Jean-Baptiste Charbonnier

DNA double-strand breaks (DSBs) are repaired by Ku-dependent non-homologous end-joining (NHEJ) or DNA end resection-dependent homologous recombination (HR). Previously, it was shown that short-range resection by MRX-pSae2 can remove Ku from DSBs and initiate resection, showing flexibility in DSB repair pathway choice. However, which NHEJ intermediates can be disrupted by resection factors remains unknown. Using reconstituted reactions with purified S. cerevisiae proteins, we show here that short-range resection by MRX-pSae2 is not directly inhibited by any of the core NHEJ factors. Instead, we find that MRX-pSae2 is inhibited structurally. DNA synapsis, a dynamic NHEJ intermediate that bridges DSBs, strongly suppresses resection. Non-ligatable ends are refractory to MRX-pSae2 once synapsis formed, and NHEJ assemblies with catalytic mutants of DNA ligase IV that permit synapsis but do not allow ligation likewise inhibit resection. We further demonstrate that removal of Ku from DSBs requires RPA, which interacts with MRX and pSae2 to synergistically stimulate MRX 3′−5′ exonuclease activity, generating free 3′ overhangs. Our data support a model in which DNA synapsis temporarily prevents resection, allowing NHEJ to execute ligation. If ligation fails and synapsis dissociates, MRX-pSae2 and RPA promote resection that removes non-productive NHEJ assemblies and channels DSB repair toward HR. The data help explain the suppression of NHEJ by RPA observed in previous genetic experiments.

Telomere capping largely depends on telomere length. Abnormally short telomeres are prone to activate DNA damage checkpoint, undergo unscheduled chromosomal fusions through nonhomologous end-joining (NHEJ) and be resected. All these processes are mediated by the Mre11–Rad50–Xrs2^NBS1 (MRX^MRN) complex. The response to telomere length is thought to correlate with the number of DNA-bound telomeric proteins but the mechanisms translating this number into functional protection remain unclear. Here, combining genetic and reconstitution biochemistry approaches, we explain why NHEJ inhibition and DNA resection suppression are proportional to the length of Rap1-bound arrays adjacent to DNA ends in Saccharomyces cerevisiae. Our findings reveal that MRX has a capacity to distinguish short from long protein blocks at DNA breaks, which translates into the ability of Rap1 protein–DNA arrays to suppress MRX on the basis of their length. Unlike the previously described direct inhibition of MRX^MRN by Rif2 and TRF2 iDDR, the process described here does not rely on direct protein–protein interactions. Instead, Rap1 inhibits MRX by steric hindrance, determined both by the length of DNA it shields and by the coverage density. These results explain how cells detect short telomeres, a crucial process for telomere length homeostasis and telomere-driven senescence.

Werner (WRN) is the only human RecQ helicase family member with DNA exonuclease activity. WRN promotes genome stability through its functions in DNA replication, repair and telomere maintenance, the deficiency of which presents clinically as Werner syndrome, causing premature aging and cancer predisposition. The main DNA double strand-break sensor Ku70/80 heterodimer (Ku) is a known partner of WRN, which stimulates its nuclease activity. However, the molecular basis of Ku-WRN interplay is currently unknown. Here, we present a high resolution cryo-EM structure of human Ku bound to DNA in complex with the N-terminal WRN exonuclease domain. This structure reveals multiple interaction sites between WRN and the Ku:DNA complex. The catalytic domain of WRN-exo engages with the DNA ends, stabilized by the vWA-like Ku80 domain interacting with the N-terminal APLF-like Ku binding motif (A-KBM) of WRN. Most surprisingly, we visualize the SAP domain of Ku70 stabilized within this complex, and we identify specific contacts mediating this interaction. These interactions are validated by assessing the impact of point mutations on either side of the Ku-WRN interfaces on exonuclease activity with purified recombinant proteins, and on live protein recruitment at biphoton laser-damaged nuclear sites. Finally, we show that disruption of WRN-Ku70 interaction results in aberrant resection of stalled replication forks. Together, we define the architecture of the Ku-WRN exonuclease domain interface and its impact on WRN exonuclease activity, recruitment and replication fork processing.

Cyclodipeptide synthases (CDPSs) sequentially use two aminoacyl-tRNAs (AA-tRNAs) as substrates to catalyze cyclodipeptide formation. We previously showed that microhelices (miHxs), which mimic the tRNAs acceptor arms, are as efficient as full-length AA-tRNAs as substrates when aminoacylated by flexizymes. We generated a diverse set of miHxs (acylated, unacylated, misacylated, mutated, or shortened miHxs) and analyzed their interactions with CDPSs. We studied the Nocardia brasiliensis CDPS (Nbra-CDPS), which synthesizes cyclo(l-Ala-l-Glu) using Ala-tRNA^Ala and Glu-tRNA^Glu as its first and second substrates, respectively. We determined the crystal structure of Nbra-CDPS bound to two analogues of its first substrate, unacylated miHx^Ala and acylated miHx^Ala, in which alanine is attached via an amide bond. We showed by cryoEM that the miHx^Ala mimics well the acceptor stem of the full-length tRNA^Ala. We determined the crystal structure of Nbra-CDPS bound to unacylated miHx^Glu, an analogue of its second substrate, and showed that, despite sequence differences, it superimposes well with miHx^Ala. This result, combined with the use of misacylated substrates, indicates that the RNA stem moieties of both substrates share a common binding mode. Together, our findings establish miHxs as powerful tools for dissecting CDPS substrate recognition and provide a framework for studying other AA-tRNA-utilizing enzymes.

Non-homologous end joining (NHEJ) repairs DNA double-strand breaks by synapsing and ligating DNA ends. In vertebrates, DNA-PKcs promotes end alignment and processing, yet several eukaryotes - including budding yeast - perform NHEJ without DNA-PKcs, and the underlying mechanism remains unclear. Here we report cryo-electron microscopy structures of reconstituted Saccharomyces cerevisiae NHEJ synaptic complexes assembled on DNA ends bearing either 4-bp terminal microhomologies or blunt termini. On the microhomology substrate with a single 5’-P, we capture a ligation-competent short-range complex in which a single Dnl4 catalytic core is fully ordered and engaged at the 5′-adenylated nick. When two 5’-P are present, we resolve two predominant, coexisting DNA-aligned protective states in which both Dnl4 DBD-NTD modules occupy the break region with reciprocal geometries, supporting an alternating engagement model for sequential sealing of the two strands while maintaining continuous end association. In contrast, blunt-ended substrates yield a non-aligned protective configuration in which two Dnl4 catalytic modules constrain the DNA ends ∼30 Å apart in a ligation-incompatible arrangement, providing a structural explanation for slow blunt-end joining in yeast. Together, these structures define architectural and mechanistic principles of DNA-PKcs-independent NHEJ.

DNA double-strand breaks (DSBs) are repaired by Ku-dependent non-homologous end-joining (NHEJ) or DNA end resection-dependent homologous recombination (HR). Previously, it was shown that short-range resection by MRX-pSae2 can remove Ku from DSBs and initiate resec-tion, showing flexibility in DSB repair pathway choice. However, which NHEJ intermediates can be disrupted by resection factors remains unknown. Using reconstituted reactions with purified S. cerevisiae proteins, we show here that short-range resection by MRX-pSae2 is not directly inhibited by any of the core NHEJ factors. Instead, we found that MRX-pSae2 is inhibited structurally. DNA synapsis, a dynamic NHEJ intermediate that bridges DSBs, strongly suppresses resection. Non-ligatable ends were refractory to MRX-pSae2 once synapsis formed, and NHEJ assemblies with catalytic mutants of DNA ligase IV that permit synapsis but do not allow ligation likewise inhibited resection. We further demonstrate that removal of Ku from DSBs requires RPA, which interacts with MRX and pSae2 to synergistically stimulate MRX 3′-5′ exonuclease activity, generating free 3′ overhangs. Our data support a model in which DNA synapsis temporarily prevents resection, allowing NHEJ to execute ligation. If ligation fails and synapsis dissociates, MRX-pSae2 and RPA promote resection that removes non-productive NHEJ assemblies and channels DSB repair toward HR. The data help explain the suppression of NHEJ by RPA observed in previous genetic experiments.

Werner (WRN) is the only human RecQ helicase family member with DNA exonuclease activity. WRN promotes genome stability through its functions in DNA replication, repair and telomere maintenance, the deficiency of which presents clinically as Werner syndrome, causing premature aging and cancer predisposition. The main DNA double strand-break sensor Ku70/80 heterodimer (Ku) is a known partner of WRN, which stimulates its nuclease activity. However, the molecular basis of Ku-WRN interplay is currently unknown. Here, we present a high resolution cryo-EM structure of human Ku bound to DNA in complex with the N-terminal WRN exonuclease domain. This structure reveals multiple interaction sites between WRN and the Ku:DNA complex. The catalytic domain of WRN-exo engages with the DNA ends, stabilized by the vWA-like Ku80 domain interacting with the N-terminal APLF-like Ku binding motif (A-KBM) of WRN. Most surprisingly, we visualize the SAP domain of Ku70 stabilized within this complex, and we identify specific contacts mediating this interaction. These interactions were validated by assessing the impact of point mutations on either side of the Ku-WRN interfaces on exonuclease activity with purified recombinant proteins, and on live protein recruitment at biphoton laser-damaged nuclear sites. Finally, we show that disruption of WRN-Ku70 interaction results in aberrant resection of stalled replication forks. Together, we define the architecture of the Ku-WRN exonuclease domain interface and its impact on WRN exonuclease activity, recruitment and replication fork processing.

DNA double-strand breaks (DSBs) are highly deleterious lesions that can trigger cell death or carcinogenesis if unrepaired or misrepaired. In mammals, most DSBs are repaired by non-homologous end joining (NHEJ), which begins when Ku70/80 binds DNA ends and recruits DNA-PKcs to form the DNA-PK holoenzyme. Although recent cryo-EM studies have resolved several NHEJ assemblies, how these factors access DSBs within nucleosomes remains unclear. Here, we present cryo-EM structures of human Ku70/80 and DNA-PK bound to nucleosomes. Ku70/80 binds the DNA end and bends it away from the nucleosome core, while the Ku70 C-terminal SAP domain makes an additional, specific DNA contact. Our DNA-PK–nucleosome structure further reveals the opening of the Ku80 vWA domain, and we show that non-hydrolysable ATP promotes synapsis by stabilising the Ku80-mediated DNA-PK dimer. These structures reveal a model for DSB recognition on nucleosomal DNA and provide insights relevant to targeting NHEJ in cancer therapy.

Safeguarding chromosome ends against fusions via nonhomologous end joining (NHEJ) is essential for genome integrity. Paradoxically, the conserved NHEJ core factor Ku binds telomere ends. How it is prevented from promoting NHEJ remains unclear, as does the mechanism that allows Ku to coexist with telomere-protective DNA binding proteins, Rap1 in Saccharomyces cerevisiae. Here, we find that Rap1 directly inhibits Ku’s NHEJ function at telomeres. A single Rap1 molecule near a double-stand break suppresses NHEJ without displacing Ku in cells. Furthermore, Rap1 and Ku form a complex on short DNA duplexes in vitro. Cryo-EM shows Rap1 blocks Ku’s inward translocation on DNA – an essential step for NHEJ at DSBs. Nanopore sequencing of telomere fusions confirms this mechanism protects native telomere ends. These findings uncover a telomere protection mechanism where Rap1 restricts Ku’s inward translocation. This switches Ku from a repair-promoting to a protective role preventing NHEJ at telomeres.

Non-homologous end joining (NHEJ) is the main repair pathway for double-strand DNA breaks (DSBs) in mammals. DNA polymerases lambda (Pol λ) and mu (Pol μ), members of the Pol X family, play a key role in this process. However, their interaction within the NHEJ complexes is unclear. Here, we present cryo-EM structures of Pol λ in complex with the DNA-PK long-range synaptic complex, and Pol μ bound to Ku70/80-DNA. These structures identify interaction sites between Ku70/80 and Pol X BRCT domains. Using mutants at the proteins interface in functional assays including cell transfection with an original gap-filling reporter, we define the role of the BRCT domain in the recruitment and activity of the two Pol X members in NHEJ and in their contribution to cell survival following DSBs. Finally, we propose a unified model for the interaction of all Pol X members with Ku70/80.