The enzyme system for duplicating the MATERIALS AND METHODSSource of Enzymes. OX174 cisA (60,000 daltons) was prepared by an improved procedure and was more than 90% pure as judged by sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gel electrophoresis (4.4 X 106 units/mg of protein) (Eisenberg et al., unpublished data). E. coli rep was prepared from JFS-19 [rha, lys, thy, polB, strr/F+/ColEl (pLC 44-7) ilv +, cya + rho +, rep + ] cells, which overproduce rep 7-to 10-fold above wild-type levels. Plasmid pLC 44-7 was obtained by screening of the collection of ColEl/E. coli hybrids prepared by Clarke and Carbon (9). Details of the screening and purification, which yields rep (68,000 daltons) more than 95% pure as judged by NaDodSO4/polyacrylamide gel electrophoresis (2.0 X 107 units/mg of protein), will be described elsewhere. DNA polymerase III holoenzyme was DEAE-Sephadex peak fraction V (5.9 X 105 units/mg of protein; approximately 60% pure) (C. S. McHenry and A. Kornberg, unpublished data

Complementary DNA clones for liver protein 56K (SLP-56) were isolated by screening a lambda Zap mouse liver library. The cloned cDNAs represented the complete message. The correct reading frame was verified by alignment of the deduced amino acid sequence with that of peptides sequenced from the purified protein. The primary sequence has not been reported previously since homologous DNA sequences were not found in GenBank. Most importantly, the DNA sequence did not contain an in-frame TGA codon that would code for seleno-cysteine, as occurs in the prototypic selenoprotein, glutathione peroxidase. Hydropathy analysis suggested the protein was not a membrane-spanning protein. SLP-56 was previously localized as a cytosolic-soluble protein on the basis of cell fractionation experiments. The results suggest that SLP-56 is different from proteins whose synthesis and concentration are dependent upon selenium and require TGA to encode for selenocysteine. In this respect, SLP-56 appears to be similar to liver fatty acid binding protein (SLP-14) for which selenium is a ligand. Our working hypothesis is that selenium exerts its inhibitory effects on cell growth by modulating the properties of existing growth regulatory proteins. The two proteins that are readily labeled by selenium in many rodent tissues, SLP-56 and SLP-14, would fit into this category.

molecules (22, 42). In addition, it has been suggested that these sequences normally serve as initiation sites for DNA replication in the intact chromosome.Replication of chromosomal DNA in yeast requires the products of a number of cell cycle (CDC) genes. The products of CDC28, CDC4, and CDC7 act sequentially before the initiation of DNA synthesis, whereas the products of CDC8 and CDC21 are required continuously t Current address: Department of Microbiology, Urbana, IL 61801. during the S phase (18)(19)(20). The product of only one of these genes (CDC21), thymidylate synthetase, has been identified (5). Replication of the multiple-copy endogenous plasmid of yeast called 2, DNA also depends on these gene products (26,32). In contrast, replication of yeast mitochondrial DNA occurs in the absence of an active CDC28, CDC4, or CDC7 gene product (13,30) and in the presence of the yeast pheromone a-factor (14, 31), which inhibits replication of both chromosomal and 2> DNAs (8,26). Moreover, both yeast chromosomal and 2> DNAs are organized in typical nucleosomal subunits (25,27,28), whereas mitochondrial DNA is not believed to be associated with histones (9,33).In this paper we describe the construction and stability properties of a multiple-copy extrachro-

Highlights d ORC self-interacts and dimerizes at the M-to-G 1 transition and de-dimerizes in S phase d ORC dimers provide symmetric platforms to load the symmetric pre-RCs at origins d Upon ORC de-dimerization, each single hexamer binds one of the two nascent origins d ORC dimerization is essential for and regulates pre-RC formation and DNA replication