63P - Clinical dysregulation of DNA repair by the Polynucleotide Kinase/Phosphatase-XRCC4-DNA Ligase IV in neurological disease

Date 11 September 2017
Event ESMO 2017 Congress
Session Poster display session
Topics Basic Science
Presenter Daniel Aceytuno
Citation Annals of Oncology (2017) 28 (suppl_5): v1-v21. 10.1093/annonc/mdx361
Authors D. Aceytuno1, C.G. Piett2, Z. Havali-Shahriari1, R.A. Edwards1, M. Rey3, R.S. Mani4, S. Fang2, M. Hammel5, M. Weinfeld4, J.A. Tainer6, D. Schriemer2, S.P. Lees-Miller2, J.N.M. Glover1
  • 1Biochemistry, University of Alberta, T6G2H7 - Edmonton/CA
  • 2Biochemistry & Molecular Biology, Arnie Charbonneau Cancer Institute, University of Calgary, Calgary/CA
  • 3Biochemistry & Molecular Biology, University of Calgary, Calgary/CA
  • 4Oncology, Cross Cancer Institute, Edmonton/CA
  • 5Molecular Biophysics & Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley/US
  • 6Molecular And Cellular Oncology, University of Texas M. D. Anderson Cancer Center, Houston/US

Abstract

Background

If not repaired, DNA double-strand breaks (DSBs) can lead to genomic instability and cell death or neoplastic transformation. The major DSB repair (DSBR) mechanism in higher eukaryotes is non-homologous end-joining (NHEJ). In NHEJ, polynucleotide kinase/phosphatase (PNKP) is the primary enzyme for processing abnormal 5'-OH and 3'-phosphate ends that prevent the final repair step by XRCC4/DNA Ligase IV (Lig IV). This processing step is thought to be mediated by an interaction between the PNKP-FHA domain and CK2-phosphorylated XRCC4 C-terminal tails.

Methods

See below.

Results

Our binding assays show tight binding between XRCC4/Lig IV and PNKP both with and without CK2-phosphorylation of XRCC4. Low-resolution ensemble structures of purified phosphorylated-XRCC4/Lig IV/PNKP ternary complex by small-angle X-ray scattering (SAXS) experiments also suggest a second phosphorylation-independent interaction between the PNKP and XRCC4/Lig IV. Hydrogen-deuterium exchange (HDX) experiments have identified a candidate for this secondary interaction site within a loop in the PNKP phosphatase domain. This site contains the clinically significant PNKP E326K mutation found in the severe form of the hereditary neurological disease MCSZ (microcephaly with early-onset intractable seizures and developmental delay). Activity assays show that the E326K mutation decreases both substrate binding and turnover in PNKP when bound to phosphorylated-XRCC4/Lig IV. Furthermore, UV laser microirradiation in cells show that the E326K mutation also disrupts recruitment of PNKP to DNA lesions.

Conclusions

We have identified a putative secondary interaction site that functionally contributes both to recruitment and catalysis of PNKP in NHEJ. Disruptions to PNKP in this region may result in decreased DNA double-strand break repair in cells and describe a molecular basis of MCSZ. Further, PNKP has other known clinical neurological significance and its presence on chromosome arm 19q has interesting implications in oligodendrogliomas. This interaction surface may prove an interesting target for small-molecule inhibition of DNA strand break repair toward novel radio- and chemo-sensitizing therapies in cancer treatment.

Clinical trial identification

Legal entity responsible for the study

University of Alberta

Funding

Canadian Institutes of Health Research, Alberta Cancer Foundation, Structural Biology of DNA Repair Machines Consortium.

Disclosure

All authors have declared no conflicts of interest.