Recruitment of terminal protein to the ends of Streptomyces linear plasmids and chromosomes by a novel telomere-binding protein essential for linear DNA replication.
Bidirectional replication of Streptomyces linear plasmids and chromosomes from a central origin produces unpaired 3'-leading-strand overhangs at the telomeres of replication intermediates. Filling in of these overhangs leaves a terminal protein attached covalently to the 5' DNA ends of mature replicons. We report here the essential role of a novel 80-kD DNA-binding protein (telomere-associated protein, Tap) in this process. Biochemical studies, yeast two-hybrid analysis, and immunoprecipitation/immunodepletion experiments indicate that Tap binds tightly to specific sequences in 3' overhangs and also interacts with Tpg, bringing Tpg to telomere termini. Using DNA microarrays to analyze the chromosomes of tap mutant bacteria, we demonstrate that survivors of Tap ablation undergo telomere deletion, chromosome circularization, and amplification of subtelomeric DNA. Microarray-based chromosome mapping at single-ORF resolution revealed common endpoints for independent deletions, identified amplified chromosomal ORFs adjacent to these endpoints, and quantified the copy number of these ORFs. Sequence analysis confirmed chromosome circularization and revealed the insertion of adventitious DNA between joined chromosome ends. Our results show that Tap is required for linear DNA replication in Streptomyces and suggest that it functions to recruit and position Tpg at the telomeres of replication intermediates. They also identify hotspots for the telomeric deletions and subtelomeric DNA amplifications that accompany chromosome circularization.
Bao
Kai
K
Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120, USA.
Cohen
Stanley N
SN
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000378:metabolism; D015153:Blotting, Western; D002874:Chromosome Mapping; D002875:Chromosomes / Q000648:ultrastructure*; D004247:DNA / Q000378:metabolism; D004261:DNA Replication; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D016366:Open Reading Frames; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000378:metabolism*; D016615:Telomere / Q000648:ultrastructure; D034501:Telomere-Binding Proteins / Q000096:biosynthesis* / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D034501:Telomere-Binding Proteins; D004247:DNA