Inactivation and proteolytic cleavage of phage lambda repressor in vitro in an ATP-dependent reaction.

Abstract

We have reproduced in vitro the inactivation of bacteriophage X repressor that occurs in vivo when a A lysogen is treated with agents such as ultraviolet radiation that attack DNA. ATP and a divalent cation are required for the inactivation reaction. The ind-repressor is insensitive to the inactivation mechanism. A proteolytic cleavage of repressor accompanies inactivation in vitro, as it does in vivo.A A prophage is induced to grow when the lysogenic bacterium that carries it receives any of several treatments that lead to inactivation of the A repressor. The primary effect of most inducing treatments, such as subjecting cells to ultraviolet irradiation or growing cells in the presence of the antibiotic mitomycin C, is to alter the structure of DNA or to interrupt its synthesis. In addition to the induction of temperate phages, such attacks upon the bacterial genome provoke other specific responses including: the activation of an important pathway for repair of damaged DNA; an increase in mutagenesis that is observed primarily during repair of damaged DNA; and an inhibition of cell division (1, 2). This set of responses has been termed "the SOS functions" (2) because it appears both to assist the bacterium in countering a threat to its genome and to allow a prophage to escape its endangered host.The biochemical pathways that lead to the expression of SOS functions are obscure. It is known that there is a common metabolic step involving the products of the Escherichia coli recA (2, 3) and lexA (4, 5) genes, because lesions in either gene can modify or prevent the expression of all SOS functions. The inactivation of A repressor is one specific biochemical event in SOS induction that has been identified. Because this modification may represent a biochemical mechanism common to the expression of all SOS functions, we have sought to understand how it occurs.We report here that inactivation of the specific DNA binding activity of A repressor can be detected in vitro. This inactivation occurs in an ATP-dependent reaction during incubation of A repressor in an extract of cells of an E. coli mutant that expresses SOS functions constitutively. In contrast, the repressor is stable in extracts from control bacteria that do not display constitutive SOS expression. Furthermore, a proteolytic cleavage identical to that which we have found to occur in vivo (6) Growth of Bacteria and Phage and Preparation of Extracts. The sources of the bacterial and bacteriophage strains are shown in Table 1. Bacteria were grown and extracts were prepared according to the procedure of Schekman et al. (10), except that the cells were collected by centrifugation at 40. Extracts used as a source of radioactive repressor were prepared by the following modification. A 5-ml culture of bacteria was grown to an A550 of 0.5 in a low-sulfur medium (6) in the presence of 35SO4 (200 uCi/ml). The cells were chilled, centrifuged, suspended in 1.0 ml of chilled 0.05 M Tris-HCI (pH 7.5), centrifuged again, suspended in 100 Il of 0.05 M Tris-HCI (pH ...

Characterization of the Pseudomonas aeruginosa recA analog and its protein product: rec-102 is a mutant allele of the P. aeruginosa PAO recA gene

“…Damage to DNA causes the RecA protein to become activated to a condition in which it promotes the rapid cleavage of the lexA-encoded repressor protein, relieving repression of the SOS network (47). Activated RecA protein also promotes the cleavage of the bacteriophage lambda cI repressor, causing induction of the lambda prophage to lytic growth (36,37).…”

Induction by UV light of the SOS function sfiA in Escherichia coli strains deficient or proficient in excision repair

“…After DNA damage, induction of all of these functions depends on the activation of the recA+ gene product to a protease able to cleave the lexA+ gene product (10), the general repressor of SOS functions, and the lambda cI+ gene product (16), the repressor of phage lambda. The SOS functions appear to contribute to the repair or processing of UV-induced DNA damage (11).…”

Cloning and characterization of the c1 repressor of Pseudomonas aeruginosa bacteriophage D3: a functional analog of phage lambda cI protein

“…The P. aeruginosa recA gene product is also capable of allowing induction of lambda prophage from recA mutants of E. coli (27,28). For E. coli, the induction of lambda prophage after exposure to DNA-damaging agents is known to be the result of the specific cleavage of the lambda cI repressor promoted by an activated form of the RecA protein (29,36,38,39). The data available to date suggest that the P. aeruginosa RecA protein may be responsible for the induction of D3 prophage by a similar mechanism.…”

“…For phage lambda of Escherichia coli, these functions are supplied by the product of the cI gene (25,26). In E. coli the induction by DNAdamaging agents of the prophages of lambda and related viruses is initiated by the specific cleavage of the repressor of vegetative functions promoted by an activated form of the recA gene product (36,38,39). This cleavage takes place at a unique Ala-Gly bond within the repressor protein (29).…”