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. 2019 Sep 20;9(18):e3377.
doi: 10.21769/BioProtoc.3377.

SIRF: A Single-cell Assay for in situ Protein Interaction with Nascent DNA Replication Forks

Sunetra Roy  1 , Katharina Schlacher  1
Affiliations

SIRF: A Single-cell Assay for in situ Protein Interaction with Nascent DNA Replication Forks

Sunetra Roy et al. Bio Protoc. .

Abstract

The duplication of DNA is a fundamental process that is required for the transfer of the genetic information from parent to daughter cells. Aberrant DNA replication processes are associated with diverse disease phenotypes, including developmental defects, ageing disorders, blood disorders such as Fanconi Anemia, increased inflammation and cancer. Therefore, the development of tools to study proteins associated with error-free DNA replication processes is of paramount importance. So far, methods to study proteins associated with nascent replication forks relied on conventional immunofluorescence and immunoprecipitation assays of 5'-ethylene-2'-deoxyuridine (EdU) labeled DNA (iPOND). While greatly informative and important, these methods lack specificities for nascent fork interactions (e.g., IF) or assay an average change of millions of cells without single-cell resolution (e.g., iPOND). The assay system described here combines proximity ligation assay (PLA) with EdU coupled click-iT chemistry, which we termed "in situ protein interaction with nascent DNA replication forks (SIRF)". This method enables sensitive and quantitative analysis of protein interactions with nascent DNA replication forks with single-cell resolution, and can further be paired with conventional immunofluorescence marker analysis for added multi-parameter analysis.

Keywords: DNA replication; Fork protection; Genome instability; IPOND; Proximity ligation assay; SIRF; Stalled replication forks.

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Conflict of interest statement

Competing interestsThe authors declare no conflicts of interest.

Figures

Figure 1.
Figure 1.. Flowchart for the SIRF assay.
Shown is the basic workflow and the major steps involved in SIRF assay.
Figure 2.
Figure 2.. Slide chamber dis-assembly.
A. dis-assembly of slide chambers. B. removal of silicone gasket (demarcating the chambers) using fine curved forceps. Apply caution to avoid scratches on the glass wells with forceps.
Figure 3.
Figure 3.. Humid slide chamber.
Preparation of humid slide chamber by placing folded paper wipes (Kimwipes) wetted with distilled water at the bottom of a slide box. Slides are laid flat, facing up during antibody and PLA solution incubations and are covered with plastic cover slips during respective incubations.
Figure 4.
Figure 4.. Representative image to distinguish SIRF signals at various stages of the cell cycle.
The above panel shows an example of RAD51 SIRF (red channel) in HAP-1 cells treated with 125 μM EdU for 8 min and 200 μM HU for 4 h. The arrows indicate cells in late S-phase based on EdU patterning. Note that the green signal for Alexa 488 azide co-click enables distinction of late S-phase versus early S-phase cells. scale bars = 20 μm.
Figure 5.
Figure 5.. Representative RPA-SIRF image in HAP-1 cells.
A. representative images of an RPA-SIRF assay in HAP-1 cells that have been treated with 125 μM EdU for 8 min. The cells were co-clicked with biotin-azide and alexa 488-azide (10:1, total 10 μM). The GFP channel shows the EdU-Alexa 488 signal of S-phase cells. PLA signals are visualized in TXRED channel. The PLA signal can be normalized to the Alexa 488 signal to account for PLA signal differences depending on EdU content of the cells. B. representative images of RPA-SIRF with no EdU condition (negative control). scale bars = 20 μm.
Figure 6.
Figure 6.. Representative EdU SIRF image in HAP-1 cells.
The above panel shows an example of EdU-EdU SIRF in HAP-1 cells treated with 125 μM EdU for 8 min. The PLA signals are too abundant to be hand counted. Mean TXRED intensity in this case can be measured for PLA quantification. The green channel represents Alexa 488-EdU signal. scale bars = 20 μm.
See this image and copyright information in PMC

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