β*, a UV-inducible Smaller Form of the β Subunit Sliding Clamp of DNA Polymerase III of Escherichia coli

Abstract

The 40.6-kDa ␤ subunit of DNA polymerase III of Escherichia coli is a sliding DNA clamp responsible for tethering the polymerase to DNA and endowing it with high processivity (Stukenberg, P. T., Studwell-Vaughan, P. S., and O'Donnell, M. (1991) J. Biol. Chem. 266, 11328 -11334). UV irradiation of E. coli induces a smaller 26-kDa form of the ␤ subunit, termed ␤*, that, when overproduced from a plasmid, increases UV resistance of E. coli (Skaliter, R., Paz-Elizur, T., and Livneh, Z. (1996) J. Biol. Chem. 271, 2478 -2481). Here we show that this protein is synthesized from a UV-inducible internal gene, termed dnaN*, that is located in-frame inside the coding region of dnaN, encoding the ␤ subunit. The initiation codon and the Shine-Dalgarno sequence of dnaN* were identified by site-directed mutagenesis. The dnaN* transcript was shown to be induced upon treatment with nalidixic acid, and transcriptional dnaN*-cat gene fusions were UV inducible, suggesting induction of dnaN* at the transcriptional level. Analysis of translational dnaN*-lacZ gene fusions revealed that UV induction was abolished in strains carrying the recA56, lexA3, or ⌬rpoH mutations, indicating involvement of both SOS and heat shock stress responses in the induction process. Expression of dnaN* represents a strategy of producing several proteins with related functional domains from a single gene.UV irradiation of Escherichia coli cells leads to the formation of both mutagenic and inactivating DNA lesions (1). The cells respond by an immediate arrest of DNA replication, followed by a period of extensive DNA repair, that operates to eliminate DNA damage in order to prevent replication obstacles (2). These processes are controlled primarily by the SOS stress regulon, which involves more than 20 genes that are commonly regulated by the LexA repressor and the RecA activator (3, 4). However, UV irradiation induces change also in heat shock genes (5) and other genes (6) which affect the post-UV physiology of the cell. We have previously found that the ␤ subunit of DNA polymerase III holoenzyme, the major replicase of the E. coli chromosome (7), limits the ability of the purified polymerase to replicate UV-irradiated single-stranded DNA (8). Consistent with this result, overproduction of the ␤ subunit from a plasmid caused a reduction in UV resistance and in UV mutagenesis of E. coli cells (9).This involvement of the ␤ subunit in UV irradiation effects prompted us to examine whether it may be present in a different form in UV-irradiated cells. We found that upon UV irradiation a smaller form of the ␤ subunit, termed ␤*, was induced. When overproduced from a plasmid under the inducible lac promoter, ␤* caused up to a 6-fold increase in UV resistance of E. coli cells, suggesting a role in recovery from UV damage, e.g. by involvement in DNA repair or reactivation of DNA replication (48).Smaller derivatives of proteins that are found in cells are frequently generated by proteolysis, as in the case of the mutagenesis protein UmuDЈ that is formed from UmuD by spe...

Genetic Interactions between the Escherichia coli umuDC Gene Products and the β Processivity Clamp of the Replicative DNA Polymerase

“…In addition, two (Q61K and S107L) of the eight missense mutations are located in the first structural domain of ␤ (domain I), which is lacking in an alternative form of the ␤ clamp referred to as ␤* (see Fig. 2 and Discussion for further details) (39,50).…”

“…2). Because each ␤* protomer corresponds almost exactly to the C-terminal two-thirds of the dnaN gene product (39,50), it retains the C-terminal two structural domains but is missing the N-terminal domain I. It is interesting that two of the mutations we identified that reduce interactions with the umuDC gene products, Q61K and S107L, both reside within the Nterminal domain (domain I), which is lacking in ␤*.…”

“…Following exposure of E. coli to various DNA-damaging agents, the level of transcription of the dnaN gene increases (42,60). In addition, expression of a truncated form of ␤ corresponding almost exactly to the C-terminal two-thirds of the protein is also induced (39,50). This protein, termed ␤*, is expressed, in a recA-and lexA-dependent fashion, from a promoter located within the coding region of the dnaN gene (Fig.…”

“…2) (39, 50). The gene for ␤*, which is contained entirely within the dnaN ϩ gene, has been termed dnaN* (39,50). Biochemical characterization of ␤* indicates that it can form a trimer in solution (49).…”

Transcription of genes encoding DNA replication proteins is coincident with cell cycle control of DNA replication in Caulobacter crescentus

“…In E. coli, at least two mechanisms, either SOS response dependent (for dnaA, dnaN, and dnaQ [43,60]) or SOS independent (dnaB [23]), appear to exist to induce expression of DNA replication genes upon detection of DNA damage. In- deed, the E. coli dnaN gene was found to encode a shorter polypeptide (␤*) whose expression is specifically induced upon UV treatment of the cells (40,55). This alternate form of the sliding clamp imparts resistance to the UV treatment in an undefined manner.…”