CtIP forms a tetrameric dumbbell-shaped particle which bridges complex DNA end structures for double-strand break repair

Abstract

CtIP is involved in the resection of broken DNA during the S and G2 phases of the cell cycle for repair by recombination. Acting with the MRN complex, it plays a particularly important role in handling complex DNA end structures by localised nucleolytic processing of DNA termini in preparation for longer range resection. Here we show that human CtIP is a tetrameric protein adopting a dumbbell architecture in which DNA binding domains are connected by long coiled-coils. The protein complex binds two short DNA duplexes with high affinity and bridges DNA molecules in trans. DNA binding is potentiated by dephosphorylation and is not specific for DNA end structures per se. However, the affinity for linear DNA molecules is increased if the DNA terminates with complex structures including forked ssDNA overhangs and nucleoprotein conjugates. This work provides a biochemical and structural basis for the function of CtIP at complex DNA breaks.

Phosphorylated CtIP bridges DNA to promote annealing of broken ends

“…For the linearized DNA, multiple complexes are observed with protein clusters associated along the contour of the DNA molecule. Furthermore, linear complexes were also observed with protein clusters localized at the ends, suggesting specific DNA end interaction, in agreement with previous reports on the CtIP function (27,37). We also performed AFM experiments for CtIP L27E , which preferentially promotes the formation of intermolecular complexes, which requires binding to free DNA ends.…”

“…The significant decrease in circularization efficiency for the structural mutants CtIP L27E and CtIP Δ160 suggests that the previously reported tetrameric structure (27) of wtCtIP imposes a key role in bridging DNA. You et al (37) reported that modifying a minimal damage recruitment (DR) motif (amino acids 509 to 557) in CtIP exerted a down-regulating effect on the damage recruitment capacity, possibly due to the reduced DNA-binding ability of the protein.…”

“…Both studies were based on AFM imaging, which gives information only on the static DNA-protein interactions for DNA deposited on a surface, leaving important features of the interaction between DNA and CtIP in solution unassessed, such as the conformational changes of genomic length DNA on CtIP binding, the transient bridging ability of the protein, potential sequence specificity of CtIP, as well as the dynamics of the DNA-CtIP interactions. Furthermore, CtIP phosphorylation, which is required for its function to promote MRN (16), was found to be inhibitory for DNA binding and annealing (27), raising questions as to whether the same population of CtIP can carry out both functions.…”

“…To promote the nuclease activity of MRN, CtIP needs to be phosphorylated (16). Although CtIP was previously shown to bind DNA by various methods, this binding was strongly inhibited by CtIP phosphorylation (16,27). This paradox raised the question of whether stimulation of MRN and DNA binding are mutually exclusive activities, mediated by two different subpopulations of CtIP.…”

CtIP promotes the motor activity of DNA2 to accelerate long-range DNA end resection

“…The activated MRN complex then preferentially cleaves the 5′terminated DNA strand past protein blocks. Therefore, this step is particularly important to process DNA ends with noncanonical structures, including bound proteins like Ku and topoisomerase-DNA cleavage complexes, as well as secondary DNA structures (14)(15)(16)(17)(18). CtIP is phosphorylated by multiple kinases including CDK, ATM, and ATR.…”