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

Rebecca L. Smith,Amanda K. McCullough,Anuradha Kumari,R. Stephen Lloyd,Irina G. Minko

doi:10.1074/jbc.m109.078964

Rebecca L. Smith, Amanda K. McCullough

Open Access

https://doi.org/10.1074/jbc.m109.078964

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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.

Highlights

UvrD helicase activity can be significantly stimulated by interactions with the NER UvrAB complex [6] or methyl-directed mismatch repair protein, MutL [7, 8]
UvrD is implicated in the processing of Okazaki fragments, such that E. coli cells that are deficient in polymerase I are unable to grow on rich media but do survive when grown on defined minimal media [10]
DNA-protein complexes (DPCs) can be produced by exposure to a variety of chemical and physical agents, including formaldehyde, transplatin, and ionizing radiation (46 – 48)

Summary

EXPERIMENTAL PROCEDURES

Design and Generation of Substrates—The oligodeoxynucleotides were synthesized by Invitrogen. The pMS2 vector [31] (100 ng/␮l) in 10 mM Tris-HCl (pH 7.0), 1 mM EDTA was exposed to 254-nm UV light at 100 microwatts/cm, and 2 ␮l aliquots of DNA were collected during the course of exposure to measure the rate of CPD formation These DNAs were incubated with T4-pdg in 25 mM sodium phosphate buffer (pH 6.8) containing 100 ␮g/ml bovine serum albumin, 100 mM NaCl, and 1 mM EDTA to generate single-stranded breaks at the CPD sites, converting plasmids from the supercoiled form (Form I) to the nicked relaxed (Form II) and linear (Form III) forms. The results of the time course experiment are shown in supplemental Fig. S3, panels A and B After these initial analyses, the pMS2 vector was exposed under the same conditions as above for 4 min to introduce ϳ8 CPDs per plasmid molecule. Catalyzed DNA unwinding was evaluated when the lesion was located in the non-translocating strand

UvrD was capable of unwinding

Given that UvrD is induced upon

The unwinding reaction of UvrD is fueled by energy derived from

These results suggest that the remarkably low efficiency of the

Findings

DISCUSSION