Cloning and characterization of DNA damage-inducible promoter regions from Bacillus subtilis

Abstract

DNA damage-inducible (din) (16,41). Regulation of the SOS system in E. coli is controlled by the products of the recA and lexA genes. The RecA protein has many functions in E. coli and is involved in the processes of recombination, DNA repair, and mutagenesis (31,41,45). The LexA protein is a repressor of as many as 20 unlinked, coordinately regulated loci which include the recA and lexA genes themselves (19,41). Following exposure of E. coli to agents that alter DNA structure or interfere with DNA replication (such as UV radiation, mitomycin, nalidixic acid, etc.), an inducing signal is generated. (4,7,17,34,35), and the UmuD protein (2, 39). As levels of LexA repressor decline, damage-inducible loci are derepressed, resulting in expression of'the physiological phenomena that compose the SOS response (16, 41).The SOS system of E. coli has served as a model for the study of similar inducible DNA repair systems in other gram-negative bacteria (14,36,37,43,44 and essentially error free (7a). This contrasts with W reactivation in E. coli, which is capable of repairing a variety of DNA lesions by an error-prone mechanism (32). Furthermore, while induction of all SOS phenomena in E. coli is dependent upon a functional RecA protein, filamentation in B. subtilis is a RecA-independent response (21). Finally, the SOB system in B. subtilis is developmentally regulated. As B. subtilis differentiates into the physiological state of natural competence (6), SOB phenomena are spontaneously induced in the absence of externally generated DNA damage (20,23,47,49,50).DNA damage-inducible loci in B. subtilis were first identified by using transposon-mediated gene fusions (20). Tn917-lacZ transposon insertions within din loci were isolated from a library of insertions by selecting those fusions that induced expression of the lacZ reporter gene after exposure to DNA-damaging agents (20). Fifteen independently isolated din gene transposon insertions were genetically mapped and localized to three loci (dinA, dinB, and dinC) on the B. subtilis chromosome (11). As mentioned above, induction of all three din loci was demonstrated to be dependent upon a functional RecA protein (20). In order to elucidate the mechanisms that regulate damage-inducible gene expression in B. subtilis, we have cloned and sequenced DNA fragments that contain the dinA, dinB, and dinC promoter regions. Described here is our initial characterization of these cloned din promoter regions and the identification of a putative SOB operator sequence.

Characterization of DinR, the Bacillus subtilis SOS repressor

“…and GBE180 (DH5␣ pcnB1) were used for routine cloning and maintenance of plasmids. GBE180 (which maintains the normally high-copy-number pUC plasmids at low copy number) was kindly provided by G. Barcak (3,4). Plasmids precA50.46, precA57.11, and pSJ02 contain the same 202-bp fragment from recA, except that each carries a single point mutation in the putative SOS box.…”

“…A number of damage inducible (din) genes have been isolated, mapped, cloned, and sequenced by reporter gene technology (20). These studies revealed a consensus sequence, GAAC-N 4 -GTTC (previously termed the Cheo box), that was found in the promoter region of each identified din gene (3). Deletion analysis of the consensus sequence provided further evidence that the sequence and/or adjacent regions are essential to the proper SOS regulation of each of the din genes (5).…”

Role of the Nfo and ExoA Apurinic/Apyrimidinic Endonucleases in Repair of DNA Damage during Outgrowth of Bacillus subtilis Spores

“…Consequently, mutations in genes encoding NER components, particularly uvrA or uvrC, make B. subtilis cells more sensitive to several factors, including UV-C light, mitomycin C, and H 2 O 2 (40,41). In B. subtilis the expression of genes involved in DNA repair, including some genes of the NER pathway, is induced by DNA damage and during competence (3,8,9). This response, which may well be triggered by single-stranded DNA, is called the SOS response and is controlled by RecA and the transcriptional repressor DinR (18,38).…”

Clp-dependent proteolysis of the LexA N-terminal domain in Staphylococcus aureus

“…Repression of SOS-regulated genes in the absence of DNA damage is mediated by LexA binding to operator sequences (SOS boxes, or Cheo boxes in Bacillus subtilis) (Cheo et al, 1991;Little et al, 1981). LexA binds to the SOS boxes as a dimer with each monomer consisting of an N-terminal DNA-binding domain (NTD) and a C-terminal dimerization domain (CTD) separated by a hinge region (Flynn et al, 2001).…”