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bioRxiv · 10.1101/2025.11.28.691181

Molecular interplay between the DNA damage checkpoint kinase Mec1-Ddc2 and its activator Dpb11 on gapped DNA

Beckwitt, E. C.·Chua, G. N. L.·Liu, S.·O'Donnell, M. E.
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Abstract

The eukaryotic DNA damage and replication stress checkpoint is initiated by activation of the apical kinase complex ATR-ATRIP on RPA-coated ssDNA. In Saccharomyces cerevisiae, the Mec1-Ddc2 (hATR-ATRIP) activator and checkpoint mediator Dpb11 (hTopBP1) is recruited to the 9-1-1 checkpoint clamp (another Mec1-Ddc2 activator) at 5 ss-dsDNA junctions. It remains unclear how Mec1-Ddc2 encounters its activators on damaged DNA due to their differential DNA binding preferences. Using real-time single-molecule imaging, we show that Dpb11 binds to ssDNA directly and localizes to ss-dsDNA junctions in an RPA-dependent manner. Furthermore, Dpb11 recruits Mec1-Ddc2 to ss-dsDNA junctions. Single-molecule force spectroscopy was used to demonstrate that Dpb11 forms bridges on ssDNA, both alone and in the presence of RPA, reducing the end-to-end distance of gapped DNA. These data support a model in which Dpb11 facilitates Mec1-Ddc2 colocalization with its activators directly by recruiting Mec1-Ddc2 to gap junctions and indirectly by decreasing the effective gap length.

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Beckwitt, E. C., Chua, G. N. L., Liu, S., O'Donnell, M. E.. 2025-11-30. Molecular interplay between the DNA damage checkpoint kinase Mec1-Ddc2 and its activator Dpb11 on gapped DNA. https://doi.org/10.1101/2025.11.28.691181

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