Nucleic Acids Research, Vol 26, Issue 4 925-931, Copyright © 1998 by Oxford University Press
SA Akbar Behjatnia, IB Dry and M Ali Rezaian
The geminiviral replication-associated protein (Rep) is the only viral
protein required for viral DNA replication. Tomato leaf curl virus (TLCV)
Rep was expressed in Escherichia coli as a histidine-tagged fusion protein
and purified to homogeneity in non-denaturing form. The fusion protein was
used in in vitro binding experiments to identify the Rep-binding elements
within the origin of replication of TLCV. Electrophoretic mobility shift
assays demonstrated that the Rep binds specifically to a 120 bp fragment
within the TLCV intergenic region. Fine resolution of the binding regions
within the 120 bp fragment, using DNase I footprinting, demonstrated two
footprints covering the sequences GCAATTGGTGTCTCTCAA and TGAATCGGTGTCTGGGG
containing a direct repeat of the motif GGTGTCT (underlined). Our results
suggest that the repeated motif is involved in virus-specific Rep-binding,
but may not constitute the entire binding element. This is the first
demonstration of geminivirus sequence elements involved in Rep-binding by
direct protein-DNA interaction assays.
ARTICLES
Identification of the replication-associated protein binding domain within the intergenic region of tomato leaf curl geminivirus
CSIRO, Plant Industry, Horticulture Research Unit, PO Box 350, Glen Osmond 5064, Adelaide, Australia.
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