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 (> 50 kb) was extracted from log phase CV-1 cells and 6-mu-g incubated with approximately 2.5-mu-g 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- > 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.