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SIRF: Quantitative in situ analysis of protein interactions at DNA replication forks
Sunetra Roy1, Jessica W Luzwick1, Katharina Schlacher2
1Department of Cancer Biology, University of Texas MD Anderson Cancer Center, Houston, TX.
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.
Insights
A new assay, SIRF, quantifies protein interactions with nascent DNA at replication forks. This method provides insights into DNA replication regulation and its role in diseases like cancer.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- DNA replication is crucial for cell function, with errors linked to diseases like cancer and developmental defects.
- Existing tools lack efficiency for analyzing protein interactions at replication forks, hindering biological understanding.
Purpose of the Study:
- To develop a sensitive, single-cell assay for quantitative analysis of protein interactions with nascent DNA at replication forks.
- To provide a tool for multiparameter analysis in heterogeneous cell populations.
Main Methods:
- Developed in situ analysis of protein interactions at DNA replication forks (SIRF) assay.
- Utilized proximity ligation coupled with 5'-ethylene-2'-deoxyuridine click chemistry.
- Validated for sensitivity, accuracy, proximity, and quantitation.
Main Results:
- Demonstrated robust, quantitative, and sensitive single-cell analysis of protein-DNA interactions at replication forks.
- Successfully applied SIRF to gain new insights into 53BP1 regulation at stalled replication forks.
- Validated the assay's suitability for multiparameter analysis in complex cell populations.
Conclusions:
- SIRF is a powerful new tool for studying DNA replication dynamics and protein interactions.
- The assay facilitates deeper understanding of replication fork regulation and its implications in disease.
- SIRF enables accurate analysis in heterogeneous cell populations, advancing biological research.
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