Modulation of UvrD helicase activity by covalent DNA-protein cross-links

Abstract

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 DNA-protein 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 cross-link-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 ( approximately 70 degrees ), 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.

FIGURE 1.

DNA substrates. Sequence and structural representation of the double-stranded DNA substrates, 50C-30C and 50T-30T, containing a covalently linked DPC either on the complementary (non-translocating) or the translocating strand. As described under “Experimental Procedures,” the 50-mer oligodeoxynucleotide (50C or 50T) was annealed to the complementary 30-mer oligodeoxynucleotide (30C or 30T) to generate a partially complemented duplex substrate. U is a uracil base and marks the site where the DPC was formed (indicated by the filled circle). To facilitate the loading of UvrD on these substrates, the length of the 3′ single-stranded arm of the fork was kept longer than the 5′ single-stranded arm.

FIGURE 2.

UvrD helicase activity on a DNA substrate containing DPC in the complementary strand. A, UvrD helicase activity on non-damaged duplex (50C-30C) and DNA substrate containing a DPC in the complementary strand is shown. The ³²P-labeled 50-mer oligodeoxynucleotide (50C) was annealed to the complementary 30-mer oligodeoxynucleotide (30C), and T4-pdg was covalently linked to the 30-mer strand. Reaction mixtures containing 1 nm concentrations of the indicated duplex DNA substrate and specified concentrations of UvrD were incubated at room temperature for 5 min under standard conditions. The asterisk indicates the 5′ of the ³²P-labeled strand of the duplex substrate. B, quantitation of the experiments in A. Percentage duplex substrate is graphically represented as a function of UvrD concentration. The error bars indicate the S.D. derived from three independent helicase reactions. The abbreviations ND, ss DNA, ds DNA (± AP site), and ds DNA-DPC correspond to the non-damaged, single-stranded DNA, double-stranded DNA with or without AP site, and double-stranded DNA containing a DPC, respectively.

FIGURE 3.

UvrD helicase activity on a DNA substrate containing DPC in the translocating strand. A, UvrD helicase activity on the non-damaged (50T-30T) and the damaged duplex substrate containing a DPC in the translocating strand. The ³²P-labeled 30-mer oligodeoxynucleotide (30T) was annealed to the complementary 50-mer oligodeoxynucleotide (50T), and T4-pdg was covalently linked to the 50-mer strand. Reaction mixtures containing 1 nm concentrations of the indicated duplex DNA substrate and specified concentrations of UvrD were incubated at room temperature for 5 min under standard conditions. The asterisk indicates the 5′ of the ³²P-labeled strand of the duplex substrate. B, quantitation of the experiments in A. Percentage duplex substrate is graphically represented as a function of UvrD concentration used. The error bars indicate the S.D. derived from three independent helicase reactions. C, reactions with UvrD helicase at higher concentrations (0–40 nm) and a longer incubation time (15 min). Standard helicase reactions were run with the non-damaged duplex (50T-30T) and damaged substrate containing a DPC in the translocating strand. The percentage of duplex substrate is graphically represented as a function of the UvrD concentration. The error bars indicate the S.D. derived from three independent helicase reactions. The abbreviations ND, ss DNA, ds DNA (±AP site), and ds DNA-DPC correspond to the non-damaged, single-stranded DNA, double-stranded DNA with or without AP site, and double-stranded DNA containing a DPC, respectively.

FIGURE 4.

UvrD dissociates from the DNA substrate containing a DPC in the translocating strand. A, reactions for sequestration assays were initiated under conditions similar to that described for the helicase reactions in the presence of specified concentrations (1–8 nm) of unlabeled non-damaged (50T-30T) or damaged competitor substrate. After helicase reactions with the competitor DNA, the ³²P-labeled tracker substrate was added to the reactions and incubated for 5 min at room temperature. B, quantitation of the experiments in A. The error bars indicate the S.D. derived from three independent sequestration experiments. The abbreviations ND, ss DNA, ds DNA (±AP site), and ds-DPC correspond to the non-damaged, single-stranded DNA, double-stranded DNA with or without AP site, and double-stranded DNA containing a DPC, respectively.

FIGURE 5.

UvrD ATPase activity is affected in the presence of DPC-containing DNA substrate. Reactions for ATPase assay containing non-damaged (50T-30T) or damaged duplex substrates were prepared under conditions similar to that described for helicase reactions. After a 5-min incubation of DNA substrate (2 nm) and specified concentrations of UvrD enzyme, ATP-containing solution (0.5 mm) was added, and reactions were incubated at room temperature for additional 5 min before terminating the reactions. A₆₅₀ values for controls containing buffer alone, UvrD enzyme alone, and DNA substrate alone were found to be comparable. The error bars indicate the S.D. derived from three independent ATPase reactions. ND, non-damaged; ds-DPC double-stranded DNA containing a DPC.

FIGURE 6.

Relative colony-forming ability of E. coli strains after transformation with DPC containing plasmids. A, a flow chart diagram shows sequential steps involved in the generation of DPC-containing plasmids. The ds pMS2 plasmid (amp^r) was irradiated with UV-C, and T4-pdg was trapped at the UV-induced cyclobutane pyrimidine dimer sites. The non-damaged plasmid pBR322 (amp^r and tet^r) was mixed with either the UV-irradiated (triangle) or DPC containing (star) pMS2 plasmid, and the resultant mixture was used to transform the wild-type and ΔuvrD E. coli strains followed by selecting the transformants for amp resistance. Randomly selected amp resistant clones were further assessed for tet resistance to discriminate between the clones carrying pMS2 versus pBR322 plasmid. An aliquot of UV-irradiated pMS2 vector was collected before and after trapping DPC and analyzed on a 1% agarose gel. Efficiency to replicate pMS2 plasmid containing DPCs (B) or UV-induced lesions (C) is shown. The relative colony forming ability of E. coli strains was assessed after transformation with a mixture of non-damaged pBR322 and pMS2 containing randomly integrated DPCs or UV-induced lesions. For both the wild-type and ΔuvrD strains, the percentage of pMS2-DPCs transformants was calculated relative to pBR322 transformants. The apparent transformation efficiency with the reference plasmid pBR322 was comparable for all the strains tested. The data collected from two independent experiments are shown.

FIGURE 7.

Cell survival after exposure to the DPC inducing agent, formaldehyde. Exponentially growing wild-type (circles), ΔuvrD (squares), and ΔuvrA (triangles) strains were exposed to formaldehyde at the indicated concentrations for 30 min at 37 °C. The mean (symbol) and S.D. (error bar) from at least three or more independent experiments are shown. WT, wild type.