Genomic Mapping of DNA-Repair Reaction Intermediates in Living Cells With Engineered DNA Structure-Trap Proteins

Authors

Jingjing Liu
Qian Mei
Sadeieh Nimer
Devon M Fitzgerald
Susan M Rosenberg

Publication Date

1-1-2021

Journal

Methods in Enzymology

DOI

10.1016/bs.mie.2021.09.015

PMID

34776211

PMCID

PMC9502303

PubMedCentral® Posted Date

9-23-2022

PubMedCentral® Full Text Version

Author MSS

Published Open-Access

yes

Keywords

DNA, DNA Repair, DNA Replication, DNA, Cruciform, Escherichia coli, Escherichia coli Proteins, Genomics

Abstract

Diverse DNA structures occur as reaction intermediates in various DNA-damage and -repair mechanisms, most of which results from replication stress. We harness the power of proteins evolutionarily optimized to bind and "trap" specific DNA reaction-intermediate structures, to quantify the structures, and discern the mechanisms of their occurrence in cells. The engineered proteins also allow genomic mapping of sites at which specific DNA structures occur preferentially, using a structure-trapping protein and ChIP-seq- or Cut-and-Tag-like methods. Genome-wide identification of sites with recurrent DNA-damage intermediates has illuminated mechanisms implicated in genome instability, replication stress, and chromosome fragility. Here, we describe X-seq, for identifying sites of recurrent four-way DNA junctions or Holliday-junctions (HJs). X-seq uses an engineered, catalysis-defective mutant of Escherichia coli RuvC HJ-specific endonuclease, RuvCDefGFP. X-seq signal indicates sites of recombinational DNA repair or replication-fork stalling and reversal. We also describe methods for genomic mapping of 3'-single-stranded DNA ends with SsEND-seq, in E. coli. Both methods allow genomic profiling of DNA-damage and -repair intermediates, which can precede genome instability, and are expected to have many additional applications including in other cells and organisms.