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Updated: Mar 20, 2026
Single Molecule Analysis of Laser Localized Psoralen Adducts
Published on: April 20, 2017
Single Molecule Analysis of Laser Localized Interstrand Crosslinks
Jing Huang1, Himabindu Gali1, Manikandan Paramasivam1
1Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health Baltimore, MD, USA.
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.
Insights
DNA interstrand crosslinks (ICLs) pose challenges to DNA replication and transcription. This study found similar adduct distribution and replication patterns regardless of the method used to induce ICLs, validating two distinct research approaches.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- DNA interstrand crosslinks (ICLs) are toxic DNA lesions that impede DNA replication and transcription.
- ICLs are a target for cancer chemotherapy due to their cytotoxic effects.
- Psoralen, a photoactive compound, can induce ICLs upon UVA light exposure.
Purpose of the Study:
- To compare two immunofluorescence-based strategies for studying cellular responses to psoralen-induced ICLs.
- To assess the applicability of findings from DNA fiber/immuno-quantum dot technology versus laser-localized ICLs.
- To determine lesion density, spacing, and replication patterns near ICLs induced by different methods.
Main Methods:
- Development of two immunofluorescence (IF) strategies using psoralen linked to an antigen tag.
- Strategy 1: DNA fiber and immuno-quantum dot technology to visualize replication fork encounters with ICLs.
- Strategy 2: Laser-localized UVA-induced psoralen ICLs in live cells, followed by IF for damage response proteins.
Main Results:
- Analysis of DNA fibers revealed minimal differences in adduct distribution between laser-localized ICLs and bulk UVA-induced ICLs.
- Replication patterns near ICLs showed considerable similarity across both experimental approaches.
- The study validated the consistency of lesion distribution and replication response irrespective of the ICL induction method.
Conclusions:
- The findings suggest that conclusions drawn from the DNA fiber/immuno-quantum dot method are applicable to laser-localized ICLs, and vice versa.
- This reconciles potential uncertainties arising from the differences between the two technological approaches.
- The study provides a robust framework for investigating cellular responses to DNA interstrand crosslinks.
