Eukaryotic genomes are constantly at risk of damage. Metabolic processes, UV exposure, DNA replication and merely packaging chromatin can introduce DNA damage at low rates. Thus, many DNA damage repair enzymes have evolved to be essential in eukaryotes. The importance of this pathways is demonstrated by breast and ovarian cancers. Hereditary mutations in DNA damage repair genes significantly increase the risk of developing these cancers. This pathway is not merely mutated in cancers but serves as a therapeutic target. In some instances overwhelming or inhibiting DNA damage repair in breast/ovarian tumors has been a successful treatment. Despite these successes and the clear importance of DNA damage repair, we do not have a complete molecular understanding of this process. Researchers in the Taniguchi Lab (Human Biology Division) recently expanded this understanding by identifying a protein kinase (NEK8) previously unknown to regulate DNA damage response. In a recent article published in Cell Cycle, Antonio Abeyta and colleagues show that NEK8 is a non-essential gene important for response to replication stresses including DNA damage.
Figure provided by Dr. Toshiyasu Taniguchi
DNA damage repair signaling is known to be mediated by multiple kinases thus researchers performed an RNAi screen to identify any kinases not previously known to regulate this process. This approach specifically identified kinases that contribute to recognizing/repairing DNA double strand breaks induced by the alkylating agent mitomycin C. Normal response to mitomycin C is the recruitment of RAD51 protein to sites of double strand breaks forming dozens of foci in the nucleus; however, when essential members of this pathway (e.g. RAD51 or BRCA2) are depleted by siRNA the number of RAD51 foci significantly decreases. Thus researchers treated cells with siRNA targeting 713 kinases and screened for cells lacking RAD51 foci. NEK8 was identified as being the most important of the 713 kinases tested for RAD31 foci formation. Importantly, researchers validated the screen results by showing that NEK8 contributes to repairing many types of DNA damage including: inter-strand crosslinks due to γ irradiation, mitomycin C mediated alkylation, and replication fork stalls by hydroxyurea.
Surprisingly, while DNA damage repair is an essential process NEK8 can be deleted in genetically engineered mouse models and embryonic fibroblasts (MEFs) grown in culture. While these cells are viable, researchers found that they are hypersensitive to replication stresses including many DNA damaging agents as well as anti-mitotic compounds. When NEK8-null and regular MEFs were treated with these drugs at the same concentrations 10-100x fewer NEK8-null MEFs survived.
While these results emphasize the importance of NEK8 in repairing DNA damage, they do not elucidate how NEK8 contributes mechanistically to the repair. NEK8 was originally identified in the screen because its depletion resulted in a loss of RAD51 foci suggesting NEK8 functions in the same pathway as RAD51. A key step in repairing DNA is the short resection of DNA strands at the site of damage. This nuclease activity is performed by MRE-11 while RAD51 protects DNA by antagonizing MRE-11 and ensuring only short stretches of DNA are digested. To test if NEK8 protects DNA from MRE-11 activity, researchers treated NEK8-null MEFs with fluorescent dNTPs to be incorporated at sites of DNA synthesis and then stalled DNA replication using hydroxyurea. These stalled replication forks recruit DNA damage repair machinery and the fluorescent DNA is resected by MRE-11. In this assay NEK8-null MEFs treated with hydroxyurea had shorter stretches of fluorescent DNA. Researchers demonstrated this was not due to slower replication rates, but more consistent with increased nuclease activity. This was further supported by simultaneously inhibiting MRE-11 in the NEK8-null MEFs and finding this partially rescued the length of fluorescent DNA tracts.
Work such as this reminds us that biology is complex and there are still many things to discover – even in well-studied processes such as DNA damage repair. This initial study from the Taniguchi lab suggests many new exciting questions; while NEK8 is a kinase protein it was not tested if kinase activity is required for RAD51 foci formation. Future studies will focus on understanding the molecular details of this process explained Dr. Toshiyasu Taniguchi, “Antonio Abeyta will follow up the work by elucidating the precise mechanisms by which NEK8 regulates replication fork stability and RAD51 recruitment.”
Abeyta A, Castella M, Jacquemont C, Taniguchi T. 2016. NEK8 regulates DNA damage-induced RAD51 foci formation and replication fork protection. Cell Cycle, 0.
Funding for this research was provided by Howard Hughes Medical Institute, the National Cancer Institute (NIH), and the National Science Foundation.