Biomedical subjects

P A Eagle

Publications and source records attributed to P A Eagle.

6 recordsLinked to original sources

PubMed1998

Multiple cis elements contribute to geminivirus origin function.

The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two circular DNA molecules which are dissimilar in sequence except for a highly conserved 200-bp common region that includes the origin for rolling circle replication. To better characterize the plus-strand origin, we analyzed the capacities of various TGMV common region sequences to support episomal replication in tobacco protoplasts when the viral replication proteins AL1 and AL3 were supplied in trans. These experiments demonstrated that the minimal origin is located in 89-bp common region fragment that includes the known AL1 binding motif and a hairpin structure containing the DNA cleavage site. Analyses of mutant origin sequences identified two additional cis elements--one that is required for origin activity and a second that greatly enhances replication. In contrast, a conserved partial copy of the AL1 binding site did not contribute to origin function. Mutational analysis of the functional AL1 binding site showed that both spacing and sequence of this motif are important for replication in vivo and AL1/DNA binding in vitro. Spacing changes between the AL1 binding site and hairpin also negatively impacted TGMV origin function in a position-dependent manner. Together, these results demonstrated that the organization of TGMV plus-strand origin is complex, involving multiple cis elements that are likely to interact with each other during initiation of replication.

Base Sequence↗

PubMed1997

Two domains of the AL1 protein mediate geminivirus origin recognition.

The geminiviruses tomato golden mosaic virus (TGMV) and bean golden mosaic virus (BGMV) have bipartite genomes. Their A and B DNA components contain cis-acting sequences that function as origins of replication, while their A components encode the trans-acting replication proteins--AL1 and AL3. Earlier experiments demonstrated that virus-specific interactions between the cis- and trans-acting functions are required for TGMV and BGMV replication and that the AL1 proteins of the two viruses specifically bind their respective origins. In the current study, characterization of AL1 and AL3 proteins produced from plant expression cassettes in transient replication assays revealed that interaction between AL1 and the origin is responsible for virus-specific replication. The AL3 protein does not contribute to specificity but can be preferred by its cognate AL1 protein when replication is impaired. Analysis of chimeric proteins showed that two regions of AL1 act as specificity determinants during replication. The first domain is located between amino acids 1 and 116 and recognizes the AL1 origin binding site. The second region, which is between amino acids 121 and 209, is not dependent on the known AL1 DNA binding site. Analysis of wild type and chimeric proteins in transient transcription assays showed that AL1 also represses its own promoter in a virus-specific manner. Transcriptional specificity is conferred primarily by AL1 amino acids 1-93 with amino acids 121-209 making a smaller contribution. Together, these results demonstrated that the virus-specific interactions of AL1 during replication and transcription are complex, involving at least two discreet domains of the protein.

Amino Acid Sequence↗

PubMed1997

cis elements that contribute to geminivirus transcriptional regulation and the efficiency of DNA replication.

The A genomic component of the geminivirus tomato golden mosaic virus (TGMV) contains a 5' intergenic sequence that includes the overlapping AL61 promoter and positive-strand origin of DNA replication. The TGMV AL1 protein negatively regulates its own transcription and mediates origin recognition by binding to a repeated motif shared by the AL61 promoter and the viral origin. We examined a series of truncated or mutated 5' intergenic regions in transient expression and replication assay to identify other DNA sequences that contribute to TGMV promoter and origin function. These experiments revealed that negative regulation of the AL61 promoter is complex, involving multiple cis-acting sequences and the AL1 and AL4 proteins, which acted through different DNA elements. We also found that mutation of the TATA box motif in the AL61 promoter reduced overall transcriptional activity and AL1-mediated repression, confirming the importance of this sequence in promoter function. Mutation of a G-box consensus sequence was highly detrimental to AL61 transcription and abolished AL1 sensitivity, suggesting that AL1 interferes with transcriptional activation. Cotransfection experiments showed that the TATA box and G-box motif mutations also impaired viral DNA replication in the presence of a wild-type origin but had no effect in its absence, demonstrating that these transcriptional motifs also function as replication efficiency elements.

Base Sequence↗

PubMed1994

Interaction between a geminivirus replication protein and origin DNA is essential for viral replication.

The geminivirus, tomato golden mosaic virus (TGMV), encodes one protein, AL1, that is absolutely required for viral DNA replication. AL1 interacts with the TGMV DNA genome by binding specifically to the viral origin of replication. We have investigated the nature and significance of AL1/origin interactions in vitro and in vivo by using competitive DNA binding and transient replication assays. Competition assays established that a 13-base pair (bp) element (5'-GGTAGTAAGGTAG) containing two 5-bp direct repeat motifs separated by a 3-bp central core constitutes a high affinity AL1 binding site. DNAs containing intact 3' repeat sequences plus core (TAAGGTAG and ccTAGTAAGGTAG) were stronger competitors for AL1 binding than DNAs containing intact 5' repeat sequences plus core (GGTAGTAA and GGTAGTA-AccTAG), thereby demonstrating that AL1 interacts differently with the repeat motifs. Replication in tobacco protoplasts established that the AL1 binding site is an essential cis-acting element for viral replication. No replication was detected for DNAs containing mutations in either of the repeat motifs of the AL1 recognition sequence when AL1 was provided in trans from a plant gene expression vector. In contrast, a DNA with a mutation in the 5' repeat motif (ccTAGTAAGGTAG) replicated when both AL1 and AL3, a TGMV protein involved in viral DNA accumulation, were provided in trans. No replication was detected for a DNA containing a mutation in the 3' repeat motif (GGTAGTAAccTAG) in the presence of AL1 and AL3. The in vitro and in vivo results suggest that binding of AL1 to the 3' repeat element is an essential step in DNA replication, while binding to the 5' repeat element may serve to enhance viral replication.

Base Sequence↗

PubMed1994

A DNA sequence required for geminivirus replication also mediates transcriptional regulation.

Tomato golden mosaic virus (TGMV), a member of the geminivirus family, requires a single virus-encoded protein for DNA replication. We show that the TGMV replication protein, AL1, also acts during transcription to specifically repress the activity of its promoter. An earlier study established that AL1 binds to a 13-bp sequence (5'-GGTAGTAAGGTAG) that is essential for activity of the TGMV replication origin. Analysis of AL1 binding site mutants in transient expression assays demonstrated that the same site, which is located between the transcription start site and TATA box in the AL1 promoter, also mediates transcriptional repression. These experiments revealed that the repeated motifs in the AL1 binding site contribute differentially to repression, as has been observed previously for AL1-DNA binding and viral replication. Introduction of the AL1 binding site into the 35S promoter of the cauliflower mosaic virus was sufficient to confer AL1-mediated repression to the heterologous promoter. Analysis of a truncated AL1 promoter and of mutant AL1 proteins showed that repression does not require a replication-competent template or a replication-competent AL1 protein. Transient expression studies using two different Nicotiana cell lines revealed that, although the two lines replicate plasmids containing the TGMV origin similarly, they support very different levels of AL1-mediated repression. These results suggest that geminivirus transcriptional repression and replication may be independent processes.

Amino Acid Sequence↗

PubMed1992

A zinc-binding motif located between amino acids 273 and 286 in the adenovirus DNA-binding protein is necessary for ssDNA binding.

The human adenovirus single-stranded (ss) DNA-binding protein (DBP) possesses a highly conserved carboxyl domain which contains a putative zinc-binding motif between amino acids (aa) 273 and 286. Using a zinc blotting technique DBP was shown to bind 65Zn at levels similar to other documented zinc metalloproteins. In competition experiments, DBP bound specifically to the zinc ion even in the presence of other divalent ions such as Ca+2, Mg+2, Cd+2, Co+2, and Mn+2. The zinc-binding ability of DBP was also confirmed by zinc affinity chromatography. Site-directed mutagenesis was utilized to construct a mutant which deleted the entire zinc region (pKZNdl 273-286) and a mutant which contained a Cys to Ser substitution at aa residue 284 (pKZNpt 284). The deletion mutant was unable to bind zinc, and the point mutant showed limited binding suggesting that aa 273-286 are responsible for the interaction of DBP with zinc. The DBP zinc mutants were also examined for their ability to bind to ssDNA. The deletion mutant was unable to bind ssDNA cellulose while the point mutant exhibited decreased affinity. Thus, the region between aa 273 and 286 which mediates zinc binding also appears fundamental for the ssDNA-binding function of DBP.

Adenoviridae↗