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J. Biol. Chem., Vol. 279, Issue 43, 99918, October 22, 2004

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Probes Shed Light on DNA Handoff

Because DNA is constantly exposed to an onslaught of mutagens, cells must have a method for excising and repairing DNA lesions. Nucleotide excision repair (NER) is a major DNA repair pathway involving the UvrABC proteins. In bacteria, UvrA dimerizes to become UvrA₂, which then interacts with UvrB to form the UvrA₂B complex. The complex then binds to DNA and searches for lesions via the -hairpin of UvrB. When a lesion is encountered, conformational changes occur, and the DNA, which is initially in contact with UvrA, is passed to UvrB via an unknown mechanism. Once this happens, UvrC binds to the UvrB·DNA complex and excises the lesion.

Putative UvrB DNA binding model showing potential points of contact for cross-linking reagents.

To capture UvrA in the act of DNA handoff to UvrB, Matthew J. DellaVecchia and colleagues designed two types of arylazido-modified photoaffinity cross-linking reagents. One type of reagent probed Uvr residues closest to the damaged DNA strand, and one probed residues closest to the non-damaged strand. The damaged strand probes, which functioned as both lesions and cross-linkers, consisted of dNTP analogues linked to terminal arylazido groups. The non-damaged strand probe consisted of an arylazido compound coupled to a phosphorothioate-modified backbone of an oligonucleotide opposite the damaged strand, which contained an internal fluorescein adduct. Using their probes, DellaVecchia et al. determined that DNA handoff occurs in three steps: 1) UvrA and UvrB bind to DNA via contact sites on UvrA; 2) a transfer reaction occurs with UvrB mostly contacting the non-damaged strand; 3) the lesion is engaged by the damage recognition pocket of UvrB, and UvrA is released. The methods developed here should be applicable to other DNA enzymes as well and are likely to be very useful to all DNA enzymologists.

FOOTNOTES

See referenced article, J. Biol. Chem. 2004, 279, 45245–45256

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This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Search for Related Content Related Collections Papers Of The Week Social Bookmarking                      What's this?