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M Botchan

Publications and source records attributed to M Botchan.

At least 19 recordsLinked to original sources
PubMed2001

Functional analysis of mutant and wild-type Drosophila origin recognition complex.

The origin recognition complex (ORC) is the DNA replication initiator protein in eukaryotes. We have reconstituted a functional recombinant Drosophila ORC and compared activities of the wild-type and several mutant ORC variants. Drosophila ORC is an ATPase, and our studies show that the ORC1 subunit is essential for ATP hydrolysis and for ATP-dependent DNA binding. Moreover, DNA binding by ORC reduces its ATP hydrolysis activity. In vitro, ORC binds to chromatin in an ATP-dependent manner, and this process depends on the functional AAA(+) nucleotide-binding domain of ORC1. Mutations in the ATP-binding domain of ORC1 are unable to support cell-free DNA replication. However, mutations in the putative ATP-binding domain of either the ORC4 or ORC5 subunits do not affect either of these functions. We also provide evidence that the Drosophila ORC6 subunit is directly required for all of these activities and that a large pool of ORC6 is present in the cytoplasm, cytologically proximal to the cell membrane. Studies reported here provide the first functional dissection of a metazoan initiator and highlight the basic conserved and divergent features among Drosophila and budding yeast ORC complexes.

Adenosine Triphosphatases↗
PubMed1999

Assembly of functionally active Drosophila origin recognition complex from recombinant proteins.

In eukaryotes the sites for the initiation of chromosomal DNA replication are believed to be determined in part by the binding of a heteromeric origin recognition complex (ORC) to DNA. We have cloned the genes encoding the subunits of the Drosophila ORC. Each of the genes is unique and can be mapped to discrete chromosomal locations implying that the pattern and developmental regulation of origin usage in Drosophila is not regulated solely by a large family of different ORC proteins. The six-subunit ORC can be reconstituted with recombinant proteins into a complex that restores DNA replication in ORC-depleted Drosophila or Xenopus egg extracts.

Amino Acid Sequence↗
PubMed1997

Interaction between replication protein A and p53 is disrupted after UV damage in a DNA repair-dependent manner.

Replication protein A (RPA) is required for both DNA replication and nucleotide excision repair. Previous studies have shown that RPA interacts with the tumor suppressor p53. Herein, we have mapped a 20-amino acid region in the N-terminal part of p53 that is essential for its binding to RPA. This region is distinct from the minimal activation domain of p53 previously identified. We also demonstrate that UV radiation of cells greatly reduces the ability of RPA to bind to p53. Interestingly, damage-induced hyperphosphorylated RPA does not associate with p53. Furthermore, down-regulation of the RPA/p53 interaction is dependent upon the capability of cells to perform global genome repair. On the basis of these data, we propose that RPA may participate in the coordination of DNA repair with the p53-dependent checkpoint control by sensing UV damage and releasing p53 to activate its downstream targets.

Animals↗
PubMed1994

The bovine papilloma virus E1 protein has ATPase activity essential to viral DNA replication and efficient transformation in cells.

The bovine papilloma virus (BPV) E1 protein essential to viral DNA replication has recently been shown to associate via direct protein-DNA interactions with the viral origin of replication and to be an ATP-dependent helicase. We show here that in accordance with the latter function, the E1 gene product has intrinsic ATPase activity. Mutations placed throughout the nucleotide binding consensus element abolish the ATPase activity of E1 and render BPV genomes harboring such mutations defective for episomal replication and impaired for oncogenic transformation.

Adenosine Triphosphatases↗
PubMed1993

The E1 protein of bovine papilloma virus 1 is an ATP-dependent DNA helicase.

For efficient DNA replication of papillomaviruses, only two viral-encoded proteins, E1 and E2, are required. Other proteins and factors are provided by the host cell. E2 is an enhancer of both transcription and replication and is known to help E1 bind cooperatively to the origin of DNA replication. E1 is sufficient for replication in extracts prepared from permissive cells, but the activity is enhanced by E2. Here we show that purified E1 can act as an ATP-dependent DNA helicase. To measure this activity, we have used strand displacement, unwinding of topologically constrained DNA, denaturation of duplex fragments, and electron microscopy. The ability of E1 to unwind circular DNA is found to be independent of origin-specific viral DNA sequences under a variety of experimental conditions. In unfractionated cellular extracts, E1-dependent viral DNA replication is origin-dependent, but at elevated E1 concentrations, replication can occur on non-origin-containing DNA templates. This conversion from an origin-dependent replication system to a nonspecific initiator system is discussed in the context of the current understanding of the initiation of chromosomal DNA replication.

Adenosine Triphosphate↗
PubMed1992

A deletion in the simian virus 40 large T antigen impairs lytic replication in monkey cells in vivo but enhances DNA replication in vitro: new complementation function of T antigen.

We describe a new complementation function within the simian virus 40 (SV40) A gene. This function is required for viral DNA replication and virus production in vivo but, surprisingly, does not affect any of the intrinsic enzymatic functions of T antigen directly required for in vitro DNA replication. Other well-characterized SV40 T-antigen mutants, whether expressed stably from integrated genomes or in cotransfection experiments, complement these mutants for in vivo DNA replication and plaque formation. These new SV40 mutants were isolated and cloned from human cells which stably carry the viral DNA. The alteration in the large-T-antigen gene was shown by marker rescue and nucleotide sequence analysis to be a deletion of 322 bp spanning the splice-donor site of the first exon, creating a 14-amino-acid deletion in the large T antigen. The mutant gene was expressed in H293 human cells from an adenovirus vector, and the protein was purified by immunoaffinity chromatography. The mutant protein directs greater levels of DNA replication in vitro than does the wild-type protein. Moreover, the mutant protein reduces the lag time for in vitro DNA synthesis and can be diluted to lower levels than wild-type T antigen and still promote good replication, which is in clear contrast to the in vivo situation. These biochemical features of the protein are independent of the source of the cellular replication factors (i.e., HeLa, H293, COS 7, or CV1 cells) and the cells from which the T antigens were purified. The mutant T antigen does not transform Rat-2 cells. Several different models which might reconcile the differences observed in vivo and in vitro are outlined. We propose that the function of T antigen affected prepares cells for SV40 replication by activation of a limiting cellular replication factor. Furthermore, a link between the induction of a cellular replication factor and transformation by SV40 is discussed.

Amino Acid Sequence↗
PubMed1991

The activation domain of the bovine papillomavirus E2 protein mediates association of DNA-bound dimers to form DNA loops.

The E2 transactivator protein of bovine papillomavirus binds its specific DNA target sequence as a dimer. We have found that E2 dimers, preformed in solution independent of DNA, exhibit substantial cooperativity of DNA binding as detected by both nitrocellulose filter retention and footprint analysis techniques. If the binding sites are widely spaced, E2 forms stable DNA loops visible by electron microscopy. When three widely separated binding sites reside on the DNA, E2 condenses the molecule into a bow-tie structure. This implies that each E2 dimer has at least two independent surfaces for multimerization. Two naturally occurring shorter forms of the protein, E2C and E8/E2, which function in vivo as repressors of transcription, do not form such loops. Thus, the looping function of E2 maps to the 161-amino acid activation domain. These results support the looping model of transcription activation by enhancers.

Binding Sites↗
PubMed1990

Identification of a 68-kilodalton nuclear ATP-binding phosphoprotein encoded by bovine papillomavirus type 1.

E1 is the largest open reading frame (ORF) of bovine papillomavirus type 1 (BPV-1) and is highly conserved among all papillomaviruses, maintaining its size, amino acid composition, and location in the viral genome with respect to other early genes. Multiple viral replication functions have been mapped to the E1 ORF of BPV-1, and evidence suggested that more than one protein was encoded by this ORF. We previously identified a small protein (M) whose gene consists of two exons, one encoded by the 5' end of the E1 ORF. We show here that a 68-kilodalton (kDa) phosphoprotein made from the E1 ORF can be detected in BPV-1-transformed cells, and we present evidence that this protein is encoded by sequences colinear with the entire E1 ORF. The full-length E1 protein immunoprecipitated from virally transformed cells and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis comigrates with a protein expressed from a recombinant DNA construct capable of producing only the complete E1 protein. In addition, two different antisera directed against polypeptides encoded from either the 3' or the 5' end of the E1 ORF both recognize the full-length E1 product. A mutation converting the first methionine codon in the ORF to an isoleucine codon abolishes BPV-1 plasmid replication and E1 protein production. Consistent with the notion that this methionine codon is the start site for E1, a mutant with a termination codon placed after the splice donor at nucleotide 1235 in E1 produces a truncated protein with the molecular mass predicted from the primary sequence as well as the previously identified M protein. When visualized by immunostaining, the E1 protein expressed in COS cells is localized to the cell nucleus. A high degree of similarity exists between the BPV-1 E1 protein and polyomavirus and simian virus 40 large-T antigens in regions of the T antigens that bind ATP. We show by ATP affinity labeling that the E1 protein produced in COS cells binds ATP and that this activity is abolished by a point mutation which converts the codon for proline 434 to serine. Furthermore, this mutation renders the viral genome defective for DNA replication, suggesting that the ATP-binding activity of E1 is necessary for its putative role in viral DNA replication.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1990

Replication of bovine papillomavirus type 1 DNA initiates within an E2-responsive enhancer element.

When bovine papillomavirus transforms cells in vitro, it maintains its genome as a multicopy nuclear plasmid. Plasmid DNA extracted from such transformed cells was analyzed by the two-dimensional gel electrophoresis technique of Brewer and Fangman (B. Brewer and W. Fangman, Cell 51:463-471, 1987). The replication intermediates detected in these assays were found to be the sums of the oligomeric and monomeric forms of the replicating plasmids. The multimeric DNAs were shown by field inversion gel electrophoresis and partial restriction digestion to be head-to-tail concatemers of the monomeric forms. Furthermore, the multimers progressed in size by steps of one monomer, indicating that they did not arise by replication segregation mistakes of the unit length, which would predict a ladder spaced by integrals of two monomers. To map the plasmid DNA replication origin, the replication intermediates of the monomers were isolated by successive sucrose gradient centrifugation and then examined by the two-dimensional gel electrophoresis method. The patterns detected show that bovine papillomavirus type 1 replicates in these cells bidirectionally and that one replication origin site in the viral genome is utilized. By employing several restriction enzymes and specific viral DNA probes to dissect the replication intermediates, we were able to map the origin of initiation site with some precision. The initiation site, which maps to bovine papillomavirus type 1 DNA position 7730 +/- 100 bp, places the origin within that region of the viral upstream regulatory region which contains the major cluster of transcription factor E2-binding sites, E2RE1. Thus, the actual viral plasmid origin of replication maps near, but outside, genetic elements previously shown to be important for plasmid maintenance.

Animals↗
PubMed1989

Specific recognition nucleotides and their DNA context determine the affinity of E2 protein for 17 binding sites in the BPV-1 genome.

The DNA context of nucleotides that a protein recognizes can influence the strength of the protein-DNA interaction. Moreover, in prokaryotes, understanding the quantitative differences in binding affinities that result in part from the DNA context is often important in describing regulatory mechanisms. Nevertheless, these issues have not been a major focus yet for the investigation of protein-DNA interactions in eukaryotes. In this study, we explored the binding specificity and the range of affinities that the BPV-1 E2 transcriptional activator has for DNA. Because E2 binding sites are positioned near several different BPV-1 promoters, such quantitative information may be important to understand transcriptional regulatory mechanisms in BPV-1. Gel retardation assays and DNA footprinting were used to quantitate the affinities of the E2 binding sites in the viral genome. In the process, five sites were discovered, which, on the basis of sequence, had not been predicted previously to interact with the E2 protein. Equilibrium and kinetic studies show that the range of E2 affinities of the 17 sites varied over 300-fold. The sequence elements responsible for E2 recognition of DNA were determined by missing contact analysis of several sites and a point mutation analysis of one site. The results presented show that the affinity of an E2 binding site is to a large extent determined by the availability of specific contacts, but the data also strongly suggest that DNA structure plays an important role.

Animals↗
PubMed1989

Bovine papillomavirus type 1 encodes two forms of a transcriptional repressor: structural and functional analysis of new viral cDNAs.

Genetic and biochemical evidence has established that the E2 open reading frame (ORF) of bovine papillomavirus type 1 encodes at least two different site-specific DNA-binding proteins, one which activates and the other which represses expression from a viral promoter (P. F. Lambert, B. A. Spalholz, and P. M. Howley, Cell 50:69-78, 1987). We have obtained data which show that a second form of the repressor gene is expressed in transformed cells harboring stable viral plasmids. The structural details of this gene have been discerned by cDNA cloning, by RNase protection, and by primer extension analysis of in vivo RNA. Moreover, data from in vitro transcription experiments support the notion that this form of the E2 repressor is expressed from a novel viral promoter and that a small exon from another ORF is linked to an active repressor domain in E2. Thus, two different forms of the repressor are expressed from different promoters and might be independently regulated either in the cell cycle or in different tissue types. We show by functional in vivo assays utilizing a cDNA vector encoding this gene that the trans-acting factor has in vivo activities similar to those of the known repressor. Our screen of a cDNA library for cDNA clones representing bovine papillomavirus transcripts has also revealed a number of other novel structures defining new donor and acceptor RNA-processing sites. Notably, clones which conceptually can be translated to yield an E7 protein, the viral M gene, and the entire E2 ORF have been characterized. Finally, truncated versions of putative E8 cDNAs were also obtained.

Bovine papillomavirus 1↗
PubMed1988

The product of the bovine papillomavirus type 1 modulator gene (M) is a phosphoprotein.

The M gene of bovine papillomavirus type 1 has been genetically defined as encoding a trans-acting product which negatively regulates bovine papillomavirus type 1 replication and is important for establishment of stable plasmids in transformed cells. The gene for this regulatory protein has been mapped in part to the 5' portion of the largest open reading frame (E1) in the virus. We constructed a trpE-E1 fusion gene and expressed this gene in Escherichia coli. Rabbits were immunized with purified fusion protein, and antisera directed against the product were used to identify the M gene product in virus-transformed cells. In this way a polypeptide with an apparent molecular mass of 23 kilodaltons was detected. The virus-encoded product is phosphorylated and can be readily detected by immunoprecipitation assays from cells transformed by the virus. Cells that harbor viral DNA without M as integrated copies do not produce this protein, whereas cells that harbor integrated viral genomes which are defective for another E1 viral gene important for plasmid replication, R, do produce this protein. The protein has an anomalously low electrophoretic mobility. An in vitro translation product of an SP6 RNA product of a sequenced cDNA predicts a molecular mass of 16 kilodaltons for the protein, and this in vitro translation product has an electrophoretic mobility identical to that of the in vivo immunoprecipitated protein. The results of these studies confirm our previous genetic studies which indicated that part of the E1 open reading frame defined a discrete gene product distinct from other putative products which may be encoded by this open reading frame.

Bovine papillomavirus 1↗
PubMed1986

The bovine papillomavirus replicon.

The bovine papillomavirus genome contains two cis-acting sequences which can serve as signals for replication. At least three virally encoded genes seem to be involved in plasmid replication: E6, E6/7 and E1. Mutations in either the E6 or the E7 open reading frame create plasmids that are maintained at a low copy number per cell. Mutations in the E1 open reading frame are absolutely lethal to replication. Complementation experiments show that these mutations define separate genes. Experiments are described which show that cells harbouring plasmids with mutations in either the E6 or the E7 open reading frame acquire an immunity to high copy-number plasmids. We suggest that either the cell or the virus encodes a repressor. The positive action of E6 and E6/7 modulates the activity of this repressor to allow for the high copy-number state. Though the viral oncogenes are capable of transforming cells separately when they are expressed as part of certain recombinant DNA expression systems, it is clear that, in the context of the entire viral replicon, interactions between the transforming functions and replication functions must exist.

Animals↗
PubMed1986

Effects of poly[d(pGpT).d(pApC)] and poly[d(pCpG).d(pCpG)] repeats on homologous recombination in somatic cells.

Sequencing studies have shown that in somatic cells alternating runs of purines and pyrimidines are frequently associated with recombination crossover points. To test whether such sequences actually promote recombination, we have examined the effects of poly[d(pGpT).d(pApC)] and poly[d(pCpG).d(pCpG)] repeats on a homologous recombination event. The parental molecule used in this study, pSVLD, is capable of generating wild-type simian virus 40 DNA via recombination across two 751-base-pair regions of homology and has been described previously (Miller et al., Proc. Natl. Acad. Sci. USA 81:7534-7538, 1984). Single inserts of either a poly[d(pGpT).d(pApC)] repeat or a poly[d(pCpG).d(pCpG)] repeat were positioned adjacent to one region of homology in such a way that the recombination product, wild-type simian virus 40 DNA, could be formed only by recombination within the homologies and not by recombination across the alternating purine-pyrimidine repeats. We have found that upon transfection of test DNAs into simian cells, a poly[d(pCpG).d(pCpG)] repeat enhanced homologous recombination 10- to 15-fold, whereas a poly[d(pGpT).d(pApC)] repeat had less effect. These results are discussed in terms of the features of these repeats that might be responsible for promoting homologous recombination.

Animals↗
PubMed1986

Complementation of a bovine papilloma virus low-copy-number mutant: evidence for a temporal requirement of the complementing gene.

We identified a bovine papilloma virus function encoded by the E6/E7 gene, which is required for both BPV high-copy-number replication and maintenance of transformation of cultured cells. A cDNA copy of this gene was isolated and expressed from a retrovirus vector. We found that complete complementation of a BPV low-copy-number mutant (dl576) by the cDNA encoding the E6/E7 gene was temporally dependent. When both the E6/E7 cDNA and dl576 were introduced together into cells, wild-type replication and stable transformation resulted. In contrast, introduction of the complementing cDNA into cells already carrying dl576 led to only partial amplification of the resident mutant DNA accompanied by a restoration of the transformed phenotype. These results, along with other findings, suggest that the establishment of BPV plasmid replication occurs in two stages: an initial amplification of the incoming DNA followed by stable homeostatic replication which maintains the existing copy number.

Animals↗