Redox signaling between DNA repair proteins for efficient lesion detection

Abstract

Base excision repair (BER) enzymes maintain the integrity of the genome, and in humans, BER mutations are associated with cancer. Given the remarkable sensitivity of DNA-mediated charge transport (CT) to mismatched and damaged base pairs, we have proposed that DNA repair glycosylases (EndoIII and MutY) containing a redox-active [4Fe4S] cluster could use DNA CT in signaling one another to search cooperatively for damage in the genome. Here, we examine this model, where we estimate that electron transfers over a few hundred base pairs are sufficient for rapid interrogation of the full genome. Using atomic force microscopy, we found a redistribution of repair proteins onto DNA strands containing a single base mismatch, consistent with our model for CT scanning. We also demonstrated in Escherichia coli a cooperativity between EndoIII and MutY that is predicted by the CT scanning model. This relationship does not require the enzymatic activity of the glycosylase. Y82A EndoIII, a mutation that renders the protein deficient in DNA-mediated CT, however, inhibits cooperativity between MutY and EndoIII. These results illustrate how repair proteins might efficiently locate DNA lesions and point to a biological role for DNA-mediated CT within the cell.DNA charge transport ͉ DNA damage ͉ iron-sulfur proteins ͉ oxidative stress

Crystal structure of a prokaryotic (6-4) photolyase with an Fe-S cluster and a 6,7-dimethyl-8-ribityllumazine antenna chromophore

“…The Fe-S cluster of human PriL is specifically required for initiation of primer synthesis, but the molecular details of its function are as yet unknown (33). For the base excision repair Mutator Y (MutY, a DNA glycosylase) and Endonuclease III (EndoIII), a charge transfer between DNA and the Fe-S cluster has been proposed to be involved in detecting DNA lesions (34). The present study does not yet provide insight into the function of the Fe-S cluster of PhrB apart from it being an integral component of the protein fold, but knowledge of the high-resolution structure of PhrB might help to solve this task in the future.…”

Surface enhanced vibrational spectroscopic evidence for an alternative DNA-independent redox activation of endonuclease III

“…5,6 More recent electrochemical studies of glycosylases immobilised on DNA-modified electrodes indicated that the [4Fe-4S] cluster is activated towards oxidation upon binding to DNA. It was proposed that the cluster exerts a function in DNAmediated signalling for detection of DNA lesions, [7][8][9][10][11][12] but based on data obtained by electrochemical approaches, which cannot provide any information about the molecular origin of the electrochemical signal. Here we employed surface enhanced resonance Raman (SERR) spectroscopy and surfaced enhanced IR absorption (SEIRA) spectro-electrochemistry to demonstrate that the cluster of the immobilised EndoIII from D. radiodurans (DrEndoIII) is prone to reduction, which is not necessarily DNA-mediated.…”

DNA charge transport as a first step in coordinating the detection of lesions by repair proteins

“…Using DNA electrochemistry and atomic force microscopy (AFM) experiments, we found that the ability of EndoIII mutants to localize in the vicinity of a base mismatch correlates with their ability to carry out DNA CT. Moreover, using a genetics assay, we found that EndoIII cooperates with MutY in vivo in finding MutY lesions, but that a CT-deficient mutant of EndoIII cannot similarly aid MutY (22). Interestingly, more proteins involved in genome maintenance that contain [4Fe-4S] clusters are emerging, many with no clear structural or enzymatic role for their clusters.…”

“…We have recently examined whether DNA CT may play some role in how these BER proteins find their site (22,23). Using DNA electrochemistry and atomic force microscopy (AFM) experiments, we found that the ability of EndoIII mutants to localize in the vicinity of a base mismatch correlates with their ability to carry out DNA CT.…”

“…Thus, in the model, DNA-mediated reduction of one protein by the other leads to the dissociation of the reduced protein from DNA, effectively giving overall dissociation of repair proteins away from a region of the genome without lesions. Our model relies on the sensitivity of CT to proper π stacking of the bases between the donor protein and the distant acceptor protein (22,23); if instead there is an intervening lesion, DNA-mediated CT does not occur, and the repair protein remains bound and can processively move to the lesion and carry out enzymatic repair. In this process, the repair proteins eventually relocalize in the vicinity of lesions or any modification that inhibits DNA CT. As a result the overall search regime for the repair proteins is made smaller than the full genome: they preferentially localize where needed for repair.…”