DNA damages compromise genomic integrity and function. There are many types of DNA damages, ranging from chemical modifications to the nitrogenous bases to breaks in the sugar-phosphate backbones.1, 2 Especially deleterious are helix-distorting, or bulky, DNA base damages that halt the activity of DNA and RNA polymerases. These include the cyclobutyl pyrimidine dimers (CPDs) and the (6-4) pyrimidine pyrimidine photoproduct [(6-4)PP] induced by ultraviolet (UV) radiation, Benzo[a]pyrene diol epoxide (BPDE) adducts induced by cigarette smoking, and adducts induced by the chemotherapy cisplatin.1, 2 While CPDs and (6-4)PPs form primarily in pyrimidine dimers,3, 4, 5 BPDE and platinum adducts occur primarily in Guanine (G) nucleotides.6, 7, 8 Due to the miscoding nature of the damaged bases, UV light exposure and smoking lead to specific mutational signatures prevalent in skin and lung cancers, respectively.9, 10, 11, 12

The major mechanism for the repair of helix-distorting damages in human cells is nucleotide excision repair (NER).13, 14, 15 NER is initiated by the recognition of a bulky DNA damage, either directly by excision repair proteins including the XPC/hRAD23b complex, or by an RNA polymerase stalled at a lesion.13, 14, 15 The two distinct damage recognition mechanisms divide NER to two sub-pathways: Global Genome repair (GG-NER), which removes lesions throughout the genome, and Transcription Coupled repair (TC-NER), which removes lesions located on the transcribed strands of active genes.16, 17, 18 Following lesion recognition, endonucleases are recruited for dual incision of the damaged strand resulting in the excision of the damage along with short flanking sequences, leaving a single stranded gap in the genome. This gap is then filled by re-synthesis, using the undamaged complementary strand as a template, completing error-free repair.13, 14, 15, 19

In general, helix-distorting damages form stochastically throughout the genome, and at different positions in each cell in the population. Even high damaging doses that result in significant cell death will still induce damages only at the range of one in a few thousand bases.3 In the mammalian genomes, damage detection and measurement can thus be quite challenging. Still, methods are available to detect and quantify helix-distorting damages in cells.20, 21 These existing methods include: 1) Immunological methods that utilize specialized anti-damage antibodies,22, 23, 24 2) In-vitro enzymatic approaches, that employ either purified DNA repair proteins to restrict DNA at sites of damage, or DNA polymerases that will synthesize DNA until blocked by a damage.25, 26, 27 While the immunological approaches can measure the total damage level in the genome, the enzymatic approaches are primarily used to measure damage at specific loci. Damages can also be measured indirectly, by following recruitment of repair proteins or the phosphorylation of the histone isoform H2AX.28, 29 Using any of these methods, repair can then be measured by following damage reduction with time.

The recent advances in DNA sequencing technologies facilitated the development of elegant, yet complicated, methods to map helix-distorting DNA damages at different resolutions across the genome.21, 30, 31, 32 These methods require strong expertise in both DNA damage and genomics and provide valuable information on the distribution and determinants of damage formation and repair across the genome.

All the methods we described so far require specialized reagents and expertise that are usually available only in DNA repair labs. As biological sciences become more interdisciplinary, many non-expert labs find that their protein of interest or process are linked to damage and repair, but face technical challenges in pursuing these research avenues. Here, we describe a simple method to measure DNA polymerase-blocking DNA damages and their repair, which we name damage-sensing PCR (dsPCR). dsPCR is an updated and improved version of the previously described long-amplicon PCR (LA-PCR) also named QPCR33, 34, 35, 36, 37, 38 that combined several aspects of the previous methodologies for a robust and relatively simple experimental assay. The method relies entirely on commercial reagents, making it easy-to-implement and reproduce in any molecular biology lab. DNA purification is performed with a new bead-based protocol that isolates high-quality and longer DNA fragments to improve long PCR consistency. We employ the well-established Qubit fluorometric DNA measurements, negating the need for gel electrophoresis or radioactive labeling. These adjustments make the method fully amenable to high-throughput screening in which multiple gene disruptions by siRNA or CRISPR-Cas9 knock-out can be tested in parallel for DNA repair defects at a selected locus. We show that dsPCR sensitively measures differences in repair efficiencies of various damage types, genomic loci and defects in DNA repair proteins.