Functional domains of a geminivirus replication protein.
Tomato golden mosaic virus, a member of the geminivirus family, has a single-stranded DNA genome that is replicated and transcribed in infected plant cells through the concerted action of viral and host factors. One viral protein, AL1, contributes to both processes by binding to a directly repeated, double-stranded DNA sequence located in the overlapping (+) strand origin of replication and AL1 promoter. The AL1 protein, which occurs as a multimeric complex in solution, also catalyzes DNA cleavage during initiation of rolling circle replication. To identify the tomato golden mosaic virus AL1 domains that mediate protein oligomerization, DNA binding, and DNA cleavage, a series of truncated AL1 proteins were produced in a baculovirus expression system and assayed for each activity. These experiments localized the AL1 oligomerization domain between amino acids 121 and 181, the DNA binding domain between amino acids 1 and 181, and the DNA cleavage domain between amino acids 1 and 120. Deletion of the first 29 amino acids of AL1 abolished DNA binding and DNA cleavage, demonstrating that an intact N terminus is required for both activities. The observation that the DNA binding domain includes the oligomerization domain suggested that AL1-AL1 protein interaction may be a prerequisite for DNA binding but not for DNA cleavage. The significance of these results for AL1 function during geminivirus replication and transcription is discussed.
Orozco
B M
BM
Department of Biochemistry, North Carolina State University, Raleigh, North Carolina 27695-7622, USA. orozco@bchserver.bch.ncsu.edu
Miller
A B
AB
Settlage
S B
SB
Hanley-Bowdoin
L
L
D000595:Amino Acid Sequence; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics; D014779:Virus Replication
D014764:Viral Proteins; C080288:replication protein AL1, Begomovirus; D004247:DNA