Colicin E2 is DNA endonuclease.

Abstract

Colicin E2 purified by conventional methods contains a tightly bound low-molecular-weight protein, as has been found with purified colicin E3 [Jakes, N. & Zinder, N. D. (1974) Proc. Nat. Acad. Sci. USA 71, 3380-33841. Such E2 preparations do not cause DNA cleavage in vitro. After separation from the low-molecular-weight protein, colicin E2 retained the original in vivo killing activity, and in addition showed a high activity in vitro in cleaving various DNA molecules, such as a ColE1 hybrid plasmid and DNAs from Escherichia coli, X phage, 4X174 phage, and simian virus 40. The low-molecular-weight protein ("E2-immunity protein") specifically prevented this in vitro DNA cleavage reaction, i.e., had an "immunity function." The results demonstrate that colicin E2 itself is a DNA endonuclease and explain the in vivo effects caused by E2 in sensitive cells as well as the mechanism of immunity in E2-colicinogenic cells. Colicin E2 (E2) is a protein antibiotic synthesized by certain strains of coliform bacteria that carry the ColE2 plasmid, and is classified in the E group of colicins along with colicins El and E3 (1, 2). Although the physical properties of E2 and E3 are similar (3) and both colicins share the same receptor (1, 2), their apparent mode of action is different. It was first discovered that E2 causes degradation of DNA, while colicin E3 (E3) causes specific inhibition of protein synthesis in sensitive Escherichia coli cells treated with these colicins (4). Subsequent work on the mode of action of E3 has demonstrated that ES inactivates ribosomes in treated cells (5) by causing the cleavage of a fragment from the 3' end of the 16S RNA (6, 7). The same cleavage reaction has also been demonstrated in vitro using "purified" E3 protein (see below) and purified ribosomes (8,9). This in vitro demonstration of E3 action has strongly indicated that E3 acts directly on ribosomes rather than indirectly and that E3 itself is an RNase with a very stringent substrate specificity.In contrast to ES, the mechanism of action of E2 has not been clear. Since the initial discovery of DNA degradation in E2-treated sensitive cells (4), several authors have confirmed and extended the original observations on DNA cleavage reactions (10-13). However, others have suggested that the primary action of E2 is not on the cellular DNA, but on some other targets such as membranes (14) or tRNA (15).Since E2 has properties similar to E3 (3), and E3 causes ribosome inactivation in vitro, one might expect some kind of effects of E2 on cellular DNA in vitro, if DNA is in fact the primary target of E2. Several attempts have been made to demonstrate such effects in vitro using purified E2 (e.g., refs. 16-19). However, published results were either negative or too uncertain to allow a definitive conclusion regarding such direct effects.Recently, Jakes and Zinder observed that the "purified" ES Colicin E2* was separated from the immunity protein by dissociation of the complexed E2 with guanidine-HCl at about 4°. Lyophilized E2 was di...

Colicin E8, a DNase which indicates an evolutionary relationship between colicins E2 and E3

“…3, lanes d to g), and an excess amount of protein B completely inhibited the cleavage of DNA (lane g). These activities are almost the same as those found in the case of purified colicin E2 and its components, the naked E2 and the immunity protein (20,22,27). That is, proteins A and B are the naked E8 and the immunity protein, respectively; E8 protein A exhibits an apparently nonspecific DNA endonuclease activity which is inhibited by protein B.…”

Release of Immunity Protein Requires Functional Endonuclease Colicin Import Machinery

“…2C). Colicin E2 is known to be a nonspecific endonuclease (29). Refolded ColE2 was tested for DNase activity against linearized pUC18 plasmid DNA (Fig.…”

Homing in on the Role of Transition Metals in the HNH Motif of Colicin Endonucleases

“…Nuclease colicins require the outer membrane vitamin B 12 receptor BtuB as well as the porin OmpF and the Tol proteins (located in both the periplasm and inner membrane) for import. This complex machinery is needed to translocate the cytotoxic domain across both membranes of Escherichia coli in an energy-independent process in order that they reach their cytosolic targets, ribosomal RNA for colicin E3 (8,9), tRNA anticodons for colicin E5 (10), and chromosomal DNA for colicins E2 and E7-E9 (11)(12)(13)(14). Little is known of the mechanisms of membrane penetration by this family of nuclease colicins.…”