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Double-strand breaks (DSBs) are DNA lesions that pose a significant threat to genomic stability. The repair of DSBs by the homologous recombination (HR) pathway is preceded by DNA end resection, the 5' to 3' nucleolytic degradation of DNA away from the DSB. We and others previously identified a role for RNF138, a really interesting new gene finger E3 ubiquitin ligase, in stimulating DNA end resection and HR. Yet, little is known about how RNF138's function is regulated in the context of DSB repair. Here, we show that RNF138 is phosphorylated at residue T27 by cyclin-dependent kinase (CDK) activity during the S and G2 phases of the cell cycle. We also observe that RNF138 is ubiquitylated constitutively, with ubiquitylation occurring in part on residue K158 and rising during the S/G2 phases. Interestingly, RNF138 ubiquitylation decreases upon genotoxic stress. By mutating RNF138 at residues T27, K158, and the previously identified S124 ataxia telangiectasia mutated phosphorylation site (Han et al., 2016, ref. 22), we find that post-translational modifications at all three positions mediate DSB repair. Cells expressing the T27A, K158R, and S124A variants of RNF138 are impaired in DNA end resection, HR activity, and are more sensitive to ionizing radiation compared to those expressing wildtype RNF138. Our findings shed more light on how RNF138 activity is controlled by the cell during HR., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Here, we present a chromatin-immunoprecipitation-based protocol to quantify the recruitment of proteins adjacent to site-specific DNA double-strand breaks (DSBs), such as proteins involved in DSB repair. We describe steps to induce DSBs in U2OS osteosarcoma cells stably expressing the restriction endonucleases FokI or AsiSI. We then detail the procedures of chromatin isolation and immunoprecipitation, followed by protein elution and quantitative-PCR-based quantification of DNA. This protocol cannot be used on DSBs generated at random loci by DNA damaging agents. For complete details on the use and execution of this protocol, please refer to Fitieh et al. (2022). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Multiple Myeloma (MM) is a B cell malignancy marked by genomic instability that arises both through pathogenesis and during disease progression. Despite recent advances in therapy, MM remains incurable. Recently, it has been reported that DNA repair can influence genomic changes and drug resistance in MM. The dysregulation of DNA repair function may provide an alternative explanation for genomic instability observed in MM cells and in cells derived from MM patients. This review provides an overview of DNA repair pathways with a special focus on their involvement in MM and discusses the role they play in MM progression and drug resistance. This review highlights how unrepaired DNA damage due to aberrant DNA repair response in MM exacerbates genomic instability and chromosomal abnormalities, enabling MM progression and drug resistance.