Proteomics

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Integrative Structural Model of DNA-PK Synapsis

ABSTRACT: In non-homologous end joining repair of DNA double strand breaks, DNA dependent protein kinase catalytic subunit (DNA-PKcs) and Ku70/80 binds the free DNA ends forming the holoenzyme DNA-PK. DNA-PK synapses across the break to tether the broken ends in the initial long range synaptic complex. We generated an integrative structural model of DNA-PK synapsis at a precision of 13.5Å with crosslinking mass spectrometry (XL-MS) restraints. While our hydrogen deuterium exchange (HX) analysis revealed an allosteric axis in DNA-PK connecting DNA binding (including the plug domain) to the kinase domain. Our model presents a symmetrical synapsis primarily through head to head interactions and protection of the DNA by previously uncharacterized a plug domain. The offset of the DNA in our model allows access to downstream processing enzymes, while the combination of DNA binding and kinase loading creates a tensed state that could have roles in the re-arrangement/dissociation of DNA-PKcs as the repair process progresses.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Morgan Hepburn  

LAB HEAD: David C. Schriemer

PROVIDER: PXD017931 | Pride | 2021-01-20

REPOSITORIES: Pride

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The active DNA-PK holoenzyme occupies a tensed state in a staggered synaptic complex.

Hepburn Morgan M   Saltzberg Daniel J DJ   Lee Linda L   Fang Shujuan S   Atkinson Claire C   Strynadka Natalie C J NCJ   Sali Andrej A   Lees-Miller Susan P SP   Schriemer David C DC  

Structure (London, England : 1993) 20210106 5

In the non-homologous end-joining (NHEJ) of a DNA double-strand break, DNA ends are bound and protected by DNA-PK, which synapses across the break to tether the broken ends and initiate repair. There is little clarity surrounding the nature of the synaptic complex and the mechanism governing the transition to repair. We report an integrative structure of the synaptic complex at a precision of 13.5 Å, revealing a symmetric head-to-head arrangement with a large offset in the DNA ends and an extens  ...[more]

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