PARP10 promotes the repair of nascent strand DNA gaps through RAD18 mediated translesion synthesis.

Replication stress compromises genomic integrity. Fork blocking lesions such as those induced by cisplatin and other chemotherapeutic agents arrest replication forks. Repriming downstream of these lesions represents an important mechanism of replication restart, however the single stranded DNA (ssDNA) gaps left behind, unless efficiently filled, can serve as entry point for nucleases. Nascent strand gaps can be repaired by BRCA-mediated homology repair. Alternatively, gaps can also be filled by translesion synthesis (TLS) polymerases. How these events are regulated is still not clear. Here, we show that PARP10, a poorly-characterized mono-ADP-ribosyltransferase, is recruited to nascent strand gaps to promote their repair. PARP10 interacts with the ubiquitin ligase RAD18 and recruits it to these structures, resulting in the ubiquitination of the replication factor PCNA. PCNA ubiquitination, in turn, recruits the TLS polymerase REV1 for gap filling. We show that PARP10 recruitment to gaps and the subsequent REV1-mediated gap filling requires both the catalytic activity of PARP10, and its ability to interact with PCNA. We moreover show that PARP10 is hyperactive in BRCA-deficient cells, and its inactivation potentiates gap accumulations and cytotoxicity in these cells. Our work uncovers PARP10 as a regulator of ssDNA gap filling, which promotes genomic stability in BRCA-deficient cells.

CRISPR knockout genome-wide screens identify the HELQ-RAD52 axis in regulating the repair of cisplatin-induced single stranded DNA gaps.

Treatment with genotoxic agents, such as platinum compounds, is still the mainstay therapeutical approach for the majority of cancers. Our understanding of the mechanisms of action of these drugs is however imperfect, and continuously evolving. Recent advances in the field highlighted single stranded DNA (ssDNA) gap accumulation as a potential determinant underlying cisplatin chemosensitivity, at least in some genetic backgrounds, such as BRCA mutations. Cisplatin-induced ssDNA gaps form upon the arrest of replication forks at sites of cisplatin adducts, and restart of DNA synthesis downstream of the lesion through repriming catalyzed by the PRIMPOL enzyme. Here, we show that PRIMPOL overexpression in otherwise wildtype cells results in accumulation of cisplatin-induced ssDNA gaps without sensitizing cells to cisplatin, suggesting that ssDNA gap accumulation does not confer cisplatin sensitivity in BRCA-proficient cells. To understand how ssDNA gaps may cause cellular sensitivity, we employed CRISPR-mediated genome-wide genetic screening to identify factors which enable the cytotoxicity of cisplatin-induced ssDNA gaps. We found that the helicase HELQ specifically suppresses cisplatin sensitivity in PRIMPOL-overexpressing cells, and this is associated with reduced ssDNA accumulation. We moreover identify RAD52 as a mediator of this pathway, and show that RAD52 promotes ssDNA gap accumulation through a BRCA-mediated mechanism. Our work identified the HELQ-RAD52-BRCA axis as a regulator of ssDNA gap processing, shedding light on the mechanisms of cisplatin sensitization in cancer therapy.

Role of Translesion DNA Synthesis in the Metabolism of Replication-associated Nascent Strand Gaps.

Translesion DNA synthesis (TLS) is a DNA damage tolerance pathway utilized by cells to overcome lesions encountered throughout DNA replication. During replication stress, cancer cells show increased dependency on TLS proteins for cellular survival and chemoresistance. TLS proteins have been described to be involved in various DNA repair pathways. One of the major emerging roles of TLS is single-stranded DNA (ssDNA) gap-filling, primarily after the repriming activity of PrimPol upon encountering a lesion. Conversely, suppression of ssDNA gap accumulation by TLS is considered to represent a mechanism for cancer cells to evade the toxicity of chemotherapeutic agents, specifically in BRCA-deficient cells. Thus, TLS inhibition is emerging as a potential treatment regimen for DNA repair-deficient tumors.

Complementary CRISPR genome-wide genetic screens in PARP10-knockout and overexpressing cells identify synthetic interactions for PARP10-mediated cellular survival.

PARP10 is a mono-ADP-ribosyltransferase with multiple cellular functions, including proliferation, apoptosis, metabolism and DNA repair. PARP10 is overexpressed in a significant proportion of tumors, particularly breast and ovarian cancers. Identifying genetic susceptibilities based on PARP10 expression levels is thus potentially relevant for finding new targets for precision oncology. Here, we performed a series of CRISPR genome-wide loss-of-function screens in isogenic control and PARP10-overexpressing or PARP10-knockout cell lines, to identify genetic determinants of PARP10-mediated cellular survival. We found that PARP10-overexpressing cells rely on multiple DNA repair genes for survival, including ATM, the master regulator of the DNA damage checkpoint. Moreover, we show that PARP10 impacts the recruitment of ATM to nascent DNA upon replication stress. Finally, we identify the CDK2-Cyclin E1 complex as essential for proliferation of PARP10-knockout cells. Our work identifies a network of functionally relevant PARP10 synthetic interactions, and reveals a set of factors which can potentially be targeted in personalized cancer therapy.

The TIP60-ATM axis regulates replication fork stability in BRCA-deficient cells.

Maintenance of replication fork stability is essential for genome preservation. Stalled replication forks can be reversed by translocases such as SMARCAL1, and unless protected through the activity of the BRCA pathway, are subsequently subjected to nucleolytic degradation. The ATM and ATR kinases are master regulators of the DNA damage response. ATM activation upon DNA damage is mediated by the acetyltransferase TIP60. Here, we show that the TIP60-ATM pathway promotes replication fork reversal by recruiting SMARCAL1 to stalled forks. This enables fork degradation in BRCA-deficient cells. We also show that this ATM activity is not shared by ATR. Moreover, we performed a series of genome-wide CRISPR knockout genetic screens to identify genetic determinants of the cellular sensitivity to ATM inhibition in wildtype and BRCA2-knockout cells, and validated the top hits from multiple screens. We provide a valuable list of common genes which regulate the response to multiple ATM inhibitors. Importantly, we identify a differential response of wildtype and BRCA2-deficient cells to these inhibitors. In BRCA2-knockout cells, DNA repair genes (including RAD17, MDC1, and USP28) were essential for survival upon ATM inhibitor treatment, which was not the case in wild-type cells. These findings may eventually help guide the way for rational deployment of ATM inhibitors in the clinic.