Proximity Ligation Assay to Study Oncogene-Derived Transcription-Replication Conflicts

Summary

Here, we present a sensitive and specific method to detect oncogene-induced transcription-replication conflicts (TRCs) using the proximity ligation assay (PLA). This approach leverages specific antibodies targeting PCNA and phospho-CTD RNAPII, enabling the assessment of TRC prevalence between RNA polymerase II transcription and DNA replication machinery.

Abstract

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. Conflicts between the replication and transcription machinery, termed transcription-replication conflicts (TRCs), pose a considerable risk to genome stability, a critical factor in cancer development. While several factors regulating these collisions have been identified, pinpointing primary causes remains difficult due to limited tools for direct visualization and clear interpretation. In this study, we directly visualize TRCs using a proximity ligation assay (PLA), leveraging antibodies specific to PCNA and phosphorylated CTD of RNA polymerase II. This approach allows precise measurement of TRCs between replication and transcription processes mediated by RNA polymerase II. The method is further enhanced through DNA primers conjugated covalently to these antibodies, coupled with PCR amplification using fluorescent probes, providing a highly sensitive and specific means of detecting endogenous TRCs. Fluorescence microscopy enables the visualization of these conflicts, offering a powerful tool to study genome instability mechanisms associated with cancer. This technique addresses the gap in direct TRC visualization, allowing for a more comprehensive analysis and understanding of the underlying processes driving genome instability in cells.

Introduction

The genome serves as a template for essential biological processes, including replication and transcription. In healthy cells, RNA transcription and DNA replication machinery typically cooperate and are spatially and temporally segregated^1,2,3,4. However, under pathological conditions, such as oncogene overexpression, this harmonious cooperation can be disrupted.

Oncogenes often drive elevated transcription levels, increasing the likelihood of collisions between the transcription and replication machinery

Protocol

1. Lentivirus production and transduction into target cell line

1. Lentivirus production²⁴​
   1. Seed 293T packaging cells at 3 × 10⁶ cells per 10 cm-plate in high glucose Dulbecco's Modified Eagle Medium (DMEM; complete medium). Grow cells at 37 °C in a humidified 5% CO₂ incubator for an additional 18-20 h.
   2. In two 1.5 mL tubes, prepare a DNA mixture of a total of 24 µg plasmid DNA in 500 µL of Opti-MEM (reduced serum medium) that contains 9.2 µg of psPAX2, 2.8 µg of pMD2.G, along with 12 µg of pLVX-KRAS plasmid or empty vector respect....

Results

γH2AX serves as a biomarker for DNA damage. Overexpression of KRAS (G12D) impairs genomic stability in these cells, as evidenced by the increased γH2AX signal in Western blot analysis (Figure 2A). Additionally, the intensified S9.6 dot blot signal in KRAS (G12D)-expressing cells compared to vector controls (Figure 2B) indicates that oncogenic KRAS expression leads to the accumulation of aberrant R-loops, which may contribute to DNA break formation.

Discussion

The RNAPII transcription machinery can create a hindrance to DNA replication fork progression^26,27, promoting TRCs and DNA damage, especially in cancer cells^28,29. Deciphering the proteins that regulate TRCs and understanding the detailed mechanisms can help us comprehend how these harmful events occur and guide the development of new therapeutic approaches in the future. Therefore, to fully understand TR.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Clarity Western ECL Substrate	Bio-Rad	1705061
Duolink In Situ Detection Reagents Green	Sigma	DUO92014
FLAG antibody	Millipore	F7425
gamma H2AX antibody	Cell Signaling	25955
Glycogen, molecular biology grade	ThermoFisher	R0561
Image J	NIH	https://imagej.net/ij/
nitrocellulose membranes	Amersham	10600004
PCNA antibody	Cell Signaling	13110
pLVX-Kras G12D	N/A	N/A
pMD2.G	Addgene	12259
Proteinase K, recombinant, PCR grade	ThermoFisher	EO0491
psPAX2	Addgene	12260
RNAPII antibody	SCBT	sc-56767
S9.6 antibody	Active motif	65683
UV crosslinkers	Fisher Scientific	FB-UVXL-1000