IDENTIFICATION OF THE REPLICATION-ASSOCIATED PROTEIN-BINDING DOMAIN WITHIN THE INTERGENIC REGION OF TOMATO LEAF CURL GEMINIVIRUS

The geminiviral replication-associated protein (Rep) is the only viral protein required for viral DNA replication. Tomato leaf curl virus (T LCV) Rep was expressed in Escherichia coil as a histidine-tagged fusio n protein and purified to homogeneity In non-denaturing form. The fusi on protein was used in in vitro binding experiments to identify the Re p-binding elements within the origin of replication of TLCV. Electroph oretic mobility shift assays demonstrated that the Rep binds specifica lly to a 120 bp fragment within the TLCV intergenic region. Fine resol ution of the binding regions within the 120 bp fragment, using DNase I footprinting, demonstrated two footprints covering the sequences GCAA TTGGTGTCTCTCAA and TGAATCGGTGTCTGGGG containing a direct repeat of the motif GGTGTCT (underlined). Our results suggest that the repeated mot if is involved in virus-specific Rep-binding, but may not constitute t he entire binding element. This is the first demonstration of geminivi rus sequence elements involved in Rep-binding by direct protein-DNA in teraction assays.