TRIP12 governs DNA Polymerase ß involvement in DNA damage response and repair.

The multitude of DNA lesion types, and the nuclear dynamic context in which they occur, present a challenge for genome integrity maintenance as this requires the engagement of different DNA repair pathways. Specific 'repair controllers' that facilitate DNA repair pathway crosstalk between double strand break (DSB) repair and base excision repair (BER), and regulate BER protein trafficking at lesion sites, have yet to be identified. We find that DNA polymerase ß (Polß), crucial for BER, is ubiquitylated in a BER complex-dependent manner by TRIP12, an E3 ligase that partners with UBR5 and restrains DSB repair signaling. Here we find that, TRIP12, but not UBR5, controls cellular levels and chromatin loading of Polß. Required for Polß foci formation, TRIP12 regulates Polß involvement after DNA damage. Notably, excessive TRIP12-mediated shuttling of Polß affects DSB formation and radiation sensitivity, underscoring its precedence for BER. We conclude that the herein discovered trafficking function at the nexus of DNA repair signaling pathways, towards Polß-directed BER, optimizes DNA repair pathway choice at complex lesion sites.

HSP90 regulates DNA repair via the interaction between XRCC1 and DNA polymerase ß.

Cellular DNA repair processes are crucial to maintain genome stability and integrity. In DNA base excision repair, a tight heterodimer complex formed by DNA polymerase ß (Polß) and XRCC1 is thought to facilitate repair by recruiting Polß to DNA damage sites. Here we show that disruption of the complex does not impact DNA damage response or DNA repair. Instead, the heterodimer formation is required to prevent ubiquitylation and degradation of Polß. In contrast, the stability of the XRCC1 monomer is protected from CHIP-mediated ubiquitylation by interaction with the binding partner HSP90. In response to cellular proliferation and DNA damage, proteasome and HSP90-mediated regulation of Polß and XRCC1 alters the DNA repair complex architecture. We propose that protein stability, mediated by DNA repair protein complex formation, functions as a regulatory mechanism for DNA repair pathway choice in the context of cell cycle progression and genome surveillance.

Abnormal centrosomal structure and duplication in Cep135-deficient vertebrate cells.

Centrosomes are key microtubule-organizing centers that contain a pair of centrioles, conserved cylindrical, microtubule-based structures. Centrosome duplication occurs once per cell cycle and relies on templated centriole assembly. In many animal cells this process starts with the formation of a radially symmetrical cartwheel structure. The centrosomal protein Cep135 localizes to this cartwheel, but its role in vertebrates is not well understood. Here we examine the involvement of Cep135 in centriole function by disrupting the Cep135 gene in the DT40 chicken B-cell line. DT40 cells that lack Cep135 are viable and show no major defects in centrosome composition or function, although we note a small decrease in centriole numbers and a concomitant increase in the frequency of monopolar spindles. Furthermore, electron microscopy reveals an atypical structure in the lumen of Cep135-deficient centrioles. Centrosome amplification after hydroxyurea treatment increases significantly in Cep135-deficient cells, suggesting an inhibitory role for the protein in centrosome reduplication during S-phase delay. We propose that Cep135 is required for the structural integrity of centrioles in proliferating vertebrate cells, a role that also limits centrosome amplification in S-phase-arrested cells.

A centrosome-autonomous signal that involves centriole disengagement permits centrosome duplication in G2 phase after DNA damage.

DNA damage can induce centrosome overduplication in a manner that requires G2-to-M checkpoint function, suggesting that genotoxic stress can decouple the centrosome and chromosome cycles. How this happens is unclear. Using live-cell imaging of cells that express fluorescently tagged NEDD1/GCP-WD and proliferating cell nuclear antigen, we found that ionizing radiation (IR)-induced centrosome amplification can occur outside S phase. Analysis of synchronized populations showed that significantly more centrosome amplification occurred after irradiation of G2-enriched populations compared with G1-enriched or asynchronous cells, consistent with G2 phase centrosome amplification. Irradiated and control populations of G2 cells were then fused to test whether centrosome overduplication is allowed through a diffusible stimulatory signal, or the loss of a duplication-inhibiting signal. Irradiated G2/irradiated G2 cell fusions showed significantly higher centrosome amplification levels than irradiated G2/unirradiated G2 fusions. Chicken-human cell fusions demonstrated that centrosome amplification was limited to the irradiated partner. Our finding that only the irradiated centrosome can duplicate supports a model where a centrosome-autonomous inhibitory signal is lost upon irradiation of G2 cells. We observed centriole disengagement after irradiation. Although overexpression of dominant-negative securin did not affect IR-induced centrosome amplification, Plk1 inhibition reduced radiation-induced amplification. Together, our data support centriole disengagement as a licensing signal for DNA damage-induced centrosome amplification.