Mutagenic repair in Escherichia coli: products of the recA gene and of the umuD and umuC genes act at different steps in UV-induced mutagenesis.

Abstract

When excision-deficient Escherichia coli carrying umuC or umuD alleles were exposed to visible light several hours after ultraviolet irradiation, base-pair-substitution mutations were induced in these normally non-UV-mutable bacteria. It is argued that delayed photoreversal of pyrimidine dimers removes blocks to DNA replication and allows the "survival" and expression of misincorporated bases. A model for UV mutagenesis is proposed with two steps: (i) misincorporation opposite a photoproduct, which can be mediated directly by RecA protein, and (ii) bypass, only the latter process requiring umuD+ and umuC+ alleles. Basal levels of gene products are sufficient for at least some misincorporation events, although induced levels of umuD and umuC gene products are necessary for the bypass step. umuC bacteria containing the recA441 allele showed a greater yield of mutants, and those containing recA430 a reduced yield, following delayed photoreversal. The lexA51 allele (which results in constitutive derepression of RecA protein production) did not significantly alter the yield of mutants but caused them to appear marginally sooner in a recA441 umuC strain. These results emphasize that the nature of the RecA protein and not its concentration is paramount in determining the level of misincorporation. Experiments with recA441 umuC bacteria at 43°C and 30°C suggest that the misincorporation effect is unlikely to be attributable to cleavage of a DNA binding protein such as a repressor or a component of the polymerase complex. Moreover, misincorporation seems to occur without the need for induced synthesis of any other protein under recA control. Evidence consistent with these ideas has recently been published by Fersht and Knill-Jones (5), who showed that deoxynucleoside monophosphates, particularly dGMP, inhibited the proofreading activity of DNA polymerase III holoenzyme with a concomitant decrease in fidelity of DNA replication. In addition, RecA protein inhibited the proofreading activity of DNA polymerase III on synthetic templates containing mismatched 3' termini, and the inhibition was increased in the presence of dGMP or dAMP; it also decreased the fidelity of DNA replication in vitro. Even the presence of both purine deoxynucleoside monophosphates and RecA protein did not, however, enable DNA polymerase III to copy past pyrimidine dimers.We have dissected the process of UV mutagenesis into two steps (6). The first is a misincorporation event, presumably opposite a photoproduct such as a pyrimidine dimer, and the second requires the UmuC protein and allows DNA synthesis to continue beyond the misincorporation (bypass). When the pyrimidine dimer is removed by photoreversal some time after UV irradiation of a umuC strain, the bypass step is made redundant and mutations arising from misincorporations are able to "survive."In the present work, in which we use strains lacking excision repair, we show that umuD is also involved in the second step of dimer bypass and that induced levels of umuC and umuD gene product...

In vivo stability of the Umu mutagenesis proteins: a major role for RecA

“…Certain situations arise, however, whereby the damage fails to be repaired by error-free repair pathways and is instead processed via error-prone repair pathways. It is believed that this so-called "SOS mutagenesis" occurs directly as a result of the ability of UmuDЈC and RecA proteins to coerce DNA polymerase III to replicate across damage-induced misinstructional lesions with a concomitant reduction in replication fidelity (3,24,28,32,41,46).…”

The enhanced mutagenic potential of the MucAB proteins correlates with the highly efficient processing of the MucA protein

“…Although the E. coli UmuD and UmuC proteins have been purified (10,63), the biochemical mechanism by which these proteins act to facilitate mutagenesis is still unclear. Genetic experiments suggest that the UmuDC proteins may promote DNA chain elongation after a misincorporation event opposite DNA lesions (8,9). The occurrence of these mutagenic DNA repair genes is widespread; some are encoded by chromosomal genes like those of E. coli and Salmonella typhimurium (2, 18, 19, 37, 45, 50-52, 56), while others are found on extrachromosomal multidrug resistance plasmids (R plasmids) (24,32,33,36,53).…”

Levels of epsilon, an essential replication subunit of Escherichia coli DNA polymerase III, are controlled by heat shock proteins

“…Since reconstituted Pol III holoenzyme is poor at synthesizing past lesions in vitro (18), it appears to have to be modified in vivo to allow translesion synthesis. Both in vivo and in vitro experiments indicate that insertion of a base opposite a lesion is not rate limiting but that extension from the mispaired terminus is (6,18). The two modifications of Pol III that appear to be required are inhibition of the 3'-*5' exonuclease activity of epsilon to prevent the complex from stalling at the mispaired terminus (54) and improving the ability of the polymerase to extend from the mispaired terminus (2,6,18).…”

“…Both in vivo and in vitro experiments indicate that insertion of a base opposite a lesion is not rate limiting but that extension from the mispaired terminus is (6,18). The two modifications of Pol III that appear to be required are inhibition of the 3'-*5' exonuclease activity of epsilon to prevent the complex from stalling at the mispaired terminus (54) and improving the ability of the polymerase to extend from the mispaired terminus (2,6,18). A number of lines of evidence support the idea that RecA and UmuDC are involved in performing these alterations: (i) overproduction * Corresponding author.…”