Initiation of baculovirus DNA replication: early promoter regions can function as infection-dependent replicating sequences in a plasmid-based replication assay.

1996 ◽  
Vol 70 (10) ◽  
pp. 6967-6972 ◽  
Author(s):  
Y Wu ◽  
E B Carstens
Keyword(s):  
Dna Replication ◽  
Promoter Regions ◽  
Replication Assay ◽  
Early Promoter

Efficient long DNA gap-filling in a mammalian cell-free system: A potential new in vitro DNA replication assay

Biochimie ◽  
2013 ◽  
Vol 95 (2) ◽  
pp. 320-328
Author(s):  
Seiki Nakao ◽  
Sufang Zhang ◽  
Markku Vaara ◽  
Juhani E. Syväoja ◽  
Marietta Y. Lee ◽  
...  
Keyword(s):  
Dna Replication ◽  
Mammalian Cell ◽  
Gap Filling ◽  
Free System ◽  
Cell Free System ◽  
Replication Assay ◽  
System A

scholarly journals Specification of Regions of DNA Replication Initiation during Embryogenesis in the 65-KilobaseDNApolα-dE2F Locus of Drosophila melanogaster

1999 ◽  
Vol 19 (1) ◽  
pp. 547-555 ◽  
Author(s):  
Takayo Sasaki ◽  
Tomoyuki Sawado ◽  
Masamitsu Yamaguchi ◽  
Tomoyuki Shinomiya
Keyword(s):  
Dna Replication ◽  
Cultured Cells ◽  
Early Stage ◽  
Replication Initiation ◽  
Replication Origins ◽  
Coding Region ◽  
Promoter Regions ◽  
Dna Replication Initiation ◽  
Differentiated Cells ◽  
Active Promoter

ABSTRACT In the early stage of Drosophila embryogenesis, DNA replication initiates at unspecified sites in the chromosome. In contrast, DNA replication initiates in specified regions in cultured cells. We investigated when and where the initiation regions are specified during embryogenesis and compared them with those observed in cultured cells by two-dimensional gel methods. In the DNA polymerase α gene (DNApolα) locus, where an initiation region,oriDα, had been identified in cultured Kc cells, repression of origin activity in the coding region was detected after formation of cellular blastoderms, and the range of the initiation region had become confined by 5 h after fertilization. During this work we identified other initiation regions between oriDα and the Drosophila E2F gene (dE2F) downstream of DNApolα. At least four initiation regions showing replication bubbles were identified in the 65-kbDNApolα-dE2F locus in 5-h embryos, but only two were observed in Kc cells. These results suggest that the specification levels of origin usage in 5-h embryos are in the intermediate state compared to those in more differentiated cells. Further, we found a spatial correlation between the active promoter regions fordE2F and the active initiation zones of replication. In 5-h embryos, two known transcripts differing in their first exons were expressed, and two regions close to the respective promoter regions for both transcripts functioned as replication origins. In Kc cells, only one transcript was expressed and functional replication origins were observed only in the region including the promoter region for this transcript.

scholarly journals Negative Regulation of DNA Replication by the Retinoblastoma Protein Is Mediated by Its Association with MCM7

1998 ◽  
Vol 18 (5) ◽  
pp. 2748-2757 ◽  
Author(s):  
Jacqueline M. Sterner ◽  
Susan Dew-Knight ◽  
Christine Musahl ◽  
Sally Kornbluth ◽  
Jonathan M. Horowitz
Keyword(s):  
Amino Acids ◽  
Dna Replication ◽  
Protein Complexes ◽  
Replication Assay ◽  
Amino Terminal ◽  
Licensing Factor ◽  
Human Proteins ◽  
Carboxy Terminal ◽  
Related Proteins

ABSTRACT A yeast two-hybrid screen was employed to identify human proteins that specifically bind the amino-terminal 400 amino acids of the retinoblastoma (Rb) protein. Two independent cDNAs resulting from this screen were found to encode the carboxy-terminal 137 amino acids of MCM7, a member of a family of proteins that comprise replication licensing factor. Full-length Rb and MCM7 form protein complexes in vitro, and the amino termini of two Rb-related proteins, p107 and p130, also bind MCM7. Protein complexes between Rb and MCM7 were also detected in anti-Rb immunoprecipitates prepared from human cells. The amino-termini of Rb and p130 strongly inhibited DNA replication in an MCM7-dependent fashion in a Xenopus in vitro DNA replication assay system. These data provide the first evidence that Rb and Rb-related proteins can directly regulate DNA replication and that components of licensing factor are targets of the products of tumor suppressor genes.

scholarly journals Identification and functional analysis of the origins of DNA replication in the Cydia pomonella granulovirus genome

2007 ◽  
Vol 88 (5) ◽  
pp. 1496-1504 ◽  
Author(s):  
Sally Hilton ◽  
Doreen Winstanley
Keyword(s):  
Dna Replication ◽  
Genomic Library ◽  
Cydia Pomonella ◽  
Single Copy ◽  
Homologous Region ◽  
Entire Genome ◽  
Replication Assay ◽  
Origins Of Replication ◽  
Pcr Method ◽  
Cydia Pomonella Granulovirus

The entire genome of Cydia pomonella granulovirus (CpGV) was systematically screened for origins of DNA replication, using an infection-dependent DNA replication assay in the granulovirus-permissive Cydia pomonella cell line, Cp14R. All seven cosmids in an overlapping library that covered the CpGV genome were found to replicate in the assay. A genomic library of 32 overlapping plasmids was subsequently screened. Plasmids that replicated were in turn subcloned into 1–2 kbp overlapping fragments. Eleven subclones replicated, each containing at least one of the 13 single-copy 74–76 bp imperfect palindromes, previously identified in the CpGV genome as possible origins of replication. Genome fragments of 156 bp, each containing one of the 13 palindromes, were cloned to verify replication and provided confirmation that these 13 palindromes are the only origins of replication in the genome. A real-time PCR method was developed for the quantification of DNA replication, which eliminated the need for Southern blotting and hybridization. A set of deletion clones allowed further quantitative characterization of one of the palindromes. The previously proposed non-homologous region origin of replication did not replicate in the assay.

scholarly journals Polarity of DNA replication through the avian alpha-globin locus.

1986 ◽  
Vol 6 (4) ◽  
pp. 976-984 ◽  
Author(s):  
C D James ◽  
M Leffak
Keyword(s):  
Dna Replication ◽  
Replication Origin ◽  
Transcriptional Activity ◽  
Blot Hybridization ◽  
Dna Polymerases ◽  
Replication Assay ◽  
Alpha Globin ◽  
Isopycnic Centrifugation

Toward understanding the controls affecting eucaryotic chromosome replication, we used a runoff replication assay to investigate whether the activity of a gene is related to its use of an upstream or downstream replication origin. When in vivo-initiated DNA polymerases are allowed to complete replication in vitro in the presence of bromodeoxyuridine triphosphate the density label is preferentially incorporated into origin-distal regions of DNA. Isopycnic centrifugation and blot hybridization analysis of the relative bromodeoxyuridine triphosphate incorporation into fragments spanning the chicken alpha-globin locus indicate that this region is replicated from an upstream origin both in chicken lymphocytes and in erythrocytes. Thus the replication polarity of these genes does not change as a function of transcriptional activity, consistent with earlier suggestions that DNA replication in the transcriptional direction may be a necessary but not sufficient condition for gene expression.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 K-bZIP Modulates Latency-Associated Nuclear Protein-Mediated Suppression of Lytic Origin-Dependent DNA Synthesis

2009 ◽  
Vol 83 (17) ◽  
pp. 8492-8501 ◽  
Author(s):  
Cyprian Rossetto ◽  
Irena Yamboliev ◽  
Gregory S. Pari
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Nuclear Protein ◽  
Human Herpesvirus ◽  
Lytic Replication ◽  
Human Herpesvirus 8 ◽  
Dependent Manner ◽  
Herpesvirus 8 ◽  
Replication Assay ◽  
Lytic Dna Replication

ABSTRACT The original cotransfection replication assay identified eight human herpesvirus 8 (HHV8)-encoded proteins required for origin-dependent lytic DNA replication. Previously, we demonstrated that under conditions where K-Rta is overexpressed, a K-bZIP knockout bacmid displayed an aberrant subcellular localization pattern for the latency-associated nuclear protein (LANA). Additionally, these same studies demonstrated that K-bZIP interacts with LANA in the absence of K-Rta and that K-bZIP does not directly participate in, but may facilitate, the initiation of lytic DNA synthesis. We developed a modification of the transient cotransfection replication assay wherein both lytic (oriLyt) and latent (terminal repeat) DNA replication are evaluated simultaneously. We now show that LANA represses origin-dependent lytic DNA replication in a dose dependent manner when added to the cotransfection replication assay. This repression was overcome by increasing amounts of a K-bZIP expression plasmid in the cotransfection mixture or by dominant-negative inhibition of the interaction of LANA with K-bZIP by the overexpression of the K-bZIP-LANA binding domain. Chromatin immunoprecipitation assays show that LANA interacts with oriLyt in the absence of K-bZIP expression, suggesting that suppression of lytic replication by LANA is mediated by direct binding. The interaction of K-bZIP with oriLyt was dependent upon the expression of LANA; however, LANA interacted with oriLyt independently of K-bZIP expression. These data suggest that the interaction of LANA with K-bZIP modulates lytic and latent replication and that K-bZIP facilitates lytic DNA replication and modulates the switch from the latent phase of the virus.

A rapid in vitro polyomavirus DNA replication assay

2004 ◽  
Vol 122 (1) ◽  
pp. 123-127 ◽  
Author(s):  
Katja Ziegler ◽  
Thomas Bui ◽  
Richard J. Frisque ◽  
Andrew Grandinetti ◽  
Vivek. R. Nerurkar
Keyword(s):  
Dna Replication ◽  
Replication Assay

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Contains Two Functional Lytic Origins of DNA Replication

2002 ◽  
Vol 76 (15) ◽  
pp. 7890-7896 ◽  
Author(s):  
David P. AuCoin ◽  
Kelly S. Colletti ◽  
Yiyang Xu ◽  
Sylvia A. Cei ◽  
Gregory S. Pari
Keyword(s):  
Dna Replication ◽  
Binding Sites ◽  
Human Herpesvirus ◽  
Human Herpesvirus 8 ◽  
Reading Frame ◽  
Herpesvirus 8 ◽  
Replication Assay ◽  
Inverted Duplication ◽  
Origin Of Dna Replication ◽  
The Right

ABSTRACT We used a transient-transfection replication assay to identify two functional copies of the human herpesvirus 8 (HHV8) lytic origin of DNA replication (oriLyt). BCLB-1 cells were transfected with HHV8 subgenomic fragments containing the putative lytic origin along with a plasmid expressing viral transactivator open reading frame (ORF) 50. The HHV8 left-end oriLyt (oriLyt-L) lies between ORFs K4.2 and K5 and is composed of a region encoding various transcription factor binding sites and an A+T-rich region and a G+C repeat region. The right-end oriLyt (oriLyt-R) maps between ORF 69 and vFLIP, a region similar to the RRV oriLyt, and is an inverted duplication of oriLyt-L.

scholarly journals Expression, purification and characterization of the Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) DNA polymerase and interaction with the SpliNPV non-hr origin of DNA replication

2001 ◽  
Vol 82 (7) ◽  
pp. 1767-1776 ◽  
Author(s):  
Jianhe Huang ◽  
David B. Levin
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Spodoptera Littoralis ◽  
Expression System ◽  
Functional Characterization ◽  
Eukaryotic Expression ◽  
Replication Assay ◽  
E Coli ◽  
Origin Of Dna Replication

The DNA polymerase from Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) was expressed in, and purified from, prokaryotic and eukaryotic expression systems. While less protein was obtained from the E. coli expression system, SpliNPV DNAPOL purified from E. coli displayed similar biochemical characteristics to DNAPOL expressed in, and subsequently purified from, insect cells (Sf9) using a baculovirus expression system. Biochemical analyses suggested that the DNA polymerase and the 3′–5′ exonuclease activities are intrinsic to the protein. Deletion of the first 80 amino acid residues from the N terminus of the DNAPOL affected neither the DNA polymerase nor the exonuclease activities of the enzyme. Replication products from single-stranded M13 DNA demonstrated that the DNA synthesis activity of SpliNPV DNAPOL is highly processive. Transient expression assays with a set of deletion clones containing the putative SpliNPV non-hr origin of DNA replication permitted functional characterization of sequence elements within the origin fragment. Purified SpliNPV DNAPOL stimulated origin-dependent DNA replication in a cell-free replication assay.

scholarly journals Simian Virus 40 DNA Replication Is Dependent on an Interaction between Topoisomerase I and the C-Terminal End of T Antigen

2007 ◽  
Vol 82 (3) ◽  
pp. 1136-1145 ◽  
Author(s):  
Sujata Khopde ◽  
Daniel T. Simmons
Keyword(s):  
Dna Replication ◽  
Topoisomerase I ◽  
Mutational Analysis ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Enzyme Linked Immunosorbent Assay ◽  
Helicase Domain ◽  
Replication Assay ◽  
Sv40 Dna

ABSTRACT Topoisomerase I (topo I) is needed for efficient initiation of simian virus 40 (SV40) DNA replication and for the formation of completed DNA molecules. Two distinct binding sites for topo I have been previously mapped to the N-terminal (residues 83 to 160) and C-terminal (residues 602 to 708) regions of T antigen. By mutational analysis, we identified a cluster of six residues on the surface of the helicase domain at the C-terminal binding site that are necessary for efficient binding to topo I in enzyme-linked immunosorbent assay and far-Western blot assays. Mutant T antigens with single substitutions of these residues were unable to participate normally in SV40 DNA replication. Some mutants were completely defective in supporting DNA replication, and replication was not enhanced in the presence of added topo I. The same mutants were the ones that were severely compromised in binding topo I. Other mutants demonstrated intermediate levels of activity in the DNA replication assay and were correspondingly only partially defective in binding topo I. Mutations of nearby residues outside this cluster had no effect on DNA replication or on the ability to bind topo I. These results strongly indicate that the association of topo I with these six residues in T antigen is essential for DNA replication. These residues are located on the back edges of the T-antigen double hexamer. We propose that topo I binds to one site on each hexamer to permit the initiation of SV40 DNA replication.

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