TY - JOUR
T1 - SIRF
T2 - A Single-cell Assay for in situ Protein Interaction with Nascent DNA Replication Forks
AU - Roy, Sunetra
AU - Schlacher, Katharina
N1 - Funding Information:
This work was supported by RP180463, RP180813 (CPRIT) and 1R01ES029680 (NIEHS) grants, and K.S is a Rita Allen Foundation fellow and a CPRIT Scholar in Cancer biology. The PLA technology was originally developed by Soderberg et al., 2008. PLA between biotin-clicked EdU and proteins of interest, reflecting the principle technology of SIRF, were also reported by Petruk et al., 2012 and 2017; Taglialatela et al., 2017, in addition to Roy et al., 2018a and 2018b.
Publisher Copyright:
© Bio-protocol. All rights reserved.
PY - 2019/9/20
Y1 - 2019/9/20
N2 - 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.
AB - 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.
KW - DNA replication
KW - Fork protection
KW - Genome instability
KW - iPOND
KW - Proximity ligation assay
KW - SIRF
KW - Stalled replication forks
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U2 - 10.21769/BioProtoc.3377
DO - 10.21769/BioProtoc.3377
M3 - Article
C2 - 33654873
AN - SCOPUS:85131911847
SN - 2331-8325
VL - 9
JO - Bio-protocol
JF - Bio-protocol
IS - 18
M1 - e3377
ER -