SummaryThe tumor suppressor protein 53BP1, a pivotal regulator of DNA double-strand break (DSB) repair, was first identified as a p53-interacting protein over two decades ago. However, its direct contributions to p53-dependent cellular activities remain undefined. Here, we reveal that 53BP1 stimulates genome-wide p53-dependent gene transactivation and repression events in response to ionizing radiation (IR) and synthetic p53 activation. 53BP1-dependent p53 modulation requires both auto-oligomerization and tandem-BRCT domain-mediated bivalent interactions with p53 and the ubiquitin-specific protease USP28. Loss of these activities results in inefficient p53-dependent cell-cycle checkpoint and exit responses. Furthermore, we demonstrate 53BP1-USP28 cooperation to be essential for normal p53-promoter element interactions and gene transactivation-associated events, yet dispensable for 53BP1-dependent DSB repair regulation. Collectively, our data provide a mechanistic explanation for 53BP1-p53 cooperation in controlling anti-tumorigenic cell-fate decisions and reveal these activities to be distinct and separable from 53BP1’s regulation of DNA double-strand break repair pathway choice.

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Homologous recombination (HR) mediates the error-free repair of DNA double-strand breaks to maintain genomic stability. Here we characterize C17orf53/MCM8IP, an OB-fold containing protein that binds ssDNA, as a DNA repair factor involved in HR. MCM8IPdeficient cells exhibit HR defects, especially in long-tract gene conversion, occurring downstream of RAD51 loading, consistent with a role for MCM8IP in HR-dependent DNA synthesis. Moreover, loss of MCM8IP confers cellular sensitivity to crosslinking agents and PARP inhibition. Importantly, we report that MCM8IP directly associates with MCM8-9, a helicase complex mutated in primary ovarian insufficiency, and RPA1. We additionally show that the interactions of MCM8IP with MCM8-9 and RPA facilitate HR and promote replication fork progression and cellular viability in response to treatment with crosslinking agents. Mechanistically, MCM8IP stimulates the helicase activity of MCM8-9. Collectively, our work identifies MCM8IP as a key regulator of MCM8-9-dependent DNA synthesis during DNA recombination and replication.

Precise editing of genomic DNA can be achieved upon repair of CRISPR-induced DNA double-stranded breaks (DSBs) by homology-directed repair (HDR). However, the efficiency of this process is limited by DSB repair pathways competing with HDR, such as non-homologous end joining (NHEJ). Here we individually express in human cells 204 open reading frames involved in the DNA damage response (DDR) and determine their impact on CRISPR-mediated HDR. From these studies, we identify RAD18 as a stimulator of CRISPR-mediated HDR. By defining the RAD18 domains required to promote HDR, we derive an enhanced RAD18 variant (e18) that stimulates CRISPR-mediated HDR in multiple human cell types, including embryonic stem cells. Mechanistically, e18 induces HDR by suppressing the localization of the NHEJ-promoting factor 53BP1 to DSBs. Altogether, this study identifies e18 as an enhancer of CRISPR-mediated HDR and highlights the promise of engineering DDR factors to augment the efficiency of precision genome editing.

53BP1 controls a specialized non-homologous end joining (NHEJ) pathway that is essential for adaptive immunity, yet oncogenic in BRCA1 mutant cancers. Intra-chromosomal DNA double-strand break (DSB) joining events during immunoglobulin class switch recombination (CSR) require 53BP1. However, in BRCA1 mutant cells, 53BP1 blocks homologous recombination (HR) and promotes toxic NHEJ, resulting in genomic instability. Here, we identify the protein dimerization hub—DYNLL1—as an organizer of multimeric 53BP1 complexes. DYNLL1 binding stimulates 53BP1 oligomerization, and promotes 53BP1’s recruitment to, and interaction with, DSB-associated chromatin. Consequently, DYNLL1 regulates 53BP1-dependent NHEJ: CSR is compromised upon deletion of Dynll1 or its transcriptional regulator Asciz, or by mutation of DYNLL1 binding motifs in 53BP1; furthermore, Brca1 mutant cells and tumours are rendered resistant to poly-ADP ribose polymerase (PARP) inhibitor treatments upon deletion of Dynll1 or Asciz. Thus, our results reveal a mechanism that regulates 53BP1-dependent NHEJ and the therapeutic response of BRCA1-deficient cancers.

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The tumor suppressor protein 53BP1 was first identified as a p53-interacting protein over two decades ago, however its direct contribution to p53-dependent cellular activities has remained enigmatic. Having reinvestigated the link between 53BP1 and p53, we now show 53BP1 plays an important role in directly stimulating genome-wide p53-dependent gene transactivation and repression events in response to ionizing radiation (IR) and synthetic p53 activation. We have also fine-mapped the domains in 53BP1 that modulate p53 activity and reveal it requires both auto-oligomerization and its tandem-BRCT domain-mediated bivalent interactions with p53 and the ubiquitin-specific protease USP28. Loss of 53BP1 or USP28 catalytic activities results in inefficient p53-dependent cell-cycle checkpoint and exit responses. Mechanistically, we show 53BP1-USP28 cooperation to be essential for stimulating normal p53-promoter element interactions and downstream gene transactivation-associated events, yet dispensable for 53BP1-dependent DSB repair regulation. Collectively, our data indicate a upstream role for 53BP1-USP28 complexes in priming p53's transcriptional potential, providing a mechanistic explanation for 53BP1-p53 cooperation in controlling anti-tumorigenic cell-fate decisions. Moreover, we reveal these activities to be distinct and separable from 53BP1's regulation of DNA double-strand break repair pathway choice, and establish the prime function for the 53BP1 BRCT domain and its interaction partner USP28. Our study therefore defines important and novel functions for 53BP1 in enforcing a vital tumor suppressor pathway that are likely to contribute to tumor suppression. In the meeting we will describe these findings and update on recent progress. This abstract is also being presented as Poster B04. Citation Format: Raquel Cuella-Martin, Catarina Oliveira, Helen E. Lockstone, Suzanne Snellenberg, Natalia Grolmusova, J Ross Chapman. A 53BP1 integrates DNA repair and p53-dependent cell fate decisions via distinct mechanisms [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr PR20.