Measuring the formation and repair of UV damage at the DNA sequence level by ligation-mediated PCR

Abstract

The formation and repair of DNA damage at specific locations in the genome is modulated by DNA sequence context, by DNA cytosine-5 methylation patterns, by the transcriptional status of the locus and by proteins associated with the DNA. The only method currently available to allow precise sequence mapping of DNA lesions in mammalian cells is the ligation-mediated polymerase chain reaction (LM-PCR) technique. We provide an update on technical details of LM-PCR. LM-PCR can be used, for example, for mapping of ultraviolet (UV) light-induced DNA photoproducts such as cyclobutane pyrimidine dimers.

Figure 1. Outline of the LM-PCR procedure

A. General scheme showing the LM-PCR approach for detection of strand breaks or DNA damage sites. DNA containing strand breaks introduced at the sites of UV damage is used in a primer extension reaction (with primer 1), followed by ligation of a linker, PCR (with primer 2) and a labeling step (using infrared-dye-labeled primer 3). B. Primer arrangement. The relative orientation of primers 1, 2, and 3 relative to the DNA template containing a pyrimidine dimer (T<>T) is shown.

Figure 2. LM-PCR of the cII transgene gene in mouse cells

A. Genomic DNA of transgenic Big Blue^® mouse embryonic fibroblasts was subjected to standard Maxam and Gilbert chemical reactions, and subsequently DNA footprinting of the cII transgene was performed using our updated LM-PCR protocol, as described in the text. Individual Maxam/Gilbert sequencing ladders are: “G”, “G + A”, “C”, and “C + T”. B. Transgenic Big Blue^® mouse embryonic fibroblasts were irradiated with ultraviolet light B (UVB) to produce CPDs. Control DNA was not irradiated. The cellular DNA was extracted and subsequently subjected to T4 endonuclease V cleavage and CPD photolyase reactivation, followed by LM-PCR to detect CPDs. M = Molecular size marker (IRDye^® 700 Sizing Standard).