Abstract

DNA damage and changes in the mitochondrial DNA content have been implicated in ageing and cancer development. To prevent genomic instability and tumorigenesis, cells must maintain the integrity of their nuclear and mitochondrial DNA. Advances in the research of DNA damage protection and genomic stability, however, also depend on the availability of techniques that can reliably quantify alterations of mitochondrial DNA copy numbers and DNA lesions in an accurate high-throughput manner. Unfortunately, no such method has been established yet. Here, we describe the high-sensitivity long-run real-time PCR technique for DNA-damage quantification (LORD-Q) and its suitability to simultaneously measure DNA damage rates and mitochondrial DNA copy numbers in cultured cells and tissue samples. Using the LORD-Q multiplex assay, we exemplarily show that the mitochondrial DNA content does not directly affect DNA damage susceptibility, but influences the efficacy of certain anticancer drugs. Hence, LORD-Q provides a fast and precise method to assess DNA lesions, DNA repair and mtDNA replication as well as their role in a variety of pathological settings.

Highlights

  • Human cells possess hundreds of mitochondria containing a circular genome of approximately 16 kilobase pairs

  • Using the long-run real-time PCR technique for DNA-damage quantification (LORD-Q) multiplex assay, we exemplarily show that the mitochondrial DNA content does not directly affect DNA damage susceptibility, but influences the efficacy of certain anticancer drugs

  • Due to an established role of mitochondrial dysfunction in aging, cancer and various other diseases as well as the recognized association between mitochondrial DNA (mtDNA) damage and replication, it is important to elucidate the mechanisms underlying the maintenance of mtDNA integrity and mtDNA synthesis

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Summary

Introduction

Human cells possess hundreds of mitochondria containing a circular genome of approximately 16 kilobase (kb) pairs. We describe the highsensitivity long-run real-time PCR technique for DNA-damage quantification (LORD-Q) and its suitability to simultaneously measure DNA damage rates and mitochondrial DNA copy numbers in cultured cells and tissue samples. We describe an optimized flexible LORD-Q procedure that can be used to simultaneously determine the number of DNA lesions and mtDNAcn. Using this adapted LORD-Q assay we investigated the relationship between mtDNAcn alterations and DNA damage in human cells following exposure to different genotoxic insults.

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