Genomic material must be completely replicated in coordination with cell cycle progression to ensure faithful segregation of DNA to daughter cells; the passage of under-replicated chromosomes through mitosis leads to the loss of genetic material and disease1. Endogenous and exogenous DNA lesions are barriers to replication and can delay DNA replication. For example, the formation of DNA adducts by UV irradiation or cisplatin stalls replication forks and uncouples DNA polymerase and helicase activities2. This uncoupling induces replication stress, which is manifested by increased levels of single-stranded DNA (ssDNA) that must be protected before being replicated once replication fork progression has been restored3. Notably, cancer cells have intrinsically elevated levels of replication stress due to oncogene-driven cell cycle progression, altered DNA replication check points, defects in the DNA damage response (DDR), or other oncogenic features4. The ataxia telangiectasia and Rad3-related (ATR) kinase is one of the three major phosphatidylinositol 3-kinase (PI3K)-like kinases that coordinate the DDR that is responsible for directing the cellular response to replication stress to ensure complete replication of the genome before entering mitosis5. Thus, targeting this pathway is a viable therapeutic strategy for the treatment of cancer and has resulted in the clinical evaluation of a plethora of ATR inhibitors (ATRi) that target the kinase domain for competitive inhibition6. This is particularly notable as recent studies implicate ssDNA gap accumulation as the initiating lethal chemotherapeutic DNA lesion that drives the sensitivity of BRCA-deficient cancers to PARP inhibition rather than the double-strand break (DSB)7–12.
ATR is recruited to stretches of ssDNA by ATR interacting protein (ATRIP), which directly binds replication protein A (RPA)-bound ssDNA13,14. At stalled replication forks, DNA topoisomerase 2-binding protein 1 (TopBP1) is loaded onto ssDNA-dsDNA junctions15,16 and stimulates ATR kinase activity through protein-protein interactions via an ATR activating domain (AAD)13,17,18. More recently, Ewing’s tumor-associated antigen 1 (ETAA1), which also possesses an AAD, has been suggested to activate ATR and bind to RPA19–21, but appears to function in mitosis rather than S phase22. The most well-established ATR phosphorylation target is checkpoint kinase 1 (CHK1), which halts S/G2 progression and suppresses origin firing, thereby reducing the generation of new ssDNA23,24. Phosphorylation of the many other ATR targets regulates function in replication fork reversal and restart, maintenance of dNTP pools, and overall replisome regulation5. Collectively, the ATR signaling pathway decreases the production of ssDNA to maintain genomic stability until normal replicative function can be restored. When the ATR pathway is pharmacologically inhibited, ssDNA accumulates and is bound by RPA resulting in no free, unbound RPA. This phenomenon was termed RPA exhaustion and induces replication catastrophe that ultimately leads to cell death25,26. The inability to protect newly generated ssDNA, results in degradation and replication fork breakage occurs25,26. Importantly, the ATR signaling pathway, which prevents ssDNA accumulation and RPA exhaustion, is also dependent upon RPA binding to ssDNA and interacting with ATRIP to serve as a platform for kinase activation13. RPA is therefore central ATR signaling, executing cell cycle checkpoints and protecting ssDNA to preserve genomic stability.
RPA is the major mammalian ssDNA-binding protein and plays a central role in DNA replication, recombination, repair, and the DDR. RPA is a heterotrimer made up of RPA70, RPA32, and RPA14 subunits that contain 6 oligonucleotide/oligosaccharide-binding (OB) domains which dictate high affinity ssDNA binding and protein-protein interactions27–29. Because RPA is abundant (~ 4 million molecules per cell)30 and possesses a diffusion-limited rate of association for ssDNA binding31, nearly all generated ssDNA is quickly bound and protected from degradation by RPA. The RPA-ssDNA complex then serves as a hub for protein-protein interactions to recruit DNA replication/repair machinery to ssDNA. In response to various forms of DNA damaging agents, RPA is subjected to numerous post-translational modifications (PTMs) that, at times, dictate specific protein-protein interactions for certain repair pathways32. Phosphorylation of the RPA32 N-terminus has been well characterized; cyclin-dependent kinases phosphorylate Ser23 and Ser29 in a cell cycle-dependent manner33. Additionally, PI3K-like kinases phosphorylate RPA32 in response to DNA damage: ATR phosphorylates Ser33 and DNA-dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated (ATM) have both been demonstrated to phosphorylate Ser4, Ser8, and Thr2134,35. These phosphorylation events impact DNA replication and repair, and function to maintain genomic stability36. Moreover, multiple acetyltransferases can acetylate RPA70 and acetylation, dependent on the particular sites, positively or negatively impacts ssDNA binding, nucleotide excision repair (NER), and homologous recombination37–41. Notably, RPA70 acetylation is increased upon UV damage; under these conditions, UV adducts stall replication forks thereby leading to ssDNA gap accumulation that can activate ATR42,43. Other RPA PTMs include methylation, ubiquitylation, SUMOylation, and ADP-ribosylation, all of which have been linked to the DDR32.
Due to its central role in DNA metabolism and the DDR, the chemical exhaustion of RPA through the use of a small molecule RPA inhibitors (RPAi) is a promising anti-cancer therapeutic avenue for single agent or combination treatment. Our groups and others have developed RPAi that function through distinct mechanisms. One class blocks RPA-ssDNA binding (RPA-DBi) and exhibits potency across a variety of cancer cell lines, and slows tumor growth in xenograft models44–47. Moreover, RPA-DBi demonstrate synergy with DNA damaging agents and other DDR inhibitors (PARP, DNA-PK, and ATR)9,47. The second class (RPA-PPIi) targets the RPA70 OB-F protein-protein interaction domain and has no effect on RPA ssDNA binding but disrupts the recruitment of DDR machinery and exhibits anti-cancer activity48–50. Taken together, inhibition of both the RPA-ssDNA complex by RPA-DBi or the RPA-ATRIP interaction by RPA-PPIi are expected to inhibit ATR kinase activity by targeting critical interactions. These hold the potential for greater selectivity and impact on the DDR/replication stress response than ATR inhibitors currently in the clinic. RPA inhibition is therefore anticipated to simultaneously abrogate ATR activity and decrease the cellular ssDNA protection threshold47.
In this work, we have employed the reconstituted ATR kinase signaling pathway in vitro using purified proteins51–53. We first establish the RPA-bound ssDNA- and TopBP1-dependence of ATR activity using p53 and RPA32 as phosphorylation targets. We then demonstrate RPA-DBi and RPA-PPIi mediated inhibition of ATR activity. Lastly, we examine the effect of two DNA damage-induced RPA PTMs, phosphorylation and acetylation, on ATR activity. We find that phosphorylated RPA and phosphorylated TopBP1 stimulate ATR activity, while acetylated RPA has no change in ATR activation relative to unmodified RPA. In addition, despite the increased ssDNA binding affinity upon RPA acetylation, both acetylated and unmodified RPA are inhibited by RPA-DBi to the same degree. Collectively, this work establishes a novel mechanism of action of two distinct classes of RPAi and provides new insights into the effects of RPA PTMs on ATR kinase activity.