Genetic analysis of the septal peptidoglycan synthase FtsWI complex supports a conserved activation mechanism for SEDS-bPBP complexes

SEDS family peptidoglycan (PG) glycosyltransferases, RodA and FtsW, require their cognate transpeptidases PBP2 and FtsI (class B penicillin binding proteins) to synthesize PG along the cell cylinder and at the septum, respectively. The activities of these SEDS-bPBPs complexes are tightly regulated to ensure proper cell elongation and division. In Escherichia coli FtsN switches FtsA and FtsQLB to the active forms that synergize to stimulate FtsWI, but the exact mechanism is not well understood. Previously, we isolated an activation mutation in ftsW (M269I) that allows cell division with reduced FtsN function. To try and understand the basis for activation we isolated additional substitutions at this position and found that only the original substitution produced an active mutant whereas drastic changes resulted in an inactive mutant. In another approach we isolated suppressors of an inactive FtsL mutant and obtained FtsWE289G and FtsIK211I and found they bypassed FtsN. Epistatic analysis of these mutations and others confirmed that the FtsN-triggered activation signal goes from FtsQLB to FtsI to FtsW. Mapping these mutations, as well as others affecting the activity of FtsWI, on the RodA-PBP2 structure revealed they are located at the interaction interface between the extracellular loop 4 (ECL4) of FtsW and the pedestal domain of FtsI (PBP3). This supports a model in which the interaction between the ECL4 of SEDS proteins and the pedestal domain of their cognate bPBPs plays a critical role in the activation mechanism.

TRIM21–SERPINB5 aids GMPS repression to protect nasopharyngeal carcinoma cells from radiation-induced apoptosis

Nasopharyngeal carcinoma (NPC) is the most prevalent head and neck malignancy in South China and Southeast Asia. The main NPC treatment strategy is radiotherapy. However, recurrence resulting from radioresistance is a leading clinical bottleneck. Revealing the mechanism of NPC radioresistance would help improve the therapeutic effect. Here, our work reveals thatTRIM21(tripartite motif–containing 21) functions as an oncogene in NPC progression, and its ablation increases NPC cell radiosensitivity. Further analysis indicated that TRIM21 represses TP53 expression by mediating GMPS (guanine monophosphate synthase) ubiquitination and degradation after ionizing radiation. Mass spectrometry and co-immunoprecipitation showed that SERPINB5 (serpin family B member 5) interacts with both TRIM21 and GMPS. Epistatic analysis showed that SERPINB5 acts as an adaptor to recruit GMPS and introduce TRIM21 for ubiquitination. The in vitro and in vivo results validated the finding that SERPINB5 promotes NPC cell radioresistance. Furthermore, immunohistochemistry indicated that radioresistant patients have higher SERPINB5 expression. Overall, our data show that TRIM21–SERPINB5-mediated GMPS degradation facilitates TP53 repression, which promotes the radioresistance of NPC cells.

Both High-Fidelity Replicative and Low-Fidelity Y-Family Polymerases Are Involved in DNA Rereplication

DNA rereplication is a major form of aberrant replication that causes genomic instabilities, such as gene amplification. However, little is known about which DNA polymerases are involved in the process. Here, we report that low-fidelity Y-family polymerases (Y-Pols), Pol η, Pol ι, Pol κ, and REV1, significantly contribute to DNA synthesis during rereplication, while the replicative polymerases, Pol δ and Pol ε, play an important role in rereplication, as expected. When rereplication was induced by depletion of geminin, these polymerases were recruited to rereplication sites in human cell lines. This finding was supported by RNA interference (RNAi)-mediated knockdown of the polymerases, which suppressed rereplication induced by geminin depletion. Interestingly, epistatic analysis indicated that Y-Pols collaborate in a common pathway, independently of replicative polymerases. We also provide evidence for a catalytic role for Pol η and the involvement of Pol η and Pol κ in cyclin E-induced rereplication. Collectively, our findings indicate that, unlike normal S-phase replication, rereplication induced by geminin depletion and oncogene activation requires significant contributions of both Y-Pols and replicative polymerases. These findings offer important mechanistic insights into cancer genomic instability.