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THE PLANT CELL, Vol 4, Issue 7 799-809, Copyright © 1992 by American Society of Plant Biologists
Sequence-Specific Interaction with the Viral AL1 Protein Identifies a Geminivirus DNA Replication Origin
S. G. Lazarowitz, L. C. Wu, S. G. Rogers and J. S. Elmer
Department of Microbiology, University of Illinois at Urbana-Champaign, 131 Burrill Hall, Urbana, Illinois 61801
The bipartite geminiviruses such as tomato golden mosaic virus (TGMV) and
squash leaf curl virus (SqLCV) have two single-stranded circular genomic
DNAs, the A and B components, thought to be replicated from double-stranded
circular DNA intermediates. Although it has been presumed that the origin
sequences for viral replication are located in the highly conserved
200-nucleotide common region (CR) present in both genomic components and
that the viral-encoded AL1 protein interacts with these sequences to effect
replication, there has been no evidence that this is in fact so. We have
investigated these questions, demonstrating selectivity and sequence
specificity in this protein-DNA interaction. Simple component switching
between the DNAs of TGMV and SqLCV and analysis of replication in leaf
discs showed that whereas the A components of both TGMV and SqLCV promote
their own replication and that of their cognate B component, neither
replicates the noncognate B component. Furthermore, using an in vivo
functional replication assay, we found that cloned viral CR sequences
function as a replication origin and direct the replication of nonviral
sequences in the presence of AL1, with both circular single-stranded and
double-stranded DNA being synthesized. Finally, by the creation of chimeric
viral CRs and specific subfragments of the viral CR, we demonstrated
sequence-specific recognition of the replication origin by the AL1 protein,
thereby localizing the origin to an ~90-nucleotide segment in the AL1
proximal side of the CR that includes the conserved geminiviral stem-loop
structure and ~60 nucleotides of 5[prime] upstream sequence. By deletional
analysis, we further demonstrated that the conserved stem-loop structure is
essential for replication. These studies identify the functional viral
origin of replication within the CR, demonstrating that sequence-specific
recognition of this origin by the AL1 protein is required for replication.
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