Finding a needle in the haystack

In the February 7, 2012 edition of Proceedings of the National Academy of Sciences USA, Sontz and colleagues published a paper¹ that provides new insight into an old problem in the DNA repair field: how is DNA damage efficiently recognized in a vast sea of undamaged bases in the genome? A critically important step in virtually all DNA repair pathways is the initial detection of a DNA lesion, which is followed by a series of steps to process the damaged strand, resulting in the ultimate removal of the lesion and its replacement with correct nucleotide/sequence.² The process of damage recognition has been studied in a number of prokaryotic and eukaryotic systems, and significant progress has been made in understanding how detection is achieved for various DNA repair pathways, including mismatch repair, nucleotide excision repair and base excision repair. However, the low copy number of certain DNA repair proteins (including ones involved in DNA damage verification) combined with their lack of preferential affinity for damaged DNA compared with undamaged DNA suggests that mechanisms must exist to optimize the hunt for DNA lesions. This conjures up the proverbial image of finding a needle in a haystack, a daunting challenge for the DNA repair machinery.

The very recent Sontz et al. paper¹ has taken our understanding of DNA damage recognition to the next level through careful elegant biochemical studies with defined DNA substrates and purified recombinant DNA repair proteins that possess an Iron-Sulfur (Fe-S) cluster with a physiologically relevant DNA-bound redox potential. In this work, the Barton lab builds on previous models that have implicated DNA charge transport as a signaling mechanism to recruit redox-active DNA repair proteins to the vicinity of DNA damage.³ Now, experimental evidence is presented that coordinated DNA charge transport between DNA repair proteins with redox-active Fe-S clusters can occur to enable efficient redistribution of the repair proteins to DNA lesions embedded in the vast excess of undamaged nucleotide sequence. In a sense, finding the needle in a haystack may have become easier through the cooperation of DNA repair proteins competent to participate in DNA charge transport.

A surprising twist in the story is that the experimental data was obtained from studies using Fe-S cluster DNA repair proteins, which traditionally operate in different pathways. EndoIII is a base excision repair DNA glycosylase that removes oxidized pyrimidine bases,⁴ whereas Xeroderma pigmentosum group D protein (XPD), a component of the general transcription factor TFIIH, is a DNA helicase that locally unwinds duplex DNA harboring a helix-distorting lesion in nucleotide excision repair.⁵ EndoIII and XPD are highly conserved in nature, and the current study¹ showed that the two DNA repair proteins from unrelated organisms can efficiently cooperate with each other to localize to a single base mismatch residing within several kilobase-pair of well-matched DNA duplex. The cooperative search by EndoIII and XPD is mediated by efficient DNA charge transport, as demonstrated by the observation that site-directed mutant variants of either EndoIII or XPD, which are defective in charge transport, fail to redistribute onto the mismatched strands. The remarkable ability of two Fe-S cluster DNA repair proteins from distinct repair pathways and organisms to work together suggests that signaling between redox-active proteins may be a general mechanism for efficient detection of DNA lesions. Although interprotein signaling in the context of chromatinized DNA has not yet been demonstrated, this seems likely given that DNA charge transport can occur within a nucleosome.⁶

Fe-S clusters with redox potential are found in a growing number of proteins that bind or catalytically process nucleic acids.⁷ This list includes glycosylases, primases, helicases, nucleases, transcription factors, RNA polymerases and RNA methyltransferases. For example, the Barton lab showed that XPD displays ATP-dependent electrochemistry, suggesting that an electrochemical signal is coupled to mechanical movement of the helicase as it translocates on DNA.⁸ It is conceivable that Fe-S cluster helicase molecules bound to DNA may communicate with each other over considerable distances by changes in redox activity that are mediated through DNA charge transport. Future studies will likely address if Fe-S cluster helicase molecules functionally cooperate during ATP-dependent DNA unwinding or protein stripping and if the redox function plays a role.⁹

It is provocative that DNA metabolic proteins conventionally thought to function in different pathways might collaborate with each other in an unexpected way by virtue of their ability to participate in DNA charge transport chemistry. In the Sontz et al. paper,¹ a nucleotide excision repair helicase was able to coordinate with a base excision repair glycosylase via DNA charge transport to detect a DNA lesion. Although there is evidence for crosstalk between DNA repair pathways,¹⁰ the ability of redox-active proteins to collaborate by DNA charge transport broadens the scope of possibilities, since the proteins do not necessarily need to physically interact with one another. In this sense, the discovery of Sontz et al. represents a paradigm shift. Although the findings emphasized the collaborative nature between redox active proteins in DNA damage detection, it seems probable that the impact of this work will extend into other important areas of DNA biology.