Endonuclease V is specific for single-stranded DNA or for duplex DNA that contains uracil or that is damaged by a variety of agents (B. Demple and S. Linn, J. Biol. Chem. 257:2848-2855, 1982). Thus, it may be a versatile DNA repair enzyme. The protein was purified to apparent homogeneity, and from its N-terminal sequence, its gene, nfi, was identified. nfi is immediately downstream of hemE, at kb 4208 (90.4 min) on the current chromosomal map of Escherichia coli K-12. This region was cloned, and plasmid insertion and deletion mutants were used to study its molecular organization. Although nfi is the third of four closely spaced, codirectional genes, it is expressed independently.Endonuclease V of Escherichia coli (11,12,14) selectively cleaves untreated single-stranded DNA, duplex DNA containing uracil in place of thymine, and DNA that has been treated with acid, alkali, OsO 4 , UV radiation, or 7-bromomethylbenz [a] anthracene. It appears to work processively, attacking at multiple lesions on one molecule before moving on to the next. Unlike the members of the glycosylase-endolyase class of repair enzymes that cleave DNA at damaged bases (28), endonuclease V does not appear to release free bases (specifically uracil) from DNA, and it produces 3Ј-hydroxyl and 5Ј-phosphoryl end groups. In contrast to the UvrABC system (28), which also recognizes lesions produced by a broad range of agents but consists of a high-molecular-weight protein complex, endonuclease V is a simple polypeptide of about 25 kDa. These properties suggest that endonuclease V may be a highly useful DNA repair enzyme that, because of its simplicity, might be primitive and therefore universal.Unfortunately, study of this interesting enzyme has been hampered by an absence of mutants and by its scarcity. Homogeneous preparations of endonuclease V have not been produced, and it has been difficult to obtain large amounts of the enzyme because of its low concentration in the cell and its instability during purification. We now report our first steps in overcoming these difficulties. We describe the identification, cloning, and molecular organization of nfi, the gene for endonuclease V.(While this manuscript was in review, we were made aware [18] that nfi was also identified as the structural gene for deoxyinosine 3Ј endonuclease [41], an enzyme that cleaves DNA near dIMP residues, mismatched bases, urea residues, or abasic sites [39,40]). MATERIALS AND METHODSMicrobial strains and methods. The E. coli strains, phage, and plasmids used are listed in Table 1. Phage PBS2 and its host, Bacillus subtilis SB19, were obtained from A. Price (26). E. coli was grown in Luria-Bertani (LB) medium supplemented with antibiotics, as required, for plasmid maintenance (23). Plasmids containing sequences counterclockwise of hemE (pGG4, pGG15, pGG6, pGG10, and pGG13) were unstable and did not survive serial propagation even in antibiotic media; therefore, cultures were prepared from colonies of freshly transformed cells. Strains with plasmids carrying the hemE gene wer...

The present study shows that COS-7 cells transiently transfected and maintained on positively charged (trimethylamine-coated) microcarrier beads synthesize recombinant protein at higher levels and for longer periods of time than cells transfected and maintained on polystyrene flasks in monolayer culture. Sustained, high-level synthesis was observed with secreted chimeric proteins (murine E-selectin- and P-selectin-human IgM chimeras) and a secreted hematopoietic growth factor (granulocyte-macrophage colony-stimulating factor). Studies with green fluorescent protein indicated that the transfected cells attached more firmly to the trimethylamine-coated microcarriers than to polystyrene flasks. After 10-14 days in culture, most of the transfected cells detached from the surface of the polystyrene flasks, whereas most transfected cells remained attached to the microcarriers. The transiently transfected microcarrier cultures produced higher levels of protein per transfected cell due to this prolonged attachment. The prolonged attachment and higher output of transfected cells on microcarriers resulted in a 5-fold increase in protein production from a single transfection over two weeks. Thus, microcarrier-based transient transfection yields quantities of recombinant proteins with a significant savings of time and reagents over monolayer culture.