Cancer Biology

DNA and RNA nucleases are wide-ranging enzymes, taking part in broad cellular processes from DNA repair to immune response control. Growing interest in the mechanisms and activities of newly discovered nucleases inspired us to share the detailed protocol of our nuclease assay (Sheppard et al., 2019). This easy and inexpensive method can provide data that enables understanding of the molecular mechanism for novel or tested nucleases, from substrate preference and cofactors involved to catalytic rate of reaction.

DNA double strand breaks (DSBs) are among the most toxic lesions affecting genome integrity. DSBs are mainly repaired through non-homologous end joining (NHEJ) and homologous recombination (HR). A crucial step of the HR process is the generation, through DNA end-resection, of a long 3′ single-strand DNA stretch, necessary to prime DNA synthesis using a homologous region as a template, following DNA strand invasion. DNA end resection inhibits NHEJ and triggers homology-directed DSB repair, ultimately guaranteeing a faithful DNA repair. Established methods to evaluate the DNA end-resection process are the immunofluorescence analysis of the phospho-S4/8 RPA32 protein foci, a marker of DNA end-resection, or of the phospho-S4/8 RPA32 protein levels by Western blot. Recently, the Single Molecule Analysis of Resection Tracks (SMART) has been described as a reliable method to visualize, by immunofluorescence, the long 3′ single-strand DNA tails generated upon cell treatment with a S-phase specific DNA damaging agent (such as camptothecin). Then, DNA tract lengths can be measured through an image analysis software (such as Photoshop), to evaluate the processivity of the DNA end-resection machinery. The preparation of DNA fibres is performed in non-denaturing conditions so that the immunofluorescence detects only the specific long 3′ single-strand DNA tails, generated from DSB processing.

The Comet assay (or Single Cell Gel Electrophoresis assay) is a sensitive technique to detect DNA damage at the level of an individual cell. This technique is based on micro-electrophoresis of cells DNA content. Briefly, cells are embedded in agarose, lysed and submitted to an electric field, before the staining step with a fluorescent DNA binding dye. Damaged DNA (charged DNA) migrates in this field, forming the tail of a “comet”, while undamaged DNA remained in the head of the “comet”. The following document describes the protocol to realize a neutral comet assay. This assay can be applied to different cell types and has been useful for numerous applications in fields of toxicology or DNA damage and repair.

Repair of double strand break by homologous recombination was examined using U2OS cells or RG37 cells harbouring specific substrate developed by Puget et al. (2005) and Dumay et al. (2006), respectively, to measure the repair of DNA double strand breaks by homologous recombination. The substrate is composed of two inactive copies of the GFP gene. The upstream copy is inactive due to the absence of promoter, the downstream copy present a promoter but is inactivated by the insertion of the sequence coding for the recognition site of the I-SceI enzyme. The substrate is stably expressed in cells after its insertion in the genome and present as a unique copy. The unique DNA double strand break is then induced by the expression of the I-SceI enzyme after cell transfection with a plasmid coding for the I-SceI enzyme.

Many environmental agents induce double-strand breaks (DSBs) in DNA. Unrepaired or improperly repaired DSBs can lead to cell death or cancer. Nonhomologous end joining is the primary DNA double-strand break repair pathway in eukaryotes. During NHEJ pathway, several proteins recognize and bind DNA ends, bring the ends in a synaptic complex and, finally, process and ligate the ends.

Briefly, NHEJ starts with Ku protein. Ku binds the broken DNA ends and recruits the catalytic subunit of DNA dependent protein kinase (DNA-PKcs) forming DNA-PK. After processing, the XRCC4/Ligase IV complex executes the final ligation stimulated by Cernunnos-XLF.

Here, we describe an end-synapsis assay. This assay can be used in order to delineate which proteins are necessary to bring the DNA ends in a stable synaptic complex during NHEJ. Briefly, NHEJ competent extracts from human cells were incubated with both a double-stranded DNA fragment bound to streptavidin-coated magnetic beads and the same soluble radio-labeled fragment. The beads were then washed in mild salt buffer and the radioactivity recovered with the beads was measured by scintillation counting. Control experiments without extracts or with DNA-free beads were run in parallel to determine unspecific background.

Homologous recombination deficiency, mainly resulted from BRCA1 or BRCA2 inactivation (so called BRCAness), is found in breast and ovarian cancers. Detection of actual inactivation of BRCA1/2 in a tumor is important for patients’ treatment and follow-up as it may help predicting response to DNA damaging agents and give indication Homologous recombination deficiency, mainly resulted from BRCA1 or BRCA2 inactivation (so called BRCAness), is found in breast and ovarian cancers. Detection of actual inactivation of BRCA1/2 in a tumor is important for pat for genetic testing. This protocol describes how to detect impairment of homologous recombination based on the tumor genomic profile measured by SNP-array. The proposed signature of BRCAness is related to the number of large-scale chromosomal breaks in a tumor genome calculated after filtering and smoothing small-scale alterations. The procedure strongly relies on good quality SNP-arrays preprocessed to absolute copy number and allelic content (allele-specific copy number) profiles. This genomic signature of homologous recombination deficiency was shown to be highly reliable in predicting BRCA1/2 inactivation in triple-negative breast carcinoma (97% accuracy; for more details, see Popova et al., 2012) and predictive of survival in ovarian carcinoma (unpublished data). Authors are grateful to Dominique Stoppa-Lyonnet, Anne Vincent-Salomon, Thierry Dubois, and Xavier Sastre-Garau for their contributions. (Patent was deposited: Reference number EP12305648.3, June 7, 2012)