Structure and mechanism in non-homologous end joining

Vogt, Alex; He, Yuan

doi:10.1016/j.dnarep.2023.103547

Cited by 12 publications

(7 citation statements)

References 127 publications

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“…The current structural models of the DNA bound DNA-PK complex suggest that an intermediate step is involved in protecting and making DNA ends ligation-compatible before the long-range synapsis complex is converted to the short-range complex for end-ligation (4,6,24). Interestingly, two distinct long-range complexes were observed, each coping with different scenarios of end processing and ligation (6), indicating that at least two types of intermediate steps may have evolved to process different types of DNA ends.…”

Section: Resultsmentioning

confidence: 99%

“…However, it is until recent years when several structural analyses have collectively painted an elegant picture on how a KU-initiated large protein complex evolves to align two DSB ends for rejoining (4–8). The DNA-bound KU dimers recruit DNA-PKcs and then slide inward for about 15 nt to allow DNA-PKcs to occupy the DNA ends (9).…”

Section: Introductionmentioning

confidence: 99%

“…After the synapsed DNA ends are processed and become ligation- compatible, autophosphorylation of DNA-PKcs results in its eviction to transform the long-range synapsis complex to a short-range one that brings the two DSB ends to a close proximity for ligation by an iterative mechanism (6). Later, a presumable LIG4 re-configuration allows its ligase domain to reach the dsDNA ends and ligate, one strand at a time by each of the two LIG4 molecules in the short-range synapsis complex (4–6).…”

Section: Introductionmentioning

confidence: 99%

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The Mammalian KU70 C-terminus SAP Domain Is Required to Repair Exogenous DNA Damage

Wang,

Czap,

Kim

et al. 2024

Preprint

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The mammalian non-homologous end joining (NHEJ) is required for V(D)J recombination as well as coping with exogenously induced DNA double strand breaks (DSBs). Initiated by the binding of KU70/KU80 (KU) dimer to DNA ends and the subsequent recruitment of the DNA- dependent protein kinase catalytic subunit (DNA-PKcs), NHEJ plays a key role in DNA repair. While there has been significant structural understandings of how KU70 participates in NHEJ, the specific function of its highly conserved C-terminal SAP domain remains elusive. In this study, we developed a novel mouse model by deleting the SAP domain but preserving the KU70 nuclear localization and its dimerization ability with KU80. We found that the KU70 SAP deletion did not affect the V(D)J recombination or animal development but significantly impaired the animals and cells in repairing exogenously induced DSBs. We further showed an inability of KU70-ΔSAP cells to retain the DNA Ligase IV (LIG4) and other NHEJ co-factors on chromatin, and a spreading pattern of DSB marker γH2AX in KU70-ΔSAP cells after DNA damage. Our findings suggest that a specific inhibition of the SAP function may offer an opportunity to modulate cell sensitivity to therapeutic DSB-inducing agents without interfering with the developmental function of KU70.KeyPointsGeneration of a novel transgenic mouse line lacking the C-terminal conserved KU70-SAP domainKU70-SAP defends against exogenous DSBs, but unessential for development and V(D)J recombinationKU70-SAP aids in recruiting and retaining NHEJ components, such as LIG4, to DSB sites

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The Mammalian KU70 C-terminus SAP Domain Is Required to Repair Exogenous DNA Damage

Wang,

Czap,

Kim

et al. 2024

Preprint

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“…Over the past 3 years, the understanding of NHEJ mechanisms has vastly improved due to the significant amount of NHEJ complex structures that have been solved. In a recent excellent review by Vogt and He, the alternative mechanisms for NHEJ were discussed in great detail based on the solved structures of NHEJ complexes [121]. Future structural work on the recruitment and mechanisms of end-processing enzymes will help to further clarify how NHEJ is controlled.…”

Section: Structural Characterization Of Nhej Complexesmentioning

confidence: 99%

New Discoveries on Protein Recruitment and Regulation during the Early Stages of the DNA Damage Response Pathways

Waters,

Spratt

2024

IJMS

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Maintaining genomic stability and properly repairing damaged DNA is essential to staying healthy and preserving cellular homeostasis. The five major pathways involved in repairing eukaryotic DNA include base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), non-homologous end joining (NHEJ), and homologous recombination (HR). When these pathways do not properly repair damaged DNA, genomic stability is compromised and can contribute to diseases such as cancer. It is essential that the causes of DNA damage and the consequent repair pathways are fully understood, yet the initial recruitment and regulation of DNA damage response proteins remains unclear. In this review, the causes of DNA damage, the various mechanisms of DNA damage repair, and the current research regarding the early steps of each major pathway were investigated.

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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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Structure and mechanism in non-homologous end joining

Cited by 12 publications

References 127 publications

The Mammalian KU70 C-terminus SAP Domain Is Required to Repair Exogenous DNA Damage

The Mammalian KU70 C-terminus SAP Domain Is Required to Repair Exogenous DNA Damage

New Discoveries on Protein Recruitment and Regulation during the Early Stages of the DNA Damage Response Pathways

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

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