The bacteriophage 434 repressor dimer preferentially undergoes autoproteolysis by an intramolecular mechanism

Abstract

Inactivation of the lambdoid phage repressor protein is necessary to induce lytic growth of a lambdoid prophage. Activated RecA, the mediator of the host SOS response to DNA damage, causes inactivation of the repressor by stimulating the repressor's nascent autocleavage activity. The repressor of bacteriophage lambda and its homolog, LexA, preferentially undergo RecA-stimulated autocleavage as free monomers, which requires that each monomer mediates its own (intramolecular) cleavage. The cI repressor of bacteriophage 434 preferentially undergoes autocleavage as a dimer specifically bound to DNA, opening the possibility that one 434 repressor subunit may catalyze proteolysis of its partner subunit (intermolecular cleavage) in the DNA-bound dimer. Here, we first identified and mutagenized the residues at the cleavage and active sites of 434 repressor. We utilized the mutant repressors to show that the DNA-bound 434 repressor dimer overwhelmingly prefers to use an intramolecular mechanism of autocleavage. Our data suggest that the 434 repressor cannot be forced to use an intermolecular cleavage mechanism. Based on these data, we propose a model in which the cleavage-competent conformation of the repressor is stabilized by operator binding.

FIG. 1.

Alignment of the sequences of the linkers and C-terminal domains of bacteriophage 434, P22, and λ repressors with the homologous regions of LexA and UmuD. The black background indicates positions where identical amino acids are found in at least three of the aligned sequences. The gray background indicates amino acids in the third and/or fourth position that are homologous to two identical residues. The alignment begins at the residues indicated. The positions of RecA cleavage and the active site serine and lysine are indicated by arrows.

FIG. 2.

In vitro autoproteolysis of the wild-type 434 repressor and the active (434^S126A) and cleavage (434^G89A) site mutant derivatives. The wild-type 434 repressor (200 ng) and its mutant derivatives were incubated in the absence of RecA at pH 7 (lanes 1 to 3) or pH 10.5 (lanes 4 to 5) for 24 h or in the absence (lanes 7, 9, and 11) or presence (lanes 8,10, 12) of active RecA at pH 7.5 (see Materials and Methods) for 4 h. Intact repressors and the cleavage products were fractionated on 15% Tris/Tricine gels and visualized by Western blotting using anti-434 repressor antibodies. The positions of the N- and C-terminal cleavage products are indicated in the middle. CTD, C-terminal domain; NTD, N-terminal domain.

FIG. 3.

Effect of added DNA on the ability of the wild-type 434 repressor and the active (434^S126A) and cleavage (434^G89A) site mutant derivatives of the 434 repressor to undergo intermolecular autocleavage. The wild-type 434 repressor (200 ng) and its mutant derivatives were incubated in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of excess 434 O_R1 DNA. Intact repressors and the cleavage products were fractionated on 15% Tris/Tricine gels and visualized by Western blotting using anti-434 repressor antibodies. The positions of the N- and C-terminal cleavage products are indicated on the left. CTD, C-terminal domain; NTD, N-terminal domain.

FIG. 4.

pH- and RecA-stimulated cleavage of mixtures of cleavage site (G89A) and active site (S126A) mutant 434 repressors. The wild-type repressor (200 ng) was incubated with active RecA for 4 h (lane 1) as a control. The cleavage site (G89A) and active site (S126A) mutant 434 repressors were mixed in various ratios (lanes 2 to 5, 7 to 10, and 12 to 15 contained 200 ng of 434^S126A plus [434^G89A; lanes 6, 11, and 16 contained 2,000 ng of 434^S126A plus 434^G89A) and were incubated at pH 10.5 for 24 h (lanes 2 to 6), in the presence of RecA for 4 h (lanes 7 to 11), or in the presence of RecA and a twofold molar excess of 434 O_R1 for 4 h (lanes 12 to 16). Intact repressors and the cleavage products were fractionated on 15% Tris/Tricine gels and visualized by Western blotting using anti-434 repressor antibodies. The positions of the intact repressor and the N- and C-terminal cleavage products are indicated on the left. CTD, C-terminal domain; NTD, N-terminal domain.

FIG. 5.

Autocleavage of the 434 repressor in vivo. Mid-log-phase cells containing plasmids that direct the expression of the wild-type and/or mutant repressors were induced for 1 h with 2 μM IPTG and grown for an additional 1 h in the absence or presence of 3.5 μg of mitomycin C and then processed for electrophoresis. The blot is a Western blot of the resulting products probed with antibodies directed against the 434 repressor. Cells contained only nonspecific DNA (lanes 1 to 8) or 434 O_R1 (lanes 9 to 12). The positions of the SOS-induced repressor fragments are indicated on the left (NTD, amino terminal domain; CTD, carboxyl-terminal domain).