Abstract
DNA interstrand crosslinks (ICLs) block unwinding of the double helix, and have always been regarded as major challenges to replication and transcription. Compounds that form these lesions are very toxic and are frequently used in cancer chemotherapy. We have developed two strategies, both based on immunofluorescence (IF), for studying cellular responses to ICLs. The basis of each is psoralen, a photoactive (by long wave ultraviolet light, UVA) DNA crosslinking agent, to which we have linked an antigen tag. In the one approach, we have taken advantage of DNA fiber and immuno-quantum dot technologies for visualizing the encounter of replication forks with ICLs induced by exposure to UVA lamps. In the other, psoralen ICLs are introduced into nuclei in live cells in regions of interest defined by a UVA laser. The antigen tag can be displayed by conventional IF, as can the recruitment and accumulation of DNA damage response proteins to the laser localized ICLs. However, substantial difference between the technologies creates considerable uncertainty as to whether conclusions from one approach are applicable to those of the other. In this report, we have employed the fiber/quantum dot methodology to determine lesion density and spacing on individual DNA molecules carrying laser localized ICLs. We have performed the same measurements on DNA fibers with ICLs induced by exposure of psoralen to UVA lamps. Remarkably, we find little difference in the adduct distribution on fibers prepared from cells exposed to the different treatment protocols. Furthermore, there is considerable similarity in patterns of replication in the vicinity of the ICLs introduced by the two techniques.
Highlights
After several decades of effort by many laboratories, we have a detailed understanding of the cellular biochemistry that removes single strand adducts and repairs single and double strand breaks (DSBs)
In order to develop an approach for imaging psoralen interstrand crosslinks (ICLs) we had to attach a reliable and effective detection tag
Laser induced DNA damage is the basis of a substantial literature on the induction of the DNA damage response (DDR) and the repair of DSBs as monitored by surrogate markers
Summary
After several decades of effort by many laboratories, we have a detailed understanding of the cellular biochemistry that removes single strand adducts and repairs single and double strand breaks (DSBs). Imaging strategies, those based on immunofluorescence (IF) microscopy, have been helpful in elucidating these pathways and the factors that drive them. Proteins that accumulate in the vicinity of breaks introduced by irradiation, replication fork breakage, or enzymatic cleavage, form foci readily visualized by IF This feature has been widely exploited and has been instrumental in defining the collective of proteins known as the DNA damage response (DDR), the subject of an enormous, and expanding, literature (Ciccia and Elledge, 2010). The appearance of foci in response to DSBs provides an important experimental tool for studying the
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