Efficient repair of hydrogen peroxide-induced DNA damage by Escherichia coli requires SOS induction of RecA and RuvA proteins.

The survival of Escherichia coli following treatment with a low dose (1-3 mM) of hydrogen peroxide (H(2)O(2)) that causes extensive mode-one killing of DNA repair mutants is stimulated by the induction of the SOS regulon. Results for various mutants indicate that induction of recA and RecA protein-mediated recombination are critical factors contributing to the repair of H(2)O(2)-induced oxidative DNA damage. However, because DNA damage activates RecA protein's coprotease activity essential to cleavage of LexA repressor protein and derepression of all SOS genes, it is unclear to what extent induction of RecA protein stimulates this repair.

Differential cleavage of LexA and UmuD mediated by recA Pro67 mutants: implications for common LexA and UmuD binding sites on RecA.

In Escherichia coli, RecA-mediated cleavage of LexA repressor is a key regulatory event required for expression of SOS genes involved in the repair of DNA damage. RecA also mediates the cleavage of UmuD protein to UmuD, a form active in SOS mutagenesis. To determine whether LexA and UmuD have common binding determinants on RecA, we have compared the ability of several recA mutants to function in the cleavage of LexA versus UmuD in vivo.

Mutations at Pro67 in the RecA protein P-loop motif differentially modify coprotease function and separate coprotease from recombination activities.

The functional significance of residues in the RecA protein P-loop motif was assessed by analyzing 100 unique mutants with single amino acid substitutions in this region. Comparison of the effects on the LexA coprotease and recombination activities shows that Pro67 is unique among these residues because only at this position did we find substitutions that caused differential effects on these functions. One mutant, Pro67-->Trp, displays high constitutive coprotease activity and a moderate inhibitory effect on recombination functions.

Functional characterization of residues in the P-loop motif of the RecA protein ATP binding site.

We have introduced a large number of single amino acid substitutions at six positions that lie within the P-loop motif of the ATP binding site in the Escherichia coli RecA protein. The activity of each recA mutant was determined using genetic assays which assess the catalytic proficiency of the RecA protein for both homologous genetic recombination and recombinational repair of damaged DNA. The six residues displayed unique patterns of allowed versus non-allowed substitutions that define the functional and structural constraints at each position.

Orientation of the myelin proteolipid protein C-terminus in oligodendroglial membranes.

The topology of the integral membrane proteolipid protein (PLP) has important structural and functional implications for central nervous system myelin. To determine the orientation of the carboxyl-terminal portion of PLP, cultured mouse oligodendrocytes were probed with polyclonal antibodies raised against a synthetic terminal peptide corresponding to PLP residues 264-276 and with ten separate monoclonal antibodies that react with this region. Cells were examined by double-label indirect immunofluorescence for the presence of the PLP C-terminus and either oligodendrocyte-specific surface or intracellular antigens.