable biochemical analyses. Expression of viral genes in cells infected with AEV mutants was examined by immunoprecipitation with antisera to erb-A and erb-B proteins. It was found that the product of erb-B is necessary for transformation of fibroblasts and induction of erythroblastosis by AEV, although a small portion of this protein at the carboxy terminus is dispensable.