A Toolbox for Site-Specific Labeling of RecQ Helicase With a Single Fluorophore Used in the Single-Molecule Assay

Overview

Journal Front Mol Biosci

Specialty Biology

Date 2020 Oct 26

PMID 33102530

Citations 4

Authors

Fang-Yuan Teng

Zong-Zhe Jiang

Ling-Yun Huang

Man Guo

Feng Chen

Xi-Miao Hou

Xu-Guang Xi

Yong Xu

Affiliations

Soon will be listed here.

Abstract

Fluorescently labeled proteins can improve the detection sensitivity and have been widely used in a variety of biological measurements. In single-molecule assays, site-specific labeling of proteins enables the visualization of molecular interactions, conformational changes in proteins, and enzymatic activity. In this study, based on a flexible linker in the RecQ helicase, we established a scheme involving a combination of fluorophore labeling and sortase A ligation to allow site-specific labeling of the HRDC domain of RecQ with a single Cy5 fluorophore, without inletting extra fluorescent domain or peptide fragment. Using single-molecule fluorescence resonance energy transfer, we visualized that Cy5-labeled HRDC could directly interact with RecA domains and could bind to both the 3' and 5' ends of the overhang DNA dynamically for the first time. The present work not only reveals the functional mechanism of the HRDC domain, but also provides a feasible method for site-specific labeling of a domain with a single fluorophore used in single-molecule assays.

Citing Articles

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The HRDC domain oppositely modulates the unwinding activity of E. coli RecQ helicase on duplex DNA and G-quadruplex.

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