Multiplexed DNA repair assays for multiple lesions and multiple doses via transcription inhibition and transcriptional mutagenesis.
The capacity to repair different types of DNA damage varies among individuals, making them more or less susceptible to the detrimental health consequences of damage exposures. Current methods for measuring DNA repair capacity (DRC) are relatively labor intensive, often indirect, and usually limited to a single repair pathway. Here, we describe a fluorescence-based multiplex flow-cytometric host cell reactivation assay (FM-HCR) that measures the ability of human cells to repair plasmid reporters, each bearing a different type of DNA damage or different doses of the same type of DNA damage. FM-HCR simultaneously measures repair capacity in any four of the following pathways: nucleotide excision repair, mismatch repair, base excision repair, nonhomologous end joining, homologous recombination, and methylguanine methyltransferase. We show that FM-HCR can measure interindividual DRC differences in a panel of 24 cell lines derived from genetically diverse, apparently healthy individuals, and we show that FM-HCR may be used to identify inhibitors or enhancers of DRC. We further develop a next-generation sequencing-based HCR assay (HCR-Seq) that detects rare transcriptional mutagenesis events due to lesion bypass by RNA polymerase, providing an added dimension to DRC measurements. FM-HCR and HCR-Seq provide powerful tools for exploring relationships among global DRC, disease susceptibility, and optimal treatment.
Department of Biological Engineering, Center for Environmental Health Sciences, Department of Biology, and The David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139.
Zachary D
ZD
Nagel
Carrie M
CM
Margulies
Isaac A
IA
Chaim
Siobhan K
SK
McRee
Patrizia
P
Mazzucato
Anwaar
A
Ahmad
Ryan P
RP
Abo
Vincent L
VL
Butty
Anthony L
AL
Forget
Leona D
LD
Samson
D002460:Cell Line; D004249:DNA Damage; D059766:DNA End-Joining Repair; D053843:DNA Mismatch Repair; D004260:DNA Repair; D005434:Flow Cytometry; D017930:Genes, Reporter; D005821:Genetic Techniques; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics; D017423:Sequence Analysis, RNA; D014158:Transcription, Genetic; D014162:Transfection
D006147:Guanine; C008449:O-(6)-methylguanine
disease prevention; flow cytometry; high-throughput sequencing; personalized medicine