Background

Defects in high fidelity DNA replication are associated with severe biological outcomes. These amongst others include developmental abnormalities, premature aging, hematological disorder and cancer (Loeb and Monnat, 2008; Magdalou et al., 2014). To avoid DNA replication instability, cells have evolved intricate mechanisms to ensure error-free propagation of its genetic material. There is an ever-growing list of disease-suppressing proteins that actively ensure DNA replication stability and function at all stages of DNA replication processes. A comprehensive understanding of the functions of DNA replication stability proteins is imperative to allow development of effective and specific agents targeting disease outcomes.

Historically, visualization of proteins localized at or near DNA replication forks was advanced by the advent of nucleoside analogs, such as 5-bromo-2’-deoxyuridine (BrdU) that is incorporated into newly replicating DNA and further detected with fluorescent-labeled antibodies (Gonchoroff et al., 1986; Leif et al., 2004). Immunofluorescence (IF) assays examining co-localization of proteins with the BrdU-label indicates that the protein is present in close vicinity to actively replicating DNA (Sengupta et al., 2003). This method provided critical information on replication processes. However, it is an indirect method that lacks the resolution that would be required to unequivocally distinguish protein-DNA interactions from proteins located near DNA. Another ground-breaking procedure was iPOND (standing for isolation of proteins on nascent DNA) that allows to detect and isolate proteins on newly replicated DNA (Petermann et al., 2010; Sirbu et al., 2011 and 2013; Dungrawala et al., 2015). In this procedure, nascent DNA is labeled with the thymidine analog EdU, which is then biotinylated using click-IT chemistry (Moses and Moorhouse, 2007). The newly synthesized biotinylated DNA is immuno-precipitated with streptavidin-coated agarose or magnetic beads. This so isolates proteins that bind to newly replicated DNA, which either can be detected by Western blotting or by mass spectrometry. While this method revolutionized the DNA replication field by enabling to directly study protein interactions with replication forks, iPOND detects the average of thousands of replication forks and cells. iPOND lacks the single-cell level resolution that is achieved by IF, which can provide valuable additional parameter information such as stage of the S-phase, or cell identity. Moreover, the technique is labor-intensive and requires copious amounts of starting material (~100,000,000 cells per condition). The assay system described here termed in situ protein interaction with nascent DNA replication forks (SIRF) (Roy et al., 2018a and 2018b) combines EdU-biotin Click-chemistry with proximity ligation assay (PLA) to overcome these challenges. The PLA technology developed by Soderberg et al. for single-molecule protein-protein interaction studies, utilizes antibodies with oligonucleotides conjugates, that can be ligated into a circular DNA molecule when two antibodies are in close proximity to each other (< 40 nm) (Soderberg et al., 2008). Once ligated, the DNA circle can be amplified by rolling circle DNA polymerase. After annealing of fluorescent DNA probes that are complementary to the DNA circle, this procedure results in a highly amplified fluorescent signal, so that even single molecule interactions are readily detectable by conventional immunofluorescence microscopy. In SIRF, PLA is used with antibodies against biotin (detecting biotinylated EdU) and a protein of interest, which results in a PLA signal only if the protein is located within 40 nm or less from the newly synthesized DNA (Figure 1). The SIRF method requires little amounts of cells (~100-1,000), preserves the single-cell resolution as seen with IF, and can be readily and accurately quantified. Moreover, the single-cell resolution provides additional valuable information including cell morphology, cell-cycle stage (early or late S-phase), spatial orientation of protein signal in the nucleus, which can be associated with changes in replication fork-protein dynamics. The SIRF assay furthermore can be combined with conventional IF for additional cell-biomarker analysis, which amongst others allows to examine replication proteins in a heterogeneous cell population that can be differentiated by lineage specific markers. In this case, primary and secondary antibody staining for IF is performed prior to the PLA reaction to avoid interference of PLA secondary antibodies from cross-reacting with IF primary antibodies. The SIRF assay can be used to study protein dynamics at ongoing and stalled DNA replication forks, as well as chromatin maturation at newly replicated DNA regions (Petruk et al., 2017, Roy et al., 2018a and 2018b).