Abstract
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.
Highlights
DNA replication and its regulations dictate outcomes of many biological processes including development, aging, and cancer etiology (Loeb and Monnat, 2008; Zeman and Cimprich, 2014)
The protocol follows the principle of the proximity ligation assay (PLA) (Söderberg et al, 2006), and cells are incubated with secondary antibodies conjugated to sequence specific DNA oligomers (Fig. 1 C, PLA antibodies)
In SIRF, we combine PLA with click chemistry of nascent DNA replication forks that are labeled with EdU for direct protein–DNA fork interaction
Summary
DNA replication and its regulations dictate outcomes of many biological processes including development, aging, and cancer etiology (Loeb and Monnat, 2008; Zeman and Cimprich, 2014). A rapidly evolving and exciting field is the direct involvement of proteins during DNA replication that are otherwise understood to repair DNA damage irrespective of DNA replication. Among others, these include BRCA1/2 and Fanconi anemia tumor suppressors, which protect stalled DNA replication forks from degradation by MRE11 and DNA2 nucleases and so suppress genome instability (Schlacher et al, 2011, 2012; Pefani et al, 2014; Higgs et al, 2015; Wang et al, 2015; Ding et al, 2016; Ray Chaudhuri et al, 2016). A body of evidence clearly delineates the importance of DNA repair proteins for mending DNA breaks after physical DNA damage (Moynahan and Jasin, 2010; Roy et al, 2011; Ceccaldi et al, 2016), this ever-growing list of classic DNA repair proteins acts directly in protecting DNA replication forks from damage
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