DNA Unwinding Enzyme II of <i>Escherichia coli</i>

Abdel-Monem, Mahmoud M.; Chanal, Marie-Christine; Hoffmann‐Berling, Hartmut

doi:10.1111/j.1432-1033.1977.tb11780.x

Cited by 77 publications

(22 citation statements)

References 19 publications

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“…The UvrA, UvrB, and UvrC subunits of UvrABC excision nuclease were purified separately as described previously (1,16). The UvrD protein (DNA helicase II) was purified according to the original protocol of AbdelMonem et al (17) using as a starting material a strain carrying the uvrD gene on a multicopy plasmid (18). The detail of this purification procedure will be published elsewhere.…”

Section: Methodsmentioning

confidence: 99%

Effect of DNA polymerase I and DNA helicase II on the turnover rate of UvrABC excision nuclease.

Husain¹,

Houten²,

Thomas³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

215

151

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UvrABC excision nuclease (UvrA, UvrB, and In this communication using proteins purified to near homogeneity we demonstrate that helicase II alone has no effect on the initial rate of excision nuclease activity and only a small effect on its turnover. Similarly we find that pol I alone does not stimulate the ABC excision nuclease. However, the combination of the two causes the enzyme to turn over such that in a complete excision repair system (UvrABC excision nuclease, helicase II, pol I, DNA ligase) the rate of the removal of nucleotide adducts approaches 0.08 adduct per UvrABC excision nuclease complex per min. This value is in reasonable agreement with the 0.12-0.50 min-1 that can be calculated from the in vivo data published by several workers (5, 13-15). MATERIALS AND METHODSEnzymes. The UvrA, UvrB, and UvrC subunits of UvrABC excision nuclease were purified separately as described previously (1,16 Substrates. Radiolabeled or unlabeled pBR322 DNA was prepared by standard methods and superhelical DNA was purified through two successive ethidium bromide/CsCl density gradient centrifugations (19). The tritiated DNA used in the incision and filter binding assays had a specific activity of 1.5 x 105 cpm/,ug. The substrate for these assays was prepared by irradiating the DNA with 254-nm UV light that produced 10 lethal hits per molecule as measured by the transformation assay (19). The DNA used for the incision and filter binding assays contained 70-75% superhelical molecules. The substrate for the excision assay was prepared as described (20).Assays. The activity and turnover rate of UvrABC excision nuclease were measured by three separate assays: incision, excision, and filter binding. All the assays were conducted in a nucleotide-excision-repawr buffer which contained 50 mM Tris-HCI, pH 7.4/50 mM KCl/10 mM MgCl2/2 mM ATP/33 ,uM each of dATP, dGTP, dTTP, and dCTP/10 mM dithiothreitol/10% glycerol (vol/vol)/bovine serum albumin at 50 ,g/ml plus DNA and repair proteins at the amounts indicated. The incision assay measures the conversion of superhelical DNA to open circles and has been described elsewhere (19 6774The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Section: Methodsmentioning

confidence: 99%

Effect of DNA polymerase I and DNA helicase II on the turnover rate of UvrABC excision nuclease.

Husain¹,

Houten²,

Thomas³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

215

151

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“…The purified uvrA, uvrB, and uvrC proteins were isolated by methods briefly described previously (10). The helicase II (uvrD) from E. coli was purified according to already described methods (19,24) and the E. coli DNA polymerase I, the Klenow fragment of DNA polymerase I, and bacterial alkaline phosphatase were purchased from Bethesda Research Laboratories. T4 DNA ligase was obtained from Collaborative Research and the E. coli DNA ligase was obtained from New England Biolabs.…”

mentioning

confidence: 99%

Involvement of helicase II (uvrD gene product) and DNA polymerase I in excision mediated by the uvrABC protein complex.

Caron¹,

Kushner²,

Grossman³

1985

Proc. Natl. Acad. Sci. U.S.A.

212

112

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The bimodal-incision nature ofthe reaction of UV-irradiated DNA catalyzed by the Escherichia coli uvrABC protein complex potentially leads to excision of a 12-to 13-nucleotide-long damaged fragment. However, the oligonucleotide fragment containing the UV-induced pyrimidine dimer is not released under nondenaturing in vitro reaction conditions. Also, the uvrABC proteins are stably bound to the incised DNA and do not turn over after the incision event. In this communication it is shown that release of the damaged fragment from the parental uvrABC-incised DNA is dependent upon either chelating conditions or the simultaneous addition of the uvrD gene product (helicase II) and thepoUl gene product (DNA polymerase I) when polymerization of deoxynucleoside triphosphate substrates is concomitantly catalyzed. The product of this multiprotein-catalyzed series of reactions serves as a substrate for polynucleotide ligase, resulting in the restoration of the integrity of the strands of DNA. The addition of the uvrD protein to the incised DNA-uvrABC complex also results in turnover of the uvrC protein. It is suggested that the repair processes of incision, excision, resynthesis, and ligation are coordinately catalyzed by a complex of proteins in a "repairosome" configuration.The removal of ultraviolet light-induced pyrimidine cyclobutane dimers from damaged DNA in bacteria is catalyzed by two essentially different mechanisms. In Micrococcus luteus (1-3) and T4 phage-infected Escherichia coli (4, 5) the 5'-pyrimidine N-glycosylic linkage of the pyrimidine dimer is initially acted upon by a dimer-specific endonuclease. This 17-kDa protein has two activities: A pyrimidine dimer DNA glycosylase activity initially hydrolyzes the N-glycosyl linkage of the 5'-pyrimidine moiety of the dimer, generating an apyrimidinic sitejuxtaposed to a thymine-thymidylate dimer. A 3'-apyrimidinic acid endonuclease activity associated with the same enzyme generates a 3'-hydroxylated apyrimidinic terminus and a 5'-phosphorylated thymine-thymidylate dimer terminus (3). Although these are seemingly different activities associated with the same polypeptide, a common modified Michael addition reaction can be invoked to explain both activities (unpublished data).The incision reaction in uninfected E. coli, which requires the uvrA, uvrB, and uvrC gene products (6-8), results in two endonucleolytic breaks: one break occurs seven nucleotides 5' to a pyrimidine dimer and a second site of hydrolysis is three to four nucleotides 3' to the same dimer (9-11). Preliminary evidence suggests a sequential mechanism in which the 5' break is made first. Although the size of the incised fragment in the DNA suggests that incision may be coordinated with excision reactions, neither fragment release under nondenaturing conditions nor turnover of the uvrABC proteins could be demonstrated (9-11). The implication of these findings is that the mixture for the in vitro reaction using purified uvrA, uvrB, and uvrC proteins lacks important components limiting the repair p...

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“…As shown in Fig. 4, A and B (43). The observed inhibitory effect of the DPC lesion in the translocating strand raised the possibility that the ATPase activity of UvrD might also be inhibited on the damaged DNA substrate.…”

Section: Resultsmentioning

confidence: 71%

Modulation of UvrD Helicase Activity by Covalent DNA-Protein Cross-links

Kumari

Minko

Smith

et al. 2010

Journal of Biological Chemistry

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UvrD (DNA helicase II) has been implicated in DNA replication, DNA recombination, nucleotide excision repair, and methyl-directed mismatch repair. The enzymatic function of UvrD is to translocate along a DNA strand in a 3 to 5 direction and unwind duplex DNA utilizing a DNA-dependent ATPase activity. In addition, UvrD interacts with many other proteins involved in the above processes and is hypothesized to facilitate protein turnover, thus promoting further DNA processing. Although UvrD interactions with proteins bound to DNA have significant biological implications, the effects of covalent DNAprotein cross-links on UvrD helicase activity have not been characterized. Herein, we demonstrate that UvrD-catalyzed strand separation was inhibited on a DNA strand to which a 16-kDa protein was covalently bound. Our sequestration studies suggest that the inhibition of UvrD activity is most likely due to a translocation block and not helicase sequestration on the crosslink-containing DNA substrate. In contrast, no inhibition of UvrD-catalyzed strand separation was apparent when the protein was linked to the complementary strand. The latter result is surprising given the earlier observations that the DNA in this covalent complex is severely bent (ϳ70°), with both DNA strands making multiple contacts with the cross-linked protein. In addition, UvrD was shown to be required for replication of plasmid DNAs containing covalent DNA-protein complexes. Combined, these data suggest a critical role for UvrD in the processing of DNA-protein cross-links.

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DNA Unwinding Enzyme II of Escherichia coli

Cited by 77 publications

References 19 publications

Effect of DNA polymerase I and DNA helicase II on the turnover rate of UvrABC excision nuclease.

Effect of DNA polymerase I and DNA helicase II on the turnover rate of UvrABC excision nuclease.

Involvement of helicase II (uvrD gene product) and DNA polymerase I in excision mediated by the uvrABC protein complex.

Modulation of UvrD Helicase Activity by Covalent DNA-Protein Cross-links

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