A DNA structure is required for geminivirus replication origin function.
The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two single-stranded circular DNAs, A and B, that replicate through a rolling-circle mechanism in nuclei of infected plant cells. The TGMV origin of replication is located in a conserved 5' intergenic region and includes at least two functional elements: the origin recognition site of the essential viral replication protein, AL1, and a sequence motif with the potential to form a hairpin or cruciform structure. To address the role of the hairpin motif during TGMV replication, we constructed a series of B-component mutants that resolved sequence changes from structural alterations of the motif. Only those mutant B DNAs that retained the capacity to form the hairpin structure replicated to wild-type levels in tobacco protoplasts when the viral replication proteins were provided in trans from a plant expression cassette. In contrast, the same B DNAs replicated to significantly lower levels in transient assays that included replicating, wild-type TGMV A DNA. These data established that the hairpin structure is essential for TGMV replication, whereas its sequence affects the efficiency of replication. We also showed that TGMV AL1 functions as a site-specific endonuclease in vitro and mapped the cleavage site to the loop of the hairpin. In vitro cleavage analysis of two TGMV B mutants with different replication phenotypes indicated that there is a correlation between the two assays for origin activity. These results suggest that the in vivo replication results may reflect structural and sequence requirements for DNA cleavage during initiation of rolling-circle replication.
Orozco
B M
BM
Department of Biochemistry, North Carolina State University, Raleigh 27695-7622, USA.
Hanley-Bowdoin
L
L
D001483:Base Sequence; D004261:DNA Replication; D004270:DNA, Circular / Q000737:chemistry / Q000235:genetics; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics; D004279:DNA, Viral / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D017791:Geminiviridae / Q000235:genetics / Q000502:physiology*; D018551:Solanum lycopersicum / Q000821:virology; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D018515:Plant Leaves / Q000821:virology; D018741:Replication Origin / Q000502:physiology*; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000502:physiology*
D004270:DNA, Circular; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D014764:Viral Proteins; C080288:replication protein AL1, Begomovirus; D004720:Endonucleases