DNA Chain Unwinding and Annealing Reactions of Lipocortin (Annexin) I Heterotetramer: Regulation by Ca 2+ and Mg 2+ Aiko Hirata* and Fusao Hirata* , † , ‡ ,1 *Department of Pharmaceutical Sciences and †Department of Pharmacology, and ‡Institute of Environmental Health Sciences, Wayne State University, Detroit, Michigan 48202 Received January 14, 2002 Lipocortin I-S100 (calcyclin) heterotetramer exhib- ited ATPase activity in the presence of dsDNA but not ssDNA. To demonstrate its helicase activity, an 80-mer polynucleotide complementary to the replication ori- gin of M13mp18 was synthesized, and the oligonucle- otide, (dC) 20 , was ligated to either its 5- or 3- end for binding to lipocortin. Lipocortin I heterotetramer dis- placed chains of the partially Y-shaped duplexes with a dC-tail at either the 5- or 3- end. The chain displace- ment required ATP and Mg 2 . Nonhydrolyzable ATP analogues were not effective. Lipocortin I heterotet- ramer also catalyzed annealing of the polynucleotides to M13mp18. Ca 2 and phospholipids but not ATP and Mg 2 were essential for this reaction. Since the chain displacing and annealing reactions were inhibited by monospecific anti-lipocortin I or anti-S100 antibodies, the present observations suggest that the lipocortin I heterotetramer regulates unwinding and annealing of DNA by Mg 2 (plus ATP) and Ca 2 (and phospholipids), respectively. © 2002 Elsevier Science (USA) Lipocortin I, also termed annexin I, is a member of the lipocortin (annexin) family of proteins that bind to phospholipids (biomembranes) in a Ca 2+ -dependent manner (1). This protein is a major cellular substrate of the oncogenic tyrosine kinases such as EGF receptors and c-met (1). Since its N-terminal domain contains a sequence motif for SH2 recognition, this protein is thought to be involved in signal transduction of growth factors for mitogenesis (2). We have previously shown that tyrosine phosphorylation of lipocortin I is involved in signaling for thymocyte mitogenesis by mitogens such as Con A (3). This proposal has been recently substantiated by the findings that the treatment of A549 cells with antisense oligonucleotides of lipocortin I results in reduction of the synthesis and subsequent phosphorylation of lipocortin I, thereby inhibiting cell proliferation (4). However, the detailed mechanism of signal transduction via phosphorylation of lipocortin I remains poorly understood. Since the appearance of lipocortin I in the nucleus is temporally related to its phosphorylation via serum factors (growth factors) (5), it was hypothesized that the phosphorylated lipocortin I is translocated into the nucleus, where lipocortin I heterotetramer regulates DNA synthesis. In accord with this working hypothesis, we have observed that lipocortin I-S100 (calcyclin) heterotetramer binds to pyrimidine clusters of ssDNA in a Ca 2+ - and phospholipid-dependent manner (6). Since lipocortin I is reported to bind ATP (7), and since plant annexin exhibits ATPase activity (8), the effect of ATP on bind- ing of lipocortin I heterotetramer to DNA was investi- gated. We found that the lipocortin I heterotetramer exhibits ATPase activity, when dsDNA but not ssDNA are included in the reaction mixture (9). In this com- munication, we report that lipocortin I heterotetramer possesses helicase-like activity, and that the unwind- ing and annealing activities of its helicase action are regulated by Mg 2+ (plus ATP) and Ca 2+ (and phospho- lipids), respectively. MATERIALS AND METHODS Materials. Monospecific antibodies against calpactin I (lipocortin or annexin II) and calpactin II (lipocortin or annexin I) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-S100 anti- body and anti-Igs antibody were products of Sigma Aldrich (St. Louis, MO) and Miles (Tarrytown, NY), respectively. Purification of lipocortin I. Lipocortin (annexin) I-S100 heterotet- ramer was purified from rat livers as described previously (6). Briefly, the membrane fraction obtained in the presence of 1 mM CaCl 2 was extracted with 50 mM Tris–HCl buffer, pH 7.4, containing 10 mM EDTA. The extracts were concentrated and were applied on Sephacryl S200, and fractions corresponding to molecular weights of 94,000 Da were collected. These fractions were applied on DEAE- Sepharose and subsequently on Sephcryl-200 and Q Sepharose col- umns as described previously (6). Purified protein had an apparent 1 To whom correspondence and reprint requests should be ad- dressed to Department of Pharmaceutical Sciences, Wayne State University, 528 Shapero Hall, Detroit, MI 48202. Biochemical and Biophysical Research Communications 291, 205–209 (2002) doi:10.1006/bbrc.2002.6422, available online at http://www.idealibrary.com on 205 0006-291X/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.