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

Skaliter, Rami; Bergstein, Moshe; Livneh, Zvi

doi:10.1074/jbc.271.5.2491

Cited by 10 publications

(8 citation statements)

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“…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. Here and in two companion studies (27,28), we report that UV irradiation induces a shorter form of the ␤ subunit that functions in vitro as an alternative DNA polymerase clamp. We suggest that this protein functions in DNA synthesis associated with post-UV recovery in E. coli.…”

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confidence: 82%

A Smaller Form of the Sliding Clamp Subunit of DNA Polymerase III Is Induced by UV Irradiation in Escherichia coli

Skaliter

Paz-Elizur

Livneh

1996

Journal of Biological Chemistry

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The ␤ subunit of DNA polymerase III holoenzyme of Escherichia coli is a 40.6-kDa protein that functions as a sliding DNA clamp (Stukenberg, P. T., StudwellVaughan, P. S., and O'Donnell, M. (1991) J. Biol. Chem. 266, 11328 -11334). It is responsible for tethering the polymerase to DNA and endowing it with the high processivity required for DNA replication. Here and in a companion study (Paz-Elizur, T., Skaliter, R., Blumenstein, S., and Livneh, Z. (1996) J. Biol. Chem. 271, 2482-2490) we report that the dnaN gene, encoding the ␤ subunit, contains an internal in-frame gene, termed dnaN*, that encodes a smaller form of the ␤ subunit. The novel 26-kDa protein, termed ␤*, is UV-inducible, and when overexpressed from a plasmid under an inducible promoter, it increases up to 6-fold the UV resistance of E. coli cells. These findings suggest that the ␤* protein functions in a reaction associated with DNA repair or recovery of DNA replication in UV-irradiated cells.UV irradiation of Escherichia coli cells produces in DNA primarily cyclobutyl pyrimidine dimers and pyrimidinepyrimidone (6 -4) 1 adducts that are responsible for most of the mutagenic and inactivating effects of UV irradiation (1). The immediate cellular response to UV irradiation is an arrest of chromosome replication in order to allow a period of elimination of DNA damage by DNA repair mechanisms (2). Many of the genes known to be involved in these processes, such as uvrA, uvrB, recA, umuD, and umuC are regulated by the SOS regulatory network, whose primary function is to help the cell in coping with DNA damage (3, 4). However, UV irradiation induces also heat shock genes (5) and other genes (6) which affect the post-UV physiology of the cell.We have previously examined in detail the replication of UV-irradiated DNA with purified proteins (7-10). These studies revealed that the ␤ subunit of DNA polymerase III holoenzyme, the major replicase of the E. coli chromosome (11), limits the ability of the purified polymerase to bypass UV lesions during in vitro replication of UV-irradiated single-stranded DNA (10). 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 (12). 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. Here and in two companion studies (27, 28), we report that UV irradiation induces a shorter form of the ␤ subunit that functions in vitro as an alternative DNA polymerase clamp. We suggest that this protein functions in DNA synthesis associated with post-UV recovery in E. coli. MATERIALS AND METHODSBacterial Strains-The following E. coli K-12 strains were used in this study: MC4100(⌬(argF-lac)205 araD139 rspL150 thiA1 relA1 flb5301 deoC1 ptsF25 rbsR); MC4100XL, same as MC4100 but also FЈ::Tn10 lacI q lacZ⌬M15 proAB; AB1157XL (argE3 his4 leuB6 proA2 thr1 ara14 galK2 lacY1 mtl1 xyl5 thi1 tsx33 rpsL31 supE44 FЈ::Tn10 lacI q lacZ⌬M15 proAB). Plasmids-Plas...

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confidence: 82%

A Smaller Form of the Sliding Clamp Subunit of DNA Polymerase III Is Induced by UV Irradiation in Escherichia coli

Skaliter

Paz-Elizur

Livneh

1996

Journal of Biological Chemistry

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“…␤* was purified from an overproducer strain that was constructed in our laboratory (49). Anti-␤ antibodies were affinitypurified on ␤* immobilized on nitrocellulose as previously described (48).…”

Section: Methodsmentioning

confidence: 99%

“…The intact ␤ subunit forms a ␤ 2 ring-shaped sliding DNA clamp, that confers high processivity on DNA polymerase III holoenzyme by tethering it to the DNA (41,42). As shown in a companion study (49), ␤* forms an alternative DNA clamp for DNA polymerase III that may have a specialized function connected to DNA synthesis in the UVirradiated cell. The increase in UV resistance caused by overproducing ␤* is consistent with such a model (48).…”

Section: Dnan* Gene Expression and Regulationmentioning

confidence: 99%

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

Paz-Elizur

Skaliter

Blumenstein

et al. 1996

Journal of Biological Chemistry

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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...

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“…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). Furthermore, in vitro studies have demonstrated that ␤* can modestly enhance the processivity of Pol III*, a form of Pol III containing all subunits except the ␤ clamp (49).…”

Section: Discussionmentioning

confidence: 99%

“…Biochemical characterization of ␤* indicates that it can form a trimer in solution (49). Furthermore, in vitro studies have demonstrated that ␤* can modestly enhance the processivity of Pol III*, a form of Pol III containing all subunits except the ␤ clamp (49). However, despite these detailed genetic and biochemical analyses, the physiological role of ␤* is presently unknown.…”

Section: Discussionmentioning

confidence: 99%

Replicative DNA Polymerase

Encyclopedia of Cancer

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The Escherichia coli umuDC gene products encode DNA polymerase V, which participates in both translesion DNA synthesis (TLS) and a DNA damage checkpoint control. These two temporally distinct roles of the umuDC gene products are regulated by RecA-single-stranded DNA-facilitated self-cleavage of UmuD (which participates in the checkpoint control) to yield UmuD (which enables TLS). In addition, even modest overexpression of the umuDC gene products leads to a cold-sensitive growth phenotype, apparently due to the inappropriate expression of the DNA damage checkpoint control activity of UmuD 2 C. We have previously reported that overexpression of the proofreading subunit of DNA polymerase III suppresses umuDC-mediated cold sensitivity, suggesting that interaction of with UmuD 2 C is important for the DNA damage checkpoint control function of the umuDC gene products. Here, we report that overexpression of the ␤ processivity clamp of the E. coli replicative DNA polymerase (encoded by the dnaN gene) not only exacerbates the cold sensitivity conferred by elevated levels of the umuDC gene products but, in addition, confers a severe cold-sensitive phenotype upon a strain expressing moderately elevated levels of the umuD C gene products. Such a strain is not otherwise normally cold sensitive. To identify mutant ␤ proteins possibly deficient for physical interactions with the umuDC gene products, we selected for novel dnaN alleles unable to confer a cold-sensitive growth phenotype upon a umuD C-overexpressing strain. In all, we identified 75 dnaN alleles, 62 of which either reduced the expression of ␤ or prematurely truncated its synthesis, while the remaining alleles defined eight unique missense mutations of dnaN. Each of the dnaN missense mutations retained at least a partial ability to function in chromosomal DNA replication in vivo. In addition, these eight dnaN alleles were also unable to exacerbate the cold sensitivity conferred by modestly elevated levels of the umuDC gene products, suggesting that the interactions between UmuD and ␤ are a subset of those between UmuD and ␤. Taken together, these findings suggest that interaction of ␤ with UmuD 2 C is important for the DNA damage checkpoint function of the umuDC gene products. Four possible models for how interactions of UmuD 2 C with the and the ␤ subunits of DNA polymerase III might help to regulate DNA replication in response to DNA damage are discussed.

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

Cited by 10 publications

References 40 publications

A Smaller Form of the Sliding Clamp Subunit of DNA Polymerase III Is Induced by UV Irradiation in Escherichia coli

A Smaller Form of the Sliding Clamp Subunit of DNA Polymerase III Is Induced by UV Irradiation in Escherichia coli

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

Replicative DNA Polymerase

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