Fluorescence-Based Detection of FEN1 Nuclease Activity and Screening of Small-Molecule Inhibitors

Summary

This protocol describes a gel shift biochemical assay for measuring FEN1 (Flap Endonuclease 1) activity and inhibitor development.

Abstract

FEN1 (Flap Endonuclease 1) is a key DNA nuclease involved in DNA replication and damage repair, playing a crucial role in maintaining genomic stability. Elevated levels of FEN1 activity are closely associated with the development of various cancers, making FEN1 a recognized biomarker and drug target for lung, breast, and prostate cancers. Conventional radioisotope tagging methods for detecting FEN1 nuclease activity present challenges in terms of convenience and safety. In this study, a DNA fragment substrate with a flap structure was synthesized in vitro. This substrate is labeled with a fluorophore at the 5' end of the flap, allowing the FEN1 nuclease to specifically recognize and cleave the designated site. FEN1 activity was then determined by detecting the fluorophore signal after gel electrophoresis. This method enables rapid and effective assessment of FEN1 nuclease activity and the screening of its inhibitors. It provides a sensitive and specific approach for analyzing FEN1 activity and evaluating the efficacy of small-molecule inhibitors. Furthermore, it contributes significantly to research on DNA damage repair and cancer development, offering a novel strategy for clinical research on disease mechanisms and the detection of genotoxic substances.

Introduction

DNA replication is not always flawless and can be disrupted by both internal and external factors. Consequently, the mechanisms of DNA replication and damage repair are vital for cell survival and genome stability. In this process, the human branching structure-specific endonuclease FEN1 (Flap Endonuclease 1), a core DNA nuclease, plays a crucial role^1,2,3.

FEN1 is primarily involved in DNA replication and is indispensable for the maturation of Okazaki fragments on the lagging strand^4,5. Th....

Protocol

The reagents and the equipment used in this study are listed in the Table of Materials.

1. FEN1 protein expression purification

1. Protein purification
   1. Remove the bacterial stock containing the target plasmid (see Table of Materials) from −80 °C. Thaw at room temperature and inoculate 200 µL from the frozen 1.5 mL microcentrifuge tubes into 20 mL LB medium containing 50 µg/mL kanamycin using a 200 µL pipette tip.
      NOTE: The strain is E. coli BL21(DE3)-FEN1.
   2. Incubate at 37 ....

Results

As shown in Figure 2A, the staining reagent binds to protein, causing the protein to appear as a blue band on the gel. The first lane contains the labeled protein used as a molecular weight reference, in which the BSA standard protein band corresponds to a molecular weight of 70 kDa, and the FEN1 nucleic acid protein band corresponds to a molecular weight of 45 kDa. The experimental results showed that the FEN1 nuclease appeared as a clear band at approximately 45 kDa, indicating successful .......

Discussion

The FEN1 nuclease activity assay and small molecule inhibitor screening are central to research on DNA damage repair and cancer development. Compared to traditional radiolabeling techniques, the present method offers a cost-effective, efficient, easy-to-operate, and safer alternative. Additionally, unlike fluorescence burst-based FEN1 assays, this approach provides a more intuitive visualization of substrate cleavage, thereby allowing a more accurate assessment of enzyme activity and offering a more reliable platform for.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 M Tris-HCL	Beyotime	ST774
30% Acr-Bis (29:1)	Beyotime	ST003-500ml
5M Nacl	Beyotime	ST347
BeyoBlue Plus	Beyotime	P0003S
BL21(DE3)-FEN1	Vazyme	C504-F1
BSA	Beyotime	P0007
Decoloring shaker	Kylin-Bell	TS-100
DL-Dithiothreitol	Beyotime	ST041
Dry Bath Incubator	RUICHENG	DH200
Electrophoresis apparatus	Bei jing Liuyi Biotechnology	DDY-6D
FEN1-IN-4	TargetMOL	T8545
IPTG	Takaba	9030
Kanamycin Slufate	Biosharp	BS152
Laboratory Centrifuge	Thermo	SL 16R
LB Broth	Solarbio	L8291
Ni-NTA His Bind Resin	7se biotech	PANF001-001C
Odyssey FC	ODYSSEY	LI-COR
SDS-PAGE	Beyotime	P0015L
Trition-x-100	Shyuanye	S15022