Summary auto-generated
This study investigates the enzymatic properties of the replication initiation protein (Rep) encoded by banana bunchy top virus (BBTV) DNA-1. Researchers expressed a maltose-binding protein fusion with BBTV Rep and demonstrated that this protein exhibits site-specific nicking and joining activities on single-stranded DNA in vitro. The protein specifically cleaves DNA between nucleotides 7 and 8 of a conserved nonanucleotide loop sequence present in BBTV genome components. These nicking and joining activities depend on magnesium or manganese ions but do not require ATP. During nicking reactions, the Rep protein becomes covalently attached to the 5' end of the 3' cleavage product. The joining activity enables ligation of two nicked DNA fragments in a sequence-specific manner. These findings demonstrate that BBTV Rep functions similarly to Rep proteins of geminiviruses and other rolling-circle replication initiators, suggesting BBTV likely employs a rolling-circle replication mechanism despite differences from the well-characterized geminiviruses.
Key findings
- BBTV Rep protein exhibits site-specific nicking activity that cleaves ssDNA between positions 7 and 8 of a conserved nonanucleotide sequence
- Nicking and joining activities require Mg2+ or Mn2+ but do not require ATP
- The Rep protein forms covalent attachment to the 5' end of the 3' cleavage product during nicking reactions
- BBTV Rep can catalyze sequence-specific joining of nicked DNA fragments without requiring stem-loop formation
- BBTV Rep protein functions are similar to geminivirus Rep proteins involved in rolling-circle DNA replication
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Abstract
The major open reading frame of banana bunchy top virus (BBTV) DNA-1 encodes a putative replication initiation protein (Rep). In vitro, a fusion protein of BBTV Rep linked to a maltose-binding protein exhibited both site-specific nicking and joining activities. These activities were dependent on the presence of Mg2+ or Mn2+, but did not require ATP. The fusion protein specifically cleaved ssDNA between bases +7 and +8 of a conserved nonanucleotide loop sequence which is present in the virion-strand of the stem-loop common region of each BBTV component. During this reaction, the fusion protein became covalently attached to the 5' end of the 3'cleavage product. After the nicking reactions, the fusion protein was also capable of catalysing the joining of two nicked ssDNA fragments in a site-specific manner. Based on these activities, BBTV Rep would appear to be very similar to the Rep proteins of the geminiviruses.