Summary
The mucAB operon carried on plasmid pKM101, which is an analogue of the umuDC operon of Escherichia coli, is involved in UV mutagenesis and mutagenesis induced by many chemicals. Mutagenesis dependent on either the umuDC or mucAB operon requires the function of the recA gene and is called SOS mutagenesis. By treating the cell with agents that damage DNA, RecA protein is activated by conversion into a form (RecA*) that mediates proteolytic cleavage of the LexA repressor and derepresses the SOS genes including mucAB. Since UmuD protein is proteolytically processed to an active form (UmuD*) in a RecA*-dependent fashion, and MucA shares extensive amino acid homology with UmuD, we examined whether MucA is similarly processed in the cell, using antiserum against a LacZ′-′MucA fusion protein. Like UmuD, MucA protein is indeed proteolytically processed in a RecA*-dependent fashion. In recA430 strains, MucAB but not UmuDC can mediate UV mutagenesis. However, MucA was not processed in the recA430 cells treated with mitomycin C. We constructed, by site-directed mutagenesis, several mutant mucA genes that encode MucA proteins with alterations in the amino acids flanking the putative cleavage site (Ala25-Gly26). MucA(Cys25) was processed and was as mutagenically active as wild-type MucA; MucA(Asp26) and MucA(Cys25,Asp26) were not processed, and were mutagenically inactive; MucA-(Thr25) was not processed, but was mutagenically as active as wild-type MucA. The mutant mucA gene that encoded the putative cleavage product of MucA was as active as mucA + in UV mutagenesis. These results raise the possibility that both the nascent MucA and the processed product are active in mutagenesis.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Amann E, Brousius J (1985) “ATG vector” for regulated highlevel expression of cloned genes in Escherichia coli. Gene 40:183–190
Blanco M, Herrera G, Collado P, Rebollo J, Botella LM (1982) Influence of RecA protein on induced mutagenesis. Biochimie 64:633–636
Blanco M, Herrera G, Aleixandre V (1986) Different efficiency of UmuDC and MucAB proteins in UV light induced mutagenesis in Escherichia coli. Mol Gen Genet 205:234–239
Burckhardt SE, Woodgate R, Scheuermann RH, Echols H (1988) The UmuD mutagenesis protein of Escherichia coli: overproduction, purification and cleavage by RecA. Proc Natl Acad Sci USA 85:1811–1815
Carroll SB, Laughon A (1987) Production and purification of polyclonal antibodies to the foreign segment of β-galactosidase fusion proteins. In: Glover DM (ed) DNA cloning, vol III. IRL Press, Oxford, pp 89–111
Davis RW, Botstein D, Roth JR (1980) Advanced bacterial genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Elledge SJ, Walker GC (1983) Proteins required for ultraviolet light and chemical mutagenesis: identification of the products of the umuC locus of Escherichia coli. J Mol Biol 164:175–192
Ennis DG, Fisher B, Edmiston S, Mount DW (1985) Dual role for Escherichia coli RecA protein in SOS mutagenesis. Proc Natl Acad Sci USA 82:3325–3329
Foster LP, Groopman JD, Eisenstadt E (1988) Identification of base substitution mutation by aflatoxin B1 is MucAB dependent in Escherichia coli. J Bacteriol 170:3415–3420
Gimble FS, Sauer RT (1986) Repressor inactivation: properties of purified ind − proteins in the autodigestion and RecA-mediated cleavage reactions. J Mol Biol 192:39–47
Iwasaki H, Shiba T, Nakata A, Shinagawa H (1988) Activation of UmuD protein for SOS mutagenesis by proteolytic processing mediated by RecA*. In: Friedberg EC, Hanawalt PC (eds) Mechanisms and consequences of DNA damage processing. Alan R. Liss, New York, pp 461–469
Kato T, Shinoura Y, Templin A, Clark AJ (1980) Analysis of ultraviolet light-induced suppressor mutation in the strain of Escherichia coli K-12 AB1157: an implication for molecular mechanisms of UV mutagenesis. Mol Gen Genet 180:283–291
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Little JW, Mount DW (1982) The SOS regulatory system of Escherichia coli. Cell 29:11–22
Markham BE, Little JW, Mount DW (1981) Nucleotide sequence of the lexA gene of Escherichia coli K-12. Nucleic Acids Res 9:4149–4161
Marcucci L, Gighani F, Battaglia PA, Bosi R, Sporeno E, Elli R (1986) Cellular response to DNA damage is enhanced by the pR plasmid in mouse cells and in Escherichia coli. Mol Cell Biol 6:586–592
Marsh L, Walker GC (1987) New phenotypes associated with mucAB: alteration of MucA sequence homologous to LexA cleavage site. J Bacteriol 169:1818–1823
Mount DW (1977) A mutant of Escherichia coli showing constitutive expression of the lysogenic induction and error-prone DNA repair pathways. Proc Natl Acad Sci USA 74:300–304
Nohmi T, Battista JR, Dodson LA, Walker GC (1988) RecA-mediated cleavage activates UmuD for mutagenesis: mechanistic relationship between transcriptional derepression and posttranslational activation. Proc Natl Acad Sci USA 85:1816–1820
Perry KL, Walker GC (1982) Identification of plasmid (pKM101)coded proteins involved in mutagenesis and UV resistance. Nature 300:278–281
Perry KL, Elledge SJ, Mitchell BB, Marsh L, Walker GC (1985) umuDC and mucAB operons whose products are required for UV light- and chemical-induced mutagenesis; UmuD, MucA and LexA proteins share homology. Proc Natl Acad Sci USA 82:4331–4335
Potter AA, Nestmann Earle R, Iyer VN (1984) Introduction of the plasmid pKM101-associated muc genes into Saccharomyces cerevisiae. Mutat Res 131:197–204
Rüther U, Müller-Hill B (1983) Easy identification of cDNA clones. EMBO J 2:1791–1794
Sancar A, Wharton RP, Seltzer S, Kacinski BM, Clarke ND, Rupp WD (1981) Identification of the uvrA gene product. J Mol Biol 148:45–62
Shinagawa H, Iwasaki H, Kato T, Nakata A (1988) RecA proteindependent cleavage of UmuD protein and SOS mutagenesis. Proc Natl Acad Sci USA 85:1806–1810
Strike P, Lodwick D (1987) Plasmid genes affecting DNA repair and mutation. J Cell Sci (Suppl) 6:303–321
Walker GC (1977) Plasmid (pKM101)-mediated enhancement of repair and mutagenesis: dependence on chromosomal genes in Escherichia coli. Mol Gen Genet 152:93–103
Walker GC (1984) Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol Rev 48:60–93
Witkin EM (1976) Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli. Bacteriol Rev 40:869–907
Witkin EM, Kogoma T (1984) Involvement of the activated form of RecA protein in SOS mutagenesis and stable DNA replication in Escherichia coli. Proc Natl Acad Sci USA 81:7539–7543
Witkin EM, MacCall JO, Volkert MR, Wermundsen IE (1982) Constitutive expression of SOS function and modulation of mutagenesis resulting from resolution of genetic instability at or near the recA locus of Escherichia coli. Mol Gen Genet 185:43–50
Woodgate R, Rajagopalan M, Lu C, Echoles H (1989) UmuC mutagenesis protein of Escherichia coli: purification and interaction with UmuD and UmuD′. Proc Natl Acad Sci USA 86:7301–7305
Author information
Authors and Affiliations
Additional information
Communicated by M. Sekiguchi
Rights and permissions
About this article
Cite this article
Shiba, T., Iwasaki, H., Nakata, A. et al. Proteolytic processing of MucA protein in SOS mutagenesis: Both processed and unprocessed MucA may be active in the mutagenesis. Molec. Gen. Genet. 224, 169–176 (1990). https://doi.org/10.1007/BF00271549
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00271549