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
Ultraviolet light (UV) inhibits DNA replication in Eschericia coli and induces the SOS response, a set of survival-enhancing phenotypes due to derepression of DNA damage-inducible genes, including recA and umuDC. Recovery of DNA synthesis after UV irradiation ("induced replisome reactivation," or IRR) is an SOS function requiring RecA protein and postirradiation synthesis of additional protein(s), but this recovery does not require UmuDC protein [Khidhir, M. A., Casaregola, S. & Holland, I. B. (1985) Mol. Gen. Genet. 199, 133-140]. IRR occurs in strains carrying either recA718 (which does not reduce recombination, SOS inducibility, or UV mutagenesis) or umuC36 (which eliminates UV mutability), but not in recA718 umuC36 double mutants. In recA430 mutant strains, IRR does not occur whether or not functional UmuDC protein is present. IRR occurs in lexA-(Ind-) (SOS noninducible) strains if they carry an operator-constitutive recA allele and are allowed to synthesize proteins after irradiation. We conclude the following: (i) that UmuDC protein corrects or complements a defect in the ability of RecA718 protein (but not of RecA430 protein) to promote IRR and (ii) that in lexA(Ind-) mutant strains, IRR requires amplification of RecA+ protein (but not of any other LexA-repressed protein) plus post-UV synthesis of at least one other protein not controlled by LexA protein. We discuss the results in relation to the essential, but unidentified, roles of RecA and UmuDC proteins in UV mutagenesis.
Table 1 compares the growth of the double-mutant strain SC18-12 with that of each of the single-mutant strains and with that of a poLA12 strain from which recA has been deleted. ThepoLA12 recA 718 strain differed from each of the single mutants at 42°C in its inability to form colonies on nutrient agar and in its extremely slow growth on minimal medium. Similar results were obtained at 37°C. The polA12 recA-deleted strain was unconditionally inviable at 42°C and grew very poorly at 30°C. Figure 1 shows that the rate of DNA synthesis on nutrient agar, which even at 30°C was slightly slower in the recA718poLA12 strain SC18-12 than in either single mutant, decreased steadily in the double mutant after 1 h at 42°C. The rates of DNA synthesis in both single mutants were indistinguishable from those in the recA+ poU+ strain SC18-RP under the same conditions (data not shown).
A B S T R A C T Inherited deficiency of the enzyme adenosine deaminase (ADA) results in a syndrome of severe combined immunodeficiency (SCID). Children with ADA--SCID lack ADA in all cells and tissues. In contrast, a "partial" deficiency of ADA has been described in six immunologically normal children from four different "families." These children lack ADA in their erythrocytes but retain variable amounts of activity in their lymphoid cells.We have examined ADA activity in lymphoid line cells from four of these children, who are unrelated, for evidence of genetic heterogeneity. One child, who is Caucasian, has an enzyme with increased electrophoretic mobility, a diminished isoelectric point (pl 4.8 vs. Nl = 4.9) and very low activity (2.3 vs. Nl = 82.9±12.9 nmol/mg protein per min); as a second child has an enzyme with normal electrophoretic mobility but increased isoelectric point (pI = 5.0), markedly diminished heat stability at 56°C (tl/2 = 4.2' vs. Nl = 40') and low activity (12.1); a third has an enzyme with only diminished heat stability (t1/2 = 6.5'), no detectable abnormality in charge and almost normal activity (41.9); while the fourth exhibits only diminished ADA activity (25.0) with no striking qualitative abnormalities.Thus, we have found evidence for three different mutations at the structural locus for ADA in three of these individuals, (a) an acidic, low activity heat stable mutation (b) a basic, somewhat higher activity, heat labile mutation, and (c) a relatively normal activity
In recA718 lexA+ strains of Escherichia coli, induction of the SOS response requires DNA damage. This implies that RecA718 protein, like RecA+ protein, must be converted, by a process initiated by the damage, to an activated form (RecA*) to promote cleavage of LexA, the cellular repressor of SOS genes. However, when LexA repressor activity was abolished by a lexA-defective mutation [lexA(Def)], strains carrying the recA718 gene (but not recA+) showed strong SOS mutator activity and were able to undergo stable DNA replication in the absence of DNA damage (two SOS functions known to require RecA* activity even when cleavage of LexA is not necessary). k lysogens of recA718 lexA(Def) strains exhibited mass induction of prophage, indicative of constitutive ability to cleave A repressor. When the cloned recA718 allele was present in a lexA+ strain on a plasmid, SOS mutator activity and (-galactosidase synthesis under LexA control were expressed in proportion to the plasmid copy number. We conclude that RecA718 is capable of becoming activated without DNA damage for cleavage of LexA and k repressor, but only if it is amplified above its base-line level in lexA+ strains. At amplified levels, RecA718 was also constitutively activated for its roles in SOS mutagenesis and stable DNA replication. The nucleotide sequence of recA718 reveals two base substitutions relative to the recA+ sequence. We propose that the first allows the protein to become activated constitutively, whereas the second partially suppresses this capability.
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