TY - JOUR
T1 - SIRF
T2 - Quantitative in situ analysis of protein interactions at DNA replication forks
AU - Roy, Sunetra
AU - Luzwick, Jessica W.
AU - Schlacher, Katharina
N1 - Publisher Copyright:
© 2018 Roy et al.
PY - 2018/4/1
Y1 - 2018/4/1
N2 - DNA replication reactions are central to diverse cellular processes including development, cancer etiology, drug treatment, and resistance. Many proteins and pathways exist to ensure DNA replication fidelity and protection of stalled or damaged replication forks. Consistently, mutations in proteins involved in DNA replication are implicated in diverse diseases that include defects during embryonic development and immunity, accelerated aging, increased inflammation, blood disease, and cancer. Thus, tools for efficient quantitative analysis of protein interactions at active and stalled replication forks are key for advanced and accurate biological understanding. Here we describe a sensitive single-cell-level assay system for the quantitative analysis of protein interactions with nascent DNA. Specifically, we achieve robust in situ analysis of protein interactions at DNA replication forks (SIRF) using proximity ligation coupled with 5'-ethylene-2'-deoxyuridine click chemistry suitable for multiparameter analysis in heterogeneous cell populations. We provide validation data for sensitivity, accuracy, proximity, and quantitation. Using SIRF, we obtained new insight on the regulation of pathway choice by 53BP1 at transiently stalled replication forks.
AB - DNA replication reactions are central to diverse cellular processes including development, cancer etiology, drug treatment, and resistance. Many proteins and pathways exist to ensure DNA replication fidelity and protection of stalled or damaged replication forks. Consistently, mutations in proteins involved in DNA replication are implicated in diverse diseases that include defects during embryonic development and immunity, accelerated aging, increased inflammation, blood disease, and cancer. Thus, tools for efficient quantitative analysis of protein interactions at active and stalled replication forks are key for advanced and accurate biological understanding. Here we describe a sensitive single-cell-level assay system for the quantitative analysis of protein interactions with nascent DNA. Specifically, we achieve robust in situ analysis of protein interactions at DNA replication forks (SIRF) using proximity ligation coupled with 5'-ethylene-2'-deoxyuridine click chemistry suitable for multiparameter analysis in heterogeneous cell populations. We provide validation data for sensitivity, accuracy, proximity, and quantitation. Using SIRF, we obtained new insight on the regulation of pathway choice by 53BP1 at transiently stalled replication forks.
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U2 - 10.1083/jcb.201709121
DO - 10.1083/jcb.201709121
M3 - Article
C2 - 29475976
AN - SCOPUS:85044779435
SN - 0021-9525
VL - 217
SP - 1521
EP - 1536
JO - Journal of Cell Biology
JF - Journal of Cell Biology
IS - 4
ER -