Anti-cruciform DNA affinity purification of active mammalian origins of replication.
A novel approach that employs anti-cruciform DNA monoclonal antibodies was used to isolate segments of cruciform-containing DNA from genomic DNA, in an effort to obtain fragments containing active origins of replication. High molecular weight DNA (greater than 50 kb) was extracted from log phase CV-1 cells and 6 micrograms incubated with approximately 2.5 micrograms of a monoclonal antibody, 2D3, specific for cruciform-containing DNA. The 2D3-bound DNA was digested with EcoRI and antibody-bound fragments were recovered using rabbit anti-mouse immunobeads. The beads were washed free of nonspecifically bound DNA and the 2D3-bound DNA was eluted with 2% sodium dodecyl sulphate (SDS). The yield of DNA recovered by 2D3 was 2000-fold less than the initial amount and was 17-20-fold more than that recovered nonspecifically using the control mAb, P3. The 2D3-bound DNA ranged from 0.15- greater than 23 kb with a major peak at approximately 12 kb. Specific enrichment of origin-containing DNA by 2D3 over P3 was suggested by a 10-100-fold greater recovery of a 9 kb fragment hybridizable to a low-copy monkey autonomously replicating sequence, ors 8. 20 ng of affinity-purified DNA was cloned into lambda Zap II and excised into Bluescript phagemids in vivo. Of nine randomly-selected clones between 0.15 and 3.2 kb, four were able to replicate autonomously when transfected into HeLa cells. Two of the nine clones contained sequences hybridizable to both monkey alpha-satellite and human Alu DNA, and two others to Alu alone. The present work provides further evidence for the involvement of cruciforms at active mammalian origins of DNA replication.
Bell
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McGill Cancer Centre, McGill University, Montreal, Canada.
Sabloff
M
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Zannis-Hadjopoulos
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M
Price
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G
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015139:Blotting, Southern; D001973:Bromodeoxyuridine; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D004261:DNA Replication; D015723:Gene Library; D012091:Repetitive Sequences, Nucleic Acid; D012689:Sequence Homology, Nucleic Acid; D014169:Transformation, Bacterial; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D004247:DNA; D001973:Bromodeoxyuridine