Identification of the replication-associated protein binding domain within the intergenic region of tomato leaf curl geminivirus

• Published in: Nucleic acids research Vol. 26; no. 4; pp. 925 - 931
• Main Authors: Akbar Behjatnia, S. A., Dry, Ian B., Rezaian, M. Ali
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 15.02.1998
• Subjects: Base Sequence; Binding Sites - genetics; Cloning, Molecular; DNA Footprinting; DNA Helicases - genetics; DNA Helicases - metabolism; DNA Primers - genetics; DNA Replication; DNA, Viral - chemistry; DNA, Viral - genetics; DNA, Viral - metabolism; DNA-Binding Proteins; Geminiviridae - genetics; Geminiviridae - metabolism; Geminiviridae - physiology; Lycopersicon esculentum - virology; Molecular Sequence Data; Nucleic Acid Conformation; Protein Binding; Repetitive Sequences, Nucleic Acid; Replication Origin; Trans-Activators - genetics; Trans-Activators - metabolism; Viral Proteins - genetics; Viral Proteins - metabolism; Virus Replication
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/26.4.925

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.