Achieving selectivity in space and time with DNA double-strand-break response and repair: molecular stages and scaffolds come with strings attached

Abstract

When double-strand breaks (DSBs) in DNA remain unrepaired, catastrophic loss of genes occurs, leading to translocations, mutations and carcinogenesis. If a sister chromatid is not available at the DNA DSB, nonhomologous end joining (NHEJ) is used to join broken ends. The NHEJ pathway comprises synapsis, end processing and ligation. Here, we ask how DSBs in DNA are repaired efficiently. We suggest that colocation of proteins is achieved over time by the following components: stages, where the main actors are assembled, scaffolds that are erected quickly around broken parts to give access, and strings that tether proteins together. In NHEJ, a stage is provided by the Ku heterodimer interacting with DSBs and several other proteins including DNA-PKcs, APLF, BRCA1 and PAXX. A further stage, DNA-PKcs, links the kinase with DNA, Ku, PARP1, BRCA1 and Artemis. A temporary scaffold facilitates repair and is constructed from XRCC4/XLF filaments that bridge Ku bound at DSB ends. LigIV bound to XRCC4 C-termini likely terminates the scaffold, bringing LigIV close to the DNA broken ends. A string, provided by the Artemis C-terminal region, is intrinsically disordered but includes short linear ''epitopes'' that recognise DNA-PKcs, LigIV and PTIP, so keeping these components nearby. We show that these stages, scaffolds and strings facilitate colocation and efficient DSB repair. Understanding these processes provides insight into the biology of DNA repair and possible therapeutic intervention in cancer and other diseases.

PAXX and its paralogs synergistically direct DNA polymerase λ activity in DNA repair

“…In addition to their interaction with core NHEJ proteins, PAXX or its paralogs also associated with a variety of NHEJ accessory factors including PNKP, APTX, WRN, PARP1 and Pol λ (Figs 1 , 2 , Supplementary Tables 6 and 7 ). Notably, PAXX bound Pol λ in the soluble chromatin fraction, which plays an important role during NHEJ to fill DNA gaps (Supplementary Tables 6 , 7 and Supplementary Data 1 ) 4 , 5 . PAXX also associated with multiple subunits of the trimeric protein phosphatase 6 holoenzyme, which directly interacts with and regulate DNA-PK function (Fig.…”

“…NHEJ occurs during all phases of the cell cycle, including the G1 phase when cells are uniquely dependent on NHEJ. NHEJ occurs in a series of stages and requires co-ordinated involvement of a repertoire of key proteins 4 – 6 . Initially, the DSB is sensed by Ku70/80 heterodimers leading to recruitment of DNA-PKcs (known together as the DNA-PK holoenzyme complex), activation of its protein kinase activity and tethering of the DNA termini.…”

“…These include the nuclease Artemis, polynucleotide kinase-phosphatase (PNKP), Werner (WRN) helicase and the family X DNA polymerases λ (Pol λ) and μ (Pol μ) 6 . DNA-bound Ku also recruits two structurally related proteins, XRCC4- and XRCC4-like factor (XLF/Cernunnos/NHEJ1), which independently from Ku can form long filaments facilitating alignment of DNA ends prior to ligation, the final step of NHEJ mediated by DNA Ligase IV (Lig IV) 4 – 9 . NHEJ reactions proceed via a variety of so-called “subpathways”, which utilise various subsets of NHEJ proteins and differ in the way distinct DNA ends are processed prior to ligation 6 .…”

Dissection of DNA double-strand-break repair using novel single-molecule forceps

“…The XLF-XRCC4 complexes interact with DNA-PK assembled at DNA ends and are believed to bridge spatially two DNA ends so as to facilitate ligation of the break by Ligase IV [ 20 , 21 ]. Although the overall process is understood with the key components identified and characterized through various approaches, critical information concerning the forces that hold together DNA ends, the kinetics of assembly and disassembly of NHEJ complexes, as well as the robustness of the repair pathway to fluctuations in complex composition remain unknown [ 22 , 23 ]. Here we used single-molecule experimentation along with a novel DNA substrate to enable mechanical and energetic analysis of molecular interactions across a DNA break and address these questions.…”

Combating mutations in genetic disease and drug resistance: understanding molecular mechanisms to guide drug design

“…However, the lack of methods to identify such inactive conformations or binding modes in kinases to drive the development of this type of inhibitor still remains a challenge 80 . Inhibitors that disrupt formation of the higher order oligomers, which play an important role in achieving high signalto-noise throughout the signal transduction process, have also proven to be effective kinase inhibitors that avoid the common ATP resistance mutations [81][82][83] . ABL001, also known as Asciminib, is a potent and selective third generation kinase inhibitor with activity against chronic myeloid leukemia and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia.…”