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M R Botchan

Publications and source records attributed to M R Botchan.

At least 37 records · Page 2Linked to original sources
PubMed1994

The cellular DNA polymerase alpha-primase is required for papillomavirus DNA replication and associates with the viral E1 helicase.

Persistent infection by papillomaviruses involves the maintenance of viral DNA as a nuclear plasmid, the replication of which requires host DNA polymerases. The role of the cellular DNA polymerase alpha-primase holoenzyme was probed by using soluble extracts from rodent cells that replicate bovine papilloma virus 1 and human papilloma virus 6b DNA in the presence of the viral E1 helicase and the E2 transcription factor. Monoclonal antibodies directed against the catalytic 180-kDa subunit of polymerase alpha inhibit DNA synthesis in this system. Addition of purified human polymerase alpha-primase holoenzyme to neutralized extracts restores their DNA synthetic activity. The amino-terminal 424 amino acids of E1 forms a specific protein complex with the p180 polymerase subunit. Immune complexes can be isolated with antibodies directed against E1 that contain a DNA polymerase activity. Moreover, this polymerase activity can be neutralized by anti-polymerase alpha antibodies. Permissivity barriers were not encountered in this in vitro system, as bovine E1 can interface with the murine and human replication apparatus. Although the large tumor antigens encoded by simian virus 40 and polyoma share limited primary sequence homology with the papillomavirus E1 proteins, the organization of functional motifs at the level of primary protein structure is remarkably similar. In addition to their origin-specific DNA-binding activity, each of these helicases may function to help recruit the cellular polymerase alpha-primase complex to the viral replication origin.

Animals↗
PubMed1994

Acidic transcription factors alleviate nucleosome-mediated repression of DNA replication of bovine papillomavirus type 1.

The papillomavirus E2 transcription factor is directly involved in viral DNA replication. Previous studies have shown that E2 interacts with both the viral E1 helicase and cellular replication proteins, and thus it may facilitate their targeting to the origin of replication. We demonstrate here that E1-mediated replication of bovine papillomavirus type 1 is repressed by nucleosomal assembly. The E2 protein counteracts this repression, and such activation requires the E2-binding sites adjacent to the origin of replication. These in vitro results are consistent with the previous in vivo findings that both E2 and specific E2 binding to DNA are absolutely required for replication of bovine papillomavirus. Furthermore, the function of E2 in preventing nucleosome-mediated repression can be achieved as well by several other acidic transcription factors. These data therefore strongly support the idea that a group of enhancer proteins may utilize similar mechanisms to stimulate transcription and replication.

Base Sequence↗
PubMed1994

Assembly of nucleosomes: do multiple assembly factors mean multiple mechanisms?

In eukaryotic cells, transcription and DNA replication occur on DNA templates associated with chromatin proteins, most notably histone octamers. Protein factors that can assemble these units have been isolated from many sources. In particular, one factor from human cells is associated with ongoing DNA synthesis; other known assembly factors are not obligately coupled to the replication process. The wide variety of histone chaperones suggests that multiple pathways for the remodeling of chromatin structure have evolved.

Animals↗
PubMed1993

The acidic transcriptional activation domains of VP16 and p53 bind the cellular replication protein A and stimulate in vitro BPV-1 DNA replication.

For papillomavirus DNA replication, the E2 enhancer protein cooperatively assists in binding of the E1 helicase to the origin. We report that, at limiting E1 and E2 levels, the enhancer proteins GAL4-VP16 and GAL4-p53(1-73) stimulate BPV in vitro DNA replication. This cell-free system was used to ascertain whether the acidic activation domains have a cellular target important for replication. Cellular extracts were depleted of replication activity by passage through a VP16 affinity column. The protein depleted was the cellular factor replication protein A. The direct interaction between replication protein A and VP16, as well as the activation of replication by VP16, is dependent upon the C-terminus of the VP16 activation domain. E2 and the activation domain of p53 also interact with replication protein A. We suggest that a link between transcription and replication involves factors that help convert a closed DNA complex to an open complex.

Adenovirus E2 Proteins↗
PubMed1993

DNA-binding domain of bovine papillomavirus type 1 E1 helicase: structural and functional aspects.

The E1 protein of bovine papillomavirus type 1 is a multifunctional enzyme required for papillomaviral DNA replication. It assists in the initiation of replication both as a site-specific DNA-binding protein and as a DNA helicase. Previous work has indicated that at limiting E1 concentrations, the E2 protein is required for efficient E1 binding to the replication origin. In this study, we have defined the domain of the E1 protein required for site-specific DNA binding. Experiments with a series of truncated proteins have shown that the first amino-terminal 299 amino acids contain the DNA-binding domain; however, the coterminal M protein, which is homologous to E1 for the first 129 amino acids, does not bind origin DNA. A series of small internal deletions and substitution mutations in the DNA-binding domain of E1 show that specific basic residues in this region of the protein, which are conserved in all E1 proteins of the papillomavirus family, likely play a direct role in binding DNA and that a flanking conserved hydrophobic subdomain is also important for DNA binding. A region of E1 that interacts with E2 for cooperative DNA binding is also retained in carboxy-terminal truncated proteins, and we show that the ability of full-length E1 to complex with E2 is sensitive to cold. The E1 substitution mutant proteins were expressed from mammalian expression vectors to ascertain whether site-specific DNA binding by E1 is required for transient DNA replication in the cell. These E1 proteins display a range of mutant phenotypes, consistent with the suggestion that site-specific binding by E1 is important. Interestingly, one E1 mutant which is defective for origin binding but can be rescued for such activity by E2 supports significant replication in the cell.

Amino Acid Sequence↗
PubMed1993

The E1 replication protein of bovine papillomavirus type 1 contains an extended nuclear localization signal that includes a p34cdc2 phosphorylation site.

Bovine papillomavirus (BPV) DNA replication occurs in the nucleus of infected cells. Most enzymatic activities are carried out by host cell proteins, with the viral E1 and E2 proteins required for the assembly of an initiation complex at the replication origin. In latently infected cells, viral DNA replication occurs in synchrony with the host cell chromosomes, maintaining a constant average copy number of BPV genomes per infected cell. By analyzing a series of mutants of the amino-terminal region of the E1 protein, we have identified the signal for transport of this protein to the cell nucleus. The E1 nuclear transport motif is highly conserved in the animal and human papillomaviruses and is encoded in a similar region in the related E1 genes. The signal is extended relative to the simple nuclear localization signals and contains two short amino acid sequences which contribute to nuclear transport, located between amino acids 85 and 108 of the BPV-1 E1 protein. Mutations in either basic region reduce nuclear transport of E1 protein and interfere with viral DNA replication. Mutations in both sequences simultaneously prevent any observable accumulation of the protein and reduce replication in transient assays to barely detectable levels. Surprisingly, these mutations had no effect on the ability of viral genomes to morphologically transform cells, although the plasmid DNA in the transformed cells was maintained at a very low copy number. Between these two basic amino acid blocks in the nuclear transport signal, at threonine 102, is a putative site for phosphorylation by the cell cycle regulated kinase p34cdc2. Utilizing an E1 protein purified from either a baculovirus vector system or Escherichia coli, we have shown that the E1 protein is a substrate for this kinase. An E1 gene mutant at threonine 102 encodes for a protein which is no longer a substrate for the p34cdc2 kinase. Mutation of this threonine to isoleucine had no observable effect on either nuclear localization of E1 or DNA replication of the intact viral genome.

Amino Acid Sequence↗
PubMed1992

A constitutive enhancer in the bovine papillomavirus upstream regulatory region shares genetic elements with the viral P1 promoter.

The bovine papillomavirus upstream regulatory region represents a common element in the regulation of transcription from the five early viral promoters. We have determined the sequences required for transcription from the viral P1 promoter, which is located at the 5' end of the upstream regulatory region. In vitro transcription from P1 requires a 123-bp fragment (nucleotides 7153 to 7275; -33 to +90) consisting of an upstream TATA-like sequence as well as an unidentified protein which binds to sequences immediately downstream of the initiation site. In vivo, this promoter requires additional downstream sequences (to position +160; nucleotide 7345) for maximal activity but does not require any additional DNA sequence upstream of a putative TATA box. Four regions within the downstream sequence from +9 to +160 are protected from DNase I digestion by proteins present in a HeLa cell extract. The presence of these sites correlates with the level of P1 activity. A constitutive enhancer maps to this same region, and mutations in this enhancer have been shown to affect downstream promoters. Deletion analysis indicates that the same sequences are required by both the P1 promoter and the constitutive enhancer, suggesting that the same proteins function in both activities.

Animals↗
PubMed1992

The retinoblastoma gene product regulates Sp1-mediated transcription.

We have demonstrated that the retinoblastoma gene product (Rb) can positively regulate transcription from the fourth promoter of the insulinlike growth factor II gene. Two copies of a motif (the retinoblastoma control element) similar to that found in the human c-fos, transforming growth factor beta 1, and c-myc promoters are responsible for conferring Rb regulation to the fourth promoter of the insulinlike growth factor II gene. We have shown that the transcription factor Sp1 can bind to and stimulate transcription from the retinoblastoma control element motif. Moreover, by using a GAL4-Sp1 fusion protein, we have directly demonstrated that Rb positively regulates Sp1 transcriptional activity in vivo. These results indicate that Rb can function as a positive regulator of transcription and that Sp1 is one potential target, either directly or indirectly, for transcriptional regulation by Rb.

3T3 Cells↗
PubMed1991

Activation of BPV-1 replication in vitro by the transcription factor E2.

Soluble extracts from uninfected murine cells supplemented with purified viral E1 and E2 proteins support the replication of exogenously added papilloma virus DNA. The E2 transactivator stimulates the binding of the E1 replication protein to the minimal origin of replication and activates DNA replication. These results support the concept that transcription factors have a direct role in the initiation of DNA replication in eukaryotes by participating in the assembly of a complex at the origin of replication.

Aphidicolin↗
PubMed1991

Direct interaction between Sp1 and the BPV enhancer E2 protein mediates synergistic activation of transcription.

The physical interaction of heterologous site-specific DNA-binding proteins is an important theme in eukaryotic transcriptional regulation. In this paper, we show that the cellular transcription factor Sp1 and the BPV-1 (bovine papillomavirus type 1) enhancer protein E2 activate transcription synergistically from two papilloma viral promoters and a series of synthetic promoter constructs in transient transfection experiments. Furthermore, Sp1 can target E2 to a promoter region even in the absence of a specific E2 DNA-binding motif. Biochemical experiments establish that Sp1 enhances E2 binding to its sites and that the two proteins form a specific complex. Sp1 sequesters distally bound E2 to the promoter region by formation of stable DNA loops, visualized by electron microscopy. These experiments substantiate the notion that enhancer binding proteins are targeted to promoter regions by direct interaction with proteins that bind proximal to the transcriptional start site.

Animals↗
PubMed1990

Targeting the E1 replication protein to the papillomavirus origin of replication by complex formation with the E2 transactivator.

The mechanism by which transcription factors stimulate DNA replication in eukaryotes is unknown. Bovine papillomavirus DNA synthesis requires the products of the viral E1 gene and the transcriptional activator protein encoded by the E2 gene. Experimental data showed that the 68-kilodalton (kD) E1 protein formed a complex with the 48-kD E2 transcription factor. This complex bound specifically to the viral origin of replication, which contains multiple binding sites for E2. Repressor proteins encoded by the E2 open reading frame failed to complex with E1 suggesting that the 162-amino acid region of E2 that participates in transactivation contained critical determinants for interaction with E1. The physical association between a replication protein and a transcription factor suggests that transcriptional activator proteins may function in targeting replication initiator proteins to their respective origins of replication.

Animals↗
PubMed1990

The E2 trans-activator can act as a repressor by interfering with a cellular transcription factor.

The E2 open reading frame (ORF) of the bovine papillomavirus (BPV-1) encodes a family of site-specific DNA-binding proteins. The full-length protein is a transcriptional activator, whereas the polypeptides that contain only the carboxy-terminal domain are repressors. Here we show that the trans-activator can work as a repressor of transcription for one of the BPV-1 promoters by binding to a DNA sequence required for basal activity of the promoter. This operator site is defined as a 12-bp sequence that lies immediately downstream of the cap site. The operator DNA contains sequences that are defined genetically and biochemically as being important for basal level promoter activity. Furthermore, this site has been shown to be protected in a DNase footprint assay using fractionated HeLa cell extracts. The repression does not simply result from E2 blocking RNA polymerase initiation or elongation, because a strong E2-binding site placed at the operator has no repressive effect on transcription when the basal target sequence is placed independently upstream of the promoter. Thus, this is an interesting parallel to a theme well known in prokaryotes, where some site-specific DNA-binding proteins can work as either activators or repressors. In this system, as well as in the prokaryotic systems, the precise position of the binding site relative to other cis signals at the promoter determines the nature of the effects.

Base Sequence↗
PubMed1990

The E2 transactivator of bovine papillomavirus type 1 is expressed from multiple promoters.

The E2 proteins of bovine papillomavirus type 1 (BPV-1) are a family of site-specific DNA-binding proteins which regulate viral transcription by repression and activation. Repressors E2-TR and E8/E2 are expressed from promoters P5 (P3080) and P3 (P890), respectively. Previous reports have provided evidence that the transcript for the 48-kilodalton transactivator is initiated from a promoter proximal to the open reading frame encoding this protein (P2440 or P4). Our studies extend these findings and show that the E2 transactivation gene is expressed from multiple promoters. We have described the isolation of a cDNA (N15-2) which represents an RNA species expressed from the P3 promoter. The major exon of this species was produced by splicing to an acceptor located at nucleotide 2558 and contained the complete E2 open reading frame. The acceptor is probably utilized by yet another more abundant mRNA expressed from the P2 promoter (A. Stenlund, J. Zabielski, H. Ahola, J. Moreno-Lopez, and U. Pettersson, J. Mol. Biol. 182:541-554, 1985). Linked to a surrogate promoter, the N15-2 cDNA can transactivate an E2-responsive reporter gene. BPV-1 plasmids containing mutations either in the 2558 splice acceptor or in the P4 promoter showed significantly reduced transforming ability and reduced ability to transactivate an E2-responsive reporter, while a double mutant was inactive in both assays. The transformation defect was complemented by an E2 expression vector, and the BPV genome absolutely required the E2 protein to transactivate in the second assay. Thus, these genetic experiments show that alternate modes of E2 expression contribute to the E2 mRNA pool. Direct analysis of cytoplasmic RNA from transformed cultured cells proves that transcripts containing the 2558 acceptor exon are approximately as abundant as the P4 type E2 mRNAs. Furthermore, analysis of the E2 proteins present in various cell lines harboring specific BPV-1 mutants, including the 2558 acceptor mutant, proves that alternate modes of E2 expression exist. The ability of the E2 activator and repressors to each be independently expressed from multiple E2-responsive promoters probably adds to the resiliency of the latent virus as a plasmid and may be important for its homeostasis within the cell in different environmental or developmental situations.

Animals↗
PubMed1987

A promoter with an internal regulatory domain is part of the origin of replication in BPV-1.

Extrachromosomal elements that are stably maintained at a constant copy number through cell doublings are a good model system for the study of the regulation of DNA replication in higher eukaryotes. Previous studies have defined both cis and trans functions required for the regulated plasmid replication of the bovine papilloma virus in stably transformed cells. Here, a sequence known to be a cis-dominant element of the replication origin of the plasmid is shown to contain a promoter for transcription. Both in vitro and in vivo assays have been used to define this promoter and show that a sequence located just 3' to the transcriptional start site is required for activity. This DNA sequence element, which has been defined through deletions, coincides with a binding site for a cellular factor and is also required for a functional origin of replication. Possible models for how a transcription factor may play a role in the regulation of DNA replication are discussed.

Acetyltransferases↗
PubMed1987

Simian virus 40-transformed human cells that express large T antigens defective for viral DNA replication.

Many types of human cells cultured in vitro are generally semipermissive for simian virus 40 (SV40) replication. Consequently, subpopulations of stably transformed human cells often carry free viral DNA, which is presumed to arise via spontaneous excision from an integrated DNA template. Stably transformed human cell lines that do not have detectable free DNA are therefore likely to harbor harbor mutant viral genomes incapable of excision and replication, or these cells may synthesize variant cellular proteins necessary for viral replication. We examined four such cell lines and conclude that for the three lines SV80, GM638, and GM639, the cells did indeed harbor spontaneous T-antigen mutants. For the SV80 line, marker rescue (determined by a plaque assay) and DNA sequence analysis of cloned DNA showed that a single point mutation converting serine 147 to asparagine was the cause of the mutation. Similarly, a point mutation converting leucine 457 to methionine for the GM638 mutant T allele was found. Moreover, the SV80 line maintained its permissivity for SV40 DNA replication but did not complement the SV40 tsA209 mutant at its nonpermissive temperature. The cloned SV80 T-antigen allele, though replication incompetent, maintained its ability to transform rodent cells at wild-type efficiencies. A compilation of spontaneously occurring SV40 mutations which cannot replicate but can transform shows that these mutations tend to cluster in two regions of the T-antigen gene, one ascribed to the site-specific DNA-binding ability of the protein, and the other to the ATPase activity which is linked to its helicase activity.

Animals↗
PubMed1986

Repression of bovine papilloma virus replication is mediated by a virally encoded trans-acting factor.

Cells transformed with bovine papilloma virus type 1 mutants in the E6 or E6/7 genes are resistant to high-copy-number amplification of wild-type DNA after supertransfection. Transient and stable replication assays demonstrate this effect. If the supertransfected DNA has a mutation in a newly defined gene (M), this cellular immunity to high-copy-number replication is overcome, resulting in transient replication of the input DNA. In contrast, the resident plasmid does not participate in amplification and is maintained at a constant low copy number. Cotransformation of M- mutants and wild-type DNA into these cells leads to shutoff of replication of both genomes. Thus, M- mutants define a trans-acting negative modulator that regulates viral replication. This function is distinct from the positive factors required for replication. We propose a model that explains why the loss of E6 and E6/7 function leads to immunity of the infected cell.

Bovine papillomavirus 1↗