RecA protein of Escherichia coli has a third essential role in SOS mutator activity

Abstract

The DNA damage-inducible SOS response of Escherichia coli includes an error-prone translesion DNA replication activity responsible for SOS mutagenesis. In certain recA mutant strains, in which the SOS response is expressed constitutively, SOS mutagenesis is manifested as a mutator activity. Like UV mutagenesis, SOS mutator activity requires the products of the umuDC operon and depends on RecA protein for at least two essential activities: facilitating cleavage of LexA repressor to derepress SOS genes and processing UmuD protein to produce a fragment (UmuD') that is active in mutagenesis. To determine whether RecA has an additional role in SOS mutator activity, spontaneous mutability (tryptophan dependence to independence) was measured in a family of nine lexA-defective strains, each having a different recA allele, transformed or not with a plasmid that overproduces either UmuD' alone or both UmuD' and UmuC. The magnitude of SOS mutator activity in these strains, which require neither of the two known roles of RecA protein, was strongly dependent on the particular recA allele that was present. We conclude that UmuD'C does not determine the mutation rate independently of RecA and that RecA has a third essential role in SOS mutator activity. SOS mutagenesis is due to an error-prone mode of DNA replication expressed in wild-type Escherichia coli after exposure to UV light or to chemical agents that block DNA replication and induce the SOS response. In certain mutant strains in which the SOS response is expressed constitutively, SOS mutagenesis is manifested as a mutator activity by which spontaneous mutation rates are elevated as much as 50-fold. In either case, SOS mutagenesis depends on the products of the recA, lexA, and umuDC genes. RecA and LexA proteins regulate the SOS response, and UmuDC proteins are believed to be essential for the actual mutagenic event (for reviews, see references 36 and 37).RecA protein has two essential roles in SOS mutagenesis. One is its regulatory role. DNA damage generates a signal that results in an altered form of RecA protein (RecA*). RecA* causes the proteolytic cleavage of LexA, the repressor of some 20 genes composing the SOS regulon, including recA itself and the umuDC operon (for reviews, see references 19 and 28). Amplification of UmuDC is necessary but not sufficient for SOS mutagenesis, as shown by the absence or weak expression of SOS mutator activity in recA+ strains carrying a lexA(Def) mutation that inactivates LexA. Such strains become strong mutators only if the recA+ allele is replaced by recA441 or recA730, which encode spontaneously activated RecA proteins, indicating that RecA* has an essential role in SOS mutagenesis other than its antirepressor function (la, 11, 38). It has recently been shown that RecA* promotes the proteolytic cleavage of UmuD protein both in vitro (5) and in vivo (32) and that only the larger COOH-terminal fragment (UmuD') is active in UV mutagenesis, whereas the unprocessed UmuD protein is not (27). SOS

Mechanism of DNA polymerase II-mediated frameshift mutagenesis

“…Current models of TLS propose that Pol V is positioned at the lesion site by means of a RecA͞single-stranded DNA filament (44,45). Our data suggest that the LT is available for Pol II extension (in its slipped conformation) in a reaction that competes with the Pol V͞RecA* pathway.…”

Isolation and characterization of novel plasmid-encoded umuC mutants

“…Umudependent error-prone translesion DNA synthesis is believed to occur as part of a multiprotein complex that requires protein interactions between UmuC and UmuD' (57), RecA (2,20,21,50), and probably subunits of DNA polymerase III holoenzyme (19,27,43,51). It is conceivable that the umuC mutants isolated here are defective in some, if not all, of the aforementioned interactions.…”

“…The ability of Escherichia coli DNA polymerase III holoenzyme to replicate across bulky misinstructional lesions in DNA, such as cyclobutane pyrimidine dimers or 6-4 pyrimidinepyrimidone photoproducts, is dependent upon the functional activity of the RecA and UmuD'C proteins (4,14,43,50,58). In addition, studies with a defined lesion in phage M13 DNA have shown that the extent and mutagenic consequences of translesion DNA synthesis vary and depend upon the specific type of DNA lesion encountered (3,4,25,(32)(33)(34).…”

A new class of errant DNA polymerases provides candidates for somatic hypermutation

“…Many laboratories are actively pursuing the identi¢cation of key transcription factors that occupy the E i and E 3' enhancers during the antigen stimulated phase in B cells (Reya & Grosschedl 1998). The transcription factors Oct-2 (Tang & Sharp 1999), TFE3 (Merrell et al 1997), C/EBPb (Hatada et al 2000) and the co-activator OBF-1/OCA-B (Schubart et al 2000) are thought to act speci¢cally during this stage, but many others remain unidenti¢ed. De¢ning a line between transcription activation and hypermutation targeting will be a crucial step to elucidate the components of the targeting mechanism for somatic hypermutation.…”

“…First and foremost is the targeting mechanism, which for Pol V is mediated by the single-stranded DNAbinding protein SSB (P. Pham and M. F. Goodman, unpublished data) and RecA (Sweasy et al 1990;Bailone et al 1991;Frank et al 1993). Without RecA, Pol V has a very low probability of using a DNA template, a feature that limits Pol V activity only to areas of severe replication fork blockage (Tang et al 2000).…”