10.5061/DRYAD.MSBCC2FVD
Howard, Sean
0000-0001-7556-1951
Institute for Research in Biomedicine
Ceppi, Ilaria
Institute for Research in Biomedicine
Anand, Roopesh
Institute for Research in Biomedicine
Geiger, Roger
0000-0002-8016-1669
Institute for Research in Biomedicine
Cejka, Petr
Institute for Research in Biomedicine
The internal region of CtIP functions as a regulator of DNA end resection
Dryad
dataset
2020
DNA repair
end resection
CtIP
MRN
Single strand annealing
Swiss National Science Foundation*
31003A_175444
European Research Council
https://ror.org/0472cxd90
681630
European Molecular Biology Organization
https://ror.org/04wfr2810
ALTF 1560-2015
Swiss National Science Foundation
https://ror.org/00yjd3n13
31003A_175444
2020-04-13T00:00:00Z
2020-04-13T00:00:00Z
en
622747576 bytes
4
CC0 1.0 Universal (CC0 1.0) Public Domain Dedication
DNA double-strand breaks are repaired by end-joining or homologous
recombination. A key-committing step of recombination is DNA end
resection. In resection, phosphorylated CtIP first promotes the
endonuclease of the MRE11-RAD50-NBS1 (MRN) complex. Subsequently, CtIP
also stimulates the BLM-DNA2 helicase-nuclease, coordinating thus both
short and long-range resection. The structure of CtIP differs from its
orthologues in yeast, as it contains a large internal region. Here we
conducted a domain analysis of the internal region of CtIP to define its
function in DNA end resection. We found that residues 350-600 were
entirely dispensable for resection in vitro. A mutant lacking these
residues was unexpectedly more efficient than full-length CtIP in DNA end
resection and homologous recombination in vivo, and consequently conferred
resistance to lesions induced by the topoisomerase poison camptothecin,
which require high MRN-CtIP-dependent resection activity for repair. This
suggested that the internal region, further mapped to residues 550-600,
may mediate a negative regulatory function to prevent over resection in
vivo. Consequently, the CtIP internal deletion mutant exhibited
sensitivity to other DNA-damaging drugs, showing that upregulated
resection may be instead toxic under other conditions. These experiments
together identify a region within the central CtIP domain that negatively
regulates DNA end resection.
This dataset was collected by a variety of methods including cell-base
flow cytometry reporters, Western blotting, RT-PCR, in vitro biochemical
assays and clonogenic survival assays.