Control elements located inside the coding sequence of dnaN, the gene encoding the ␤ subunit of DNA polymerase III holoenzyme, direct the synthesis of a shorter and UV-inducible form of the ␤ subunit (Skaliter, R., Paz-Elizur, T., and Livneh, Z. (1996) J. Biol. Chem. 271, 2278 -2281, and Paz-Elizur, T., Skaliter, R., Blumenstein, S., and Livneh, Z. (1996) J. Biol. Chem. 271, 2282-2290). The protein, termed ␤*, was overproduced using the phage T7 expression system, leading to its accumulation as inclusion bodies at 5-10% of the total cellular proteins. ␤* was purified in denatured form, followed by refolding to yield a preparation >95% pure. Denatured ␤* had a molecular mass of 26 kDa and contained two isoforms when analyzed by two-dimensional gel electrophoresis. The major isoform had a pI of 5.45, and comigrated with cellular ␤*. Size exclusion high performance liquid chromatography under nondenaturing conditions and chemical cross-linking experiments indicate that ␤* is a homotrimer. DNA synthesis by DNA polymerase III* was stimulated up to 10-fold by ␤*, primarily due to an increase in the processivity of polymerization. It is suggested that ␤* functions as an alternative sliding DNA clamp in a process associated with DNA synthesis in UV-irradiated cells.DNA-damaging agents in general, and UV radiation in particular, affect dramatically the physiology of Escherichia coli (1). These changes are regulated at the molecular level by several stress regulons, most notably the SOS and heat shock responses (2-4). The immediate response is a transient arrest of DNA replication (5-7) which provides time for the repair of the damaged DNA. DNA replication than recovers in a process that requires SOS-inducible proteins (5-7), but its mechanism is largely unknown. During the post-UV recovery period mutations are formed, primarily at sites of DNA damage. This mutagenesis pathway is believed to occur by polymerization through DNA lesions, a process termed bypass synthesis (7-9).A detailed biochemical analysis of in vitro replication of UV-irradiated or depurinated DNA with purified DNA polymerase III (Pol III) 1 holoenzyme (10 -13) led us to concentrate on the ␤ subunit of the polymerase and to conclude that it modulates UV mutagenesis in vivo (14) and bypass of UV lesions in vitro (15). The ␤ subunit is the major processivity factor of Pol III holoenzyme (16). It is a homodimer that forms a ring structure (17), and once loaded on DNA by the ␥ complex it functions as a sliding DNA clamp that tethers the polymerase to the DNA, thus endowing it with high processivity (18 -20). In companion studies (39, 40) we reported that in UV-irradiated E. coli cells a shorter form of the ␤ subunit, termed ␤*, is produced, which corresponds to the C-terminal two-thirds of the ␤ subunit. This study describes the overproduction, purification, and characterization of ␤* and its activity as an alternative processivity clamp for DNA polymerase III. MATERIALS AND METHODSPlasmids-Plasmid pSK11 that overproduces ␤* was constructed as follows. The s...

Exposure of Escherichia coli to UV irradiation or nalidixic acid, which induce both the SOS and heat shock responses, led to a 3-4-fold increase in the amount of the beta subunit of DNA polymerase III holoenzyme, as assayed by Western blot analysis using anti-beta antibodies. Such an induction was observed also in a delta rpoH mutant lacking the heat shock-specific sigma 32 subunit of RNA polymerase, but it was not observed in recA13 or lexA3 mutants, in which the SOS response cannot be induced. Mapping of transcription initiation sites of the dnaN gene, encoding the beta subunit, using the S1 nuclease protection assay showed essentially no induction of transcription upon UV irradiation, indicating that induction is regulated primarily at the post-transcriptional level. Analysis of translational gene fusions of the dnaN gene, encoding the beta subunit, to the lacZ reporter gene showed induction of beta-galactosidase activity upon UV irradiation of cells harboring the fusion plasmids. Elimination of a 5' flanking DNA sequence in which the dnaN promoters P1 and P2 were located, did not affect the UV inducibility of the gene fusions. Thus, element(s) present from P3 downstream were sufficient for the UV induction. The induction of the dnaN-lacZ gene fusions was dependent on the recA and lexA gene products, but not on the rpoH gene product, in agreement with the immunoblot analysis. The dependence of dnaN induction on the SOS regulators was not mediated via classical repression by the LexA repressor, since the dnaN promoter does not contain a sequence homologous to the LexA binding site, and dnaN mRNA was not inducible by UV light. This suggests that SOS control may be imposed indirectly, by a post-transcriptional mechanism. The increased amount of the beta subunit is needed, most likely, for increased replication and repair activities in cells which have been exposed to UV radiation.