Deciphering the functions of individual domains of the Tomato Golden Mosaic Virus Replication-initiator protein using fluorescently labelled DNA

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Circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses belong to the phylum of Cressdnaviricota with an extremely broad host range including animals and plants. They share a circular single-stranded DNA genome structure and encode the Replication initiator protein (Rep) as a common denominator of this group of viruses [1-3]. The CRESS-DNA virus family of Geminiviridae poses a serious economic threat to important crops including cassava, cucurbits, maize, tomato and wheat [4]. Rep is the only viral protein critical for viral DNA replication via rolling-circle replication (RCR) and fulfils multiple functions in this DNA replication process [5, 6]. Rep proteins comprise three functional domains: (i) an N-terminal HUH- (histidine (H) – hydrophobic residue (U) – histidine (H)-) endonuclease domain which recognises the origin of replication (ori) of the viral DNA, nicks the DNA, and covalently attaches to the 5’-end of the nicked site [7, 8]; (ii) a central oligomerisation domain that promotes Rep oligomer formation [9]; and (iii) a C-terminal SF3 (Superfamily 3) AAA+ (ATPases Associated with diverse cellular Activities) domain with helicase activity [10, 11]. Oligomerisation of Rep is a prerequisite to exert its helicase activity [10]. In the geminivirus Tomato Golden Mosaic Virus (TGMV), the boundaries and properties of the domains of Rep (also called AL1), including the endonuclease activity of the HUH domain, have been studied earlier [9, 11, 24]. However, the DNA-binding capacity of the C-terminal ATPase domain has not been addressed in previous studies. Here we describe a protocol to study the interaction of the individual domains of TGMV-Rep with DNA using fluorescently labelled DNA probes. Applying this protocol, we confirm the endonuclease activity of the N-terminal domain of TGMV-Rep. In addition, we show that (i) DNA binding of the C-terminal domain is independent of the DNA sequence, but that (ii) a minimum length (number of nucleotides) is required for binding and that (iii) ssDNA is likely preferred over dsDNA.

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