Repair of DNA double-stranded breaks in human cells

Abstract

DNA is continuously subjected to degradation. Therefore, our cells need to constantly repair its DNA to prevent mutations and in the long run cancer. In mammalian cells, when DNA is broken right off, a double-stranded break (DSB) is produced, and the ends are ligated by a process called non-homologous end-joining (NHEJ). NHEJ involves a protein kinase, DNA-PK that binds and becomes activated by the DNA ends. DNA-PK is composed of the DNA-binding subunit Ku and the catalytic subunit DNA-PKcs. The overall aim of the present investigation was to take a closer look at the repair of DSBs in human cells and the involvement of the DNA-PK in this process. Using purified enzymes and kinase assays, we studied the activation of DNA-PK by different forms of broken DNA ends. DNA-PK was activated by DNA-ends containing a wide variety of modifications. This indicated that DSBs that remain undetected by DNA-PK are rare. The only exception was DSBs generated by the cancer drug etoposide. In this case the DSB is covered by topoisomerase II and can therefore not be recognized by DNA-PK.We also studied DNA-PK activation by DNA strand breaks generated by agents used in cancer treatment. DSBs produced by bleomycin, calicheamicin gamma1, and different kinds of radiation activated DNA-PK to maximal levels. We also compared the cleavage of cellular DNA and purified plasmid DNA by calicheamicin gamma1 (CLM), a drug used in cancer treatment. Our findings show that the ratio of DNA DSBs to single-stranded breaks (SSB) in cellular DNA was 1:3, close to the 1:2 ratio observed when CLM cleaved purified plasmid DNA. CLM-induced DSBs were repaired slowly but completely and resulted in a normal and strong DSB-response in cells. The high DSB/SSB ratio, specificity for DNA and the even damage distribution makes CLM a superior drug for studies of the DSB-response. Finally, we identified and characterized a DNA-PK-inhibitor, SU11752. SU11752 was a potent and selective inhibitor of DNA-PK. Results showed that SU11752 inhibited DNA-PK by ATP competition and sensitized cells 5-fold to ionizing radiation (IR) by inhibition of DSB repair. SU11752 defines a new class of drugs that serves as a starting point for development of specific DNA-PK inhibitors. Loss of DNA-PK results in increased sensitivity to IR due to inefficient repair of DNA DSBs. Over-expression of DNA-PK in tumor cells conversely results in resistance to IR, because DSBs are repaired quickly. It is therefore possible that inhibition of DNA-PK will enhance the preferential killing of tumor cells by radiotherapy.