Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

Summary

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

Abstract

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Protocol

In this protocol we describe the method for analysis of DNA DSB repair with chromosomally integrated reporter constructs^1,2 where DSBs are induced in vivo by transient expression a rare cutting endonuclease I-SceI³. The integrated assay provides the advantage of analyzing DSB repair within the chromosomal context. However, this protocol requires prolonged cell passaging, which may be problematic when working with primary cells.

An alternative approach is an extrachromosomal assay where reporter constructs are digested in vitro with I-SceI or HindIII endonucleases and then transfected into cells as li....

Discussion

The fluorescent NHEJ and HR reporter assays provide a quantitative way for separately measuring each DSB repair pathway in vivo. The assays are very sensitive, as FACS can reliably detect 10 GFP+ cells in 20,000 cells. The assays may be adapted for measuring repair events in "real time" by detecting the appearance of GFP+ cells within minutes or hours after induction of DSBs². Furthermore, the analysis of GFP+ cells does not rely on additional cell proliferation, allowing DSB repair to be analyzed a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
EndoFree Plasmid Maxi kit	Qiagen	12362
Qiaex II Gel Extraction Kit	Qiagen	20021
Amaxa Nucleofector	Lonza Inc.	AAD-1001
Geneticin (G418)	Invitrogen	11811-031
pDsRed2-N1	Clontech Laboratories	632406
Round bottom tubes	BD Biosciences	352058	FACS tubes