Holding it together: DNA end synapsis during non-homologous end joining

Loparo, Joseph J.

doi:10.1016/j.dnarep.2023.103553

Cited by 4 publications

(3 citation statements)

References 121 publications

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“…Upon binding to the broken DNA ends, Ku subsequently recruits most of the c-NHEJ factors, including enzymes responsible for end processing (nucleases, kinases and phosphatases), for nucleotide addition (pol X DNA polymerases) and for ligation (the XRCC4/Lig4 complex together with XLF and PAXX co-factors) ( 4 , 7 ). Important advances on molecular understanding of the c-NHEJ process have been gained recently thanks to single-molecule approaches and structural studies (reviewed in ( 8 , 9 )). The Ku heterodimer is composed of Ku70 and Ku80 proteins, which have a similarly arrangement of vWA, β-barrel and ARM domains, but differ at the C-terminus where Ku70 encodes a SAP domain and Ku80 a globular domain that is stabilised upon interaction with DNA-PK (Figure 1A ).…”

Section: Introductionmentioning

confidence: 99%

Structural and functional basis of inositol hexaphosphate stimulation of NHEJ through stabilization of Ku-XLF interaction

Kefala Stavridi,

Gontier,

Morin

et al. 2023

Nucleic Acids Research

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The classical Non-Homologous End Joining (c-NHEJ) pathway is the predominant process in mammals for repairing endogenous, accidental or programmed DNA Double-Strand Breaks. c-NHEJ is regulated by several accessory factors, post-translational modifications, endogenous chemical agents and metabolites. The metabolite inositol-hexaphosphate (IP6) stimulates c-NHEJ by interacting with the Ku70–Ku80 heterodimer (Ku). We report cryo-EM structures of apo- and DNA-bound Ku in complex with IP6, at 3.5 Å and 2.74 Å resolutions respectively, and an X-ray crystallography structure of a Ku in complex with DNA and IP6 at 3.7 Å. The Ku-IP6 interaction is mediated predominantly via salt bridges at the interface of the Ku70 and Ku80 subunits. This interaction is distant from the DNA, DNA-PKcs, APLF and PAXX binding sites and in close proximity to XLF binding site. Biophysical experiments show that IP6 binding increases the thermal stability of Ku by 2°C in a DNA-dependent manner, stabilizes Ku on DNA and enhances XLF affinity for Ku. In cells, selected mutagenesis of the IP6 binding pocket reduces both Ku accrual at damaged sites and XLF enrolment in the NHEJ complex, which translate into a lower end-joining efficiency. Thus, this study defines the molecular bases of the IP6 metabolite stimulatory effect on the c-NHEJ repair activity.

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Section: Introductionmentioning

confidence: 99%

Structural and functional basis of inositol hexaphosphate stimulation of NHEJ through stabilization of Ku-XLF interaction

Kefala Stavridi,

Gontier,

Morin

et al. 2023

Nucleic Acids Research

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“…The core NHEJ factors include Ku70, Ku80, DNA-PKcs, XLF, XRCC4 and Ligase 4 2,7 . The highly abundant heterodimer of Ku70 and Ku80 (Ku70/80) forms a ring structure that binds double-stranded DNA ends with a high affinity 8 .…”

Section: Introductionmentioning

confidence: 99%

“…Ku70/80 serves as the regulatory subunit of the DNA-dependent protein kinase (DNA-PK), which is activated when Ku70/80 binds DNA ends, resulting in recruitment of DNA-PKcs, the catalytic subunit of DNA-PK 9 . Ku70/80, and to a lesser extent DNA-PKcs, serve as a scaffold to target the other core NHEJ factors and numerous end-processing enzymes, many of which possess Ku-binding motifs that facilitate assembly of various NHEJ complexes 7,8,10 .…”

Section: Introductionmentioning

confidence: 99%

Single-molecule imaging reveals the kinetics of non-homologous end-joining in living cells

Mikhova

Heyza

Meek

et al. 2023

Preprint

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Non-homologous end joining (NHEJ) is the predominant path-way that repairs DNA double-stranded breaks (DSBs) in vertebrates. The DNA termini of many DSBs must be processed to allow ligation while minimizing genetic changes that result from break repair. Emerging models propose that DNA termini are first synapsed approximately 115Å apart in one of two long-range synaptic complexes. The first long-range complex can be formed with only the KU70/80 heterodimer and DNA-PKcs while the second long-range complex also includes XRCC4, XLF, and Ligase 4. Both long-range complexes inefficiently progress to short-range synaptic complexes that juxtapose DNA ends to facilitate ligation. Here we perform singlemolecule analyses of the recruitment of Halo-tagged NHEJ factors to DSBs. Our results provide direct evidence for stepwise maturation of NHEJ complex and precisely define kinetics of core NHEJ factor binding to DSBs in living cells.

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