DNA break induces rapid transcription repression mediated by proteasome-dependent RNAPII removal

Shuaixin He; Zhiyuan Huang; Yang Liu; Taekjip Ha; Bin Wu

doi:10.1016/j.celrep.2024.114420

DNA break induces rapid transcription repression mediated by proteasome-dependent RNAPII removal

Cell Rep. 2024 Jul 23;43(7):114420. doi: 10.1016/j.celrep.2024.114420. Epub 2024 Jul 1.

Authors

Shuaixin He¹, Zhiyuan Huang¹, Yang Liu², Taekjip Ha³, Bin Wu⁴

Affiliations

¹ Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; The Center for Cell Dynamics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
² Department of Biochemistry, The University of Utah, Salt Lake City, UT 84112, USA.
³ Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; The Center for Cell Dynamics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA.
⁴ Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; The Center for Cell Dynamics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; The Solomon H Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Electronic address: bwu20@jhmi.edu.

Abstract

A DNA double-strand break (DSB) jeopardizes genome integrity and endangers cell viability. Actively transcribed genes are particularly detrimental if broken and need to be repressed. However, it remains elusive how fast the repression is initiated and how far it influences the neighboring genes on the chromosome. We adopt a recently developed, very fast CRISPR to generate a DSB at a specific genomic locus with precise timing, visualize transcription in live cells, and measure the RNA polymerase II (RNAPII) occupancy near the broken site. We observe that a single DSB represses the transcription of the damaged gene in minutes, which coincides with the recruitment of a damage repair protein. Transcription repression propagates bi-directionally along the chromosome from the DSB for hundreds of kilobases, and proteasome is evoked to remove RNAPII in this process. Our method builds a foundation to measure the rapid kinetic events around a single DSB and elucidate the molecular mechanism.

Keywords: CP: Molecular biology; CRISPR-Cas9; DNA damage repair; proteasome; single molecule; transcription.

MeSH terms

DNA Breaks, Double-Stranded*
DNA Repair
Humans
Proteasome Endopeptidase Complex* / metabolism
RNA Polymerase II* / metabolism
Transcription, Genetic*

Substances

RNA Polymerase II
Proteasome Endopeptidase Complex

Abstract

MeSH terms

Substances

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