Biomedical subjects

Ellen Fanning

Publications and source records attributed to Ellen Fanning.

23 records · Page 2Linked to original sources

PubMed2002

A dominant-negative mutant of human DNA helicase B blocks the onset of chromosomal DNA replication.

A cDNA encoding a human ortholog of mouse DNA helicase B, which may play a role in DNA replication, has been cloned and expressed as a recombinant protein. The predicted human DNA helicase B (HDHB) protein contains conserved helicase motifs (superfamily 1) that are strikingly similar to those of bacterial recD and T4 dda proteins. The HDHB gene is expressed at low levels in liver, spleen, kidney, and brain and at higher levels in testis and thymus. Purified recombinant HDHB hydrolyzed ATP and dATP in the presence of single-stranded DNA, displayed robust 5'-3' DNA helicase activity, and interacted physically and functionally with DNA polymerase alpha-primase. HDHB proteins with mutations in the Walker A or B motif lacked ATPase and helicase activity but retained the ability to interact with DNA polymerase alpha-primase, suggesting that the mutants might be dominant over endogenous HDHB in human cells. When purified HDHB protein was microinjected into the nucleus of cells in early G(1), the mutant proteins inhibited DNA synthesis, whereas the wild type protein had no effect. Injection of wild type or mutant protein into cells at G(1)/S did not prevent DNA synthesis. The results suggest that HDHB function is required for S phase entry.

Adenosine Triphosphatases↗

PubMed2002

Hepatitis C virus NS3 and simian virus 40 T antigen helicases displace streptavidin from 5'-biotinylated oligonucleotides but not from 3'-biotinylated oligonucleotides: evidence for directional bias in translocation on single-stranded DNA.

Helicases are enzymes that use energy from nucleoside triphosphate hydrolysis to unwind double-stranded (ds) DNA, a process vital to virtually every phase of DNA metabolism. Helicases have been classified as either 5'-to-3' or 3'-to-5' on the basis of their ability to unwind duplex DNA adjacent to either a 5' or 3' single-stranded (ss) DNA overhang. However, there has been debate as to whether this substrate preference is indicative of unidirectional translocation on ssDNA. We developed an assay that monitors the ability of a helicase to displace streptavidin from biotinylated oligonucleotides [Morris, P. D., and Raney, K. D. (1999) Biochemistry 38, 5164-5171]. Two helicases identified as having 5'-to-3' polarity displaced streptavidin from the 3'-end of biotinylated oligonucleotides but not from the 5'-end. We performed similar experiments using the 3'-to-5' helicases from the hepatitis C virus (NS3) and SV40 virus (SV40 T antigen). NS3 and SV40 T antigen were able to displace streptavidin from a 5'-biotinylated oligonucleotide but not from a 3'-biotinylated oligonucleotide. NS3 and SV40 T antigen enhanced the spontaneous rate of dissociation of streptavidin from biotin 340-fold and 1700-fold, respectively. The ssDNA binding protein, gp32, did not enhance dissociation of streptavidin from either end of an oligonucleotide. For NS3, the rate of displacement was faster from a 5'-biotinylated 60mer than from a 5'-biotinylated 30mer. The strong directional bias in streptavidin displacement activity exhibited by each helicase is consistent with a directional bias in translocation on ssDNA. The dependence of the reaction with NS3 on the oligonucleotide length suggests that multiple NS3 monomers are necessary for optimal activity.

Antigens, Polyomavirus Transforming↗

PubMed2002

Mutational analysis of simian virus 40 T-antigen primosome activities in viral DNA replication.

The recruitment of DNA polymerase alpha-primase (pol-prim) is a crucial step in the establishment of a functional replication complex in eukaryotic cells, but the mechanism of pol-prim loading and the composition of the eukaryotic primosome are poorly understood. In the model system for simian virus 40 (SV40) DNA replication in vitro, synthesis of RNA primers at the origin of replication requires only the viral tumor (T) antigen, replication protein A (RPA), pol-prim, and topoisomerase I. On RPA-coated single-stranded DNA (ssDNA), T antigen alone mediates priming by pol-prim, constituting a relatively simple primosome. T-antigen activities proposed to participate in its primosome function include DNA helicase and protein-protein interactions with RPA and pol-prim. To test the role of these activities of T antigen in mediating priming by pol-prim, three replication-defective T antigens with mutations in the ATPase or helicase domain have been characterized. All three mutant proteins interacted physically and functionally with RPA and pol-prim and bound ssDNA, and two of them displayed some helicase activity. However, only one of these, 5030, mediated primer synthesis and elongation by pol-prim on RPA-coated ssDNA. The results suggest that a novel activity, present in 5030 T antigen and absent in the other two mutants, is required for T-antigen primosome function.

Adenosine Triphosphatases↗

PubMed2002

Role of the p68 subunit of human DNA polymerase alpha-primase in simian virus 40 DNA replication.

DNA polymerase alpha-primase (pol-prim) is a heterotetramer with DNA polymerase and primase activities. The polymerase (p180) and primase (p48 and p58) subunits synthesize primers and extend them, but the function of the remaining subunit (p68) is poorly understood. Genetic studies in yeast suggested an essential role for the p68 ortholog in early S phase prior to the hydroxyurea-sensitive step, possibly a regulatory role in initiation of DNA replication, but found no evidence for an essential function of p68 later in S phase. To investigate whether the human p68 subunit has an essential role in DNA replication, we examined the ability of a purified trimeric human pol-prim lacking p68 to initiate simian virus 40 DNA replication in vitro and to synthesize and elongate primers on single-stranded DNA in the presence of T antigen and replication protein A (RPA). Both activities of trimeric pol-prim were defective, but activity was recovered upon addition of separately purified p68. Phosphorylation of p68 by cyclin A-dependent protein kinase also inhibited both activities of pol-prim. The data strongly suggest that the p68 subunit is required for priming activity of pol-prim in the presence of RPA and T antigen, both during initiation at the origin and during lagging strand replication.

Animals↗

PubMed2001

Reconstitution of human DNA polymerase delta using recombinant baculoviruses: the p12 subunit potentiates DNA polymerizing activity of the four-subunit enzyme.

Eukaryotic DNA polymerase delta is thought to consist of three (budding yeast) or four subunits (fission yeast, mammals). Four human genes encoding polypeptides p125, p50, p66, and p12 have been assigned as subunits of DNA polymerase delta. However, rigorous purification of human or bovine DNA polymerase delta from natural sources has usually yielded two-subunit preparations containing only p125 and p50 polypeptides. To reconstitute an intact DNA polymerase delta, we have constructed recombinant baculoviruses encoding the p125, p50, p66, and p12 subunits. From insect cells infected with four baculoviruses, protein preparations containing the four polypeptides of expected sizes were isolated. The four-subunit DNA polymerase delta displayed a specific activity comparable with that of the human, bovine, and fission yeast proteins isolated from natural sources. Recombinant DNA polymerase delta efficiently replicated singly primed M13 DNA in the presence of replication protein A, proliferating cell nuclear antigen, and replication factor C and was active in the SV40 DNA replication system. A three-subunit subcomplex consisting of the p125, p50, and p66 subunits, but lacking the p12 subunit, was also isolated. The p125, p50, and p66 polypeptides formed a stable complex that displayed DNA polymerizing activity 15-fold lower than that of the four-subunit polymerase. p12, expressed and purified individually, stimulated the activity of the three-subunit complex 4-fold on poly(dA)-oligo(dT) template-primer but had no effect on the activity of the four-subunit enzyme. Therefore, the p12 subunit is required to reconstitute fully active recombinant human DNA polymerase delta.

Baculoviridae↗