The Replication Protein A （RPA） complex is an essential regulator of eukaryotic DNA metabolism. RPA avidly binds to single-stranded DNA （ssDNA） through multiple ...oligonucleotide/oligosaccharide-binding folds and coordinates the recruitment and exchange of genome maintenance factors to regulate DNA replication, recombination and repair. The RPA-ssDNA platform also constitutes a key physiological signal which activates the master ATR kinase to protect and repair stalled or collapsed replication forks during replication stress. In recent years, the RPA com- plex has emerged as a key target and an important regulator of post-translational modifications in response to DNA damage, which is critical for its genome guardian functions. Phosphorylation and SUMOylation of the RPA complex, and more recently RPA-regulated ubiquitination, have all been shown to control specific aspects of DNA damage signaling and repair by modulating the interactions between RPA and its partners. Here, we review our current un- derstanding of the critical functions of the RPA-ssDNA platform in the maintenance of genome stability and its regulation through an elaborate network of covalent modifications.
The ATR kinase is a master regulator of replication stress responses. Four new studies show that the protein ETAA1 is an important activator of ATR in human cells, providing insights into how the ATR ...pathway reacts to replication stress.
The ATR kinase is a master regulator of replication stress responses. Four new studies show that the protein ETAA1 is an important activator of ATR in human cells, providing insights into how the ATR pathway reacts to replication stress.
The human ATR gene encodes a kinase that is activated by DNA damage and replication stress as a central transducer of a checkpoint signaling pathway. Once activated, ATR phosphorylates multiple ...substrates, including the kinase Chk1, to regulate cell-cycle progression, replication fork stability, and DNA repair. These events promote cell survival during replication stress and in cells with DNA damage. Accordingly, there has been the tantalizing possibility that ATR inhibitors would be therapeutically useful, especially if they were more effective in tumor versus normal cells. Indeed, multiple studies have demonstrated that alterations that promote tumorigenesis, such as defects in the ATM-p53 pathway, constitutive oncogene activation, and acquisition of the alternative lengthening of telomeres pathway, render tumor cells sensitive to ATR inhibitor monotherapy and/or increase the synergy between ATR inhibitors and genotoxic chemotherapies. Now, nearly two decades after the discovery of ATR, two highly selective and potent ATR inhibitors, AZD6738 and VX-970, are in early-phase clinical trials either as monotherapies or paired with a variety of genotoxic chemotherapies. These trials will generate important insights into the effects of ATR inhibition in humans and the potential role of inhibiting this kinase in the treatment of human malignancies.
ATM and ATR are two master checkpoint kinases activated by double-stranded DNA breaks (DSBs). ATM is critical for the initial response and the subsequent ATR activation. Here we show that ATR ...activation is coupled with loss of ATM activation, an unexpected ATM-to-ATR switch during the biphasic DSB response. ATM is activated by DSBs with blunt ends or short single-stranded overhangs (SSOs). Surprisingly, the activation of ATM in the presence of SSOs, like that of ATR, relies on single- and double-stranded DNA junctions. In a length-dependent manner, SSOs attenuate ATM activation and potentiate ATR activation through a swap of DNA-damage sensors. Progressive resection of DSBs directly promotes the ATM-to-ATR switch in vitro. In cells, the ATM-to-ATR switch is driven by both ATM and the nucleases participating in DSB resection. Thus, single-stranded DNA orchestrates ATM and ATR to function in an orderly and reciprocal manner in two distinct phases of DSB response.
The ATR (ATM and rad3-related) pathway is crucial for proliferation, responding to DNA replication stress and DNA damage. This critical signaling pathway is carefully orchestrated through a multistep ...process requiring initial priming of ATR prior to damage, recruitment of ATR to DNA damage lesions, activation of ATR signaling, and, finally, modulation of ATR activity through a variety of post-translational modifications. Following activation, ATR functions in several vital cellular processes, including suppression of replication origin firing, promotion of deoxynucleotide synthesis and replication fork restart, prevention of double-stranded DNA break formation, and avoidance of replication catastrophe and mitotic catastrophe. In many cancers, tumor cells have increased dependence on ATR signaling for survival, making ATR a promising target for cancer therapy. Tumor cells compromised in DNA repair pathways or DNA damage checkpoints, cells reliant on homologous recombination, and cells with increased replication stress are particularly sensitive to ATR inhibition. Understanding ATR signaling and modulation is essential to unraveling which tumors have increased dependence on ATR signaling as well as how the ATR pathway can best be exploited for targeted cancer therapy.
The ataxia telangiectasia mutated and Rad3-related (ATR) kinase is crucial for DNA damage and replication stress responses. Here, we describe an unexpected role of ATR in mitosis. Acute inhibition or ...degradation of ATR in mitosis induces whole-chromosome missegregation. The effect of ATR ablation is not due to altered cyclin-dependent kinase 1 (CDK1) activity, DNA damage responses, or unscheduled DNA synthesis but to loss of an ATR function at centromeres. In mitosis, ATR localizes to centromeres through Aurora A-regulated association with centromere protein F (CENP-F), allowing ATR to engage replication protein A (RPA)-coated centromeric R loops. As ATR is activated at centromeres, it stimulates Aurora B through Chk1, preventing formation of lagging chromosomes. Thus, a mitosis-specific and R loop-driven ATR pathway acts at centromeres to promote faithful chromosome segregation, revealing functions of R loops and ATR in suppressing chromosome instability.
The proper spatial organization of DNA, RNA, and proteins is critical for a variety of cellular processes. The genome is organized into numerous functional units, such as topologically associating ...domains (TADs), the formation of which is regulated by both proteins and RNA. In addition, a group of chromatin-bound proteins with the ability to undergo liquid-liquid phase separation (LLPS) can affect the spatial organization and compartmentalization of chromatin, RNA, and proteins by forming condensates, conferring unique properties to specific chromosomal regions. Although the regulation of DNA repair by histone modifications and chromatin accessibility is well established, the impacts of higher-order chromatin and protein organization on the DNA damage response (DDR) have not been appreciated until recently. In this review, we will focus on the movement of chromatin during the DDR, the compartmentalization of DDR proteins via LLPS, and the roles of membraneless nuclear bodies and transcription in DNA repair. With this backdrop, we will discuss the importance of the spatial organization of chromatin and proteins for the maintenance of genome integrity.
To escape replicative senescence, cancer cells have to overcome telomere attrition during DNA replication. Most of cancers rely on telomerase to extend and maintain telomeres, but 4-11% of cancers ...use a homologous recombination-based pathway called alternative lengthening of telomeres (ALT). ALT is prevalent in cancers from the mesenchymal origin and usually associates with poor clinical outcome. Given its critical role in protecting telomeres and genomic integrity in tumor cells, ALT is an Achilles heel of tumors and an attractive target for cancer therapy. Here, we review the recent progress in the mechanistic studies of ALT, and discuss the emerging therapeutic strategies to target ALT-positive cancers.
R loop, a transcription intermediate containing RNA:DNA hybrids and displaced single-stranded DNA (ssDNA), has emerged as a major source of genomic instability. RNaseH1, which cleaves the RNA in ...RNA:DNA hybrids, plays an important role in R loop suppression. Here we show that replication protein A (RPA), an ssDNA-binding protein, interacts with RNaseH1 and colocalizes with both RNaseH1 and R loops in cells. In vitro, purified RPA directly enhances the association of RNaseH1 with RNA:DNA hybrids and stimulates the activity of RNaseH1 on R loops. An RPA binding-defective RNaseH1 mutant is not efficiently stimulated by RPA in vitro, fails to accumulate at R loops in cells, and loses the ability to suppress R loops and associated genomic instability. Thus, in addition to sensing DNA damage and replication stress, RPA is a sensor of R loops and a regulator of RNaseH1, extending the versatile role of RPA in suppression of genomic instability.
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•RPA interacts with RNaseH1 and colocalizes with RNaseH1 and R loops in cells•RPA stimulates RNaseH1 activity on R loops in vitro•RPA enhances the association of RNaseH1 with RNA:DNA hybrids•RPA binding-defective RNaseH1 fails to recognize and suppress R loops in cells
R loop, a transcription intermediate containing RNA:DNA hybrids and displaced single-stranded DNA (ssDNA), is a major source of genomic instability. Nguyen et al. show that the ssDNA-binding protein RPA interacts with and stimulates RNaseH1, an enzyme removing the RNA in RNA:DNA hybrids, thereby suppressing R loops and associated genomic instability.
Cancer drivers require statistical modeling to distinguish them from passenger events, which accumulate during tumorigenesis but provide no fitness advantage to cancer cells. The discovery of driver ...genes and mutations relies on the assumption that exact positional recurrence is unlikely by chance; thus, the precise sharing of mutations across patients identifies drivers. Examining the mutation landscape in cancer genomes, we found that many recurrent cancer mutations previously designated as drivers are likely passengers. Our integrated bioinformatic and biochemical analyses revealed that these passenger hotspot mutations arise from the preference of APOBEC3A, a cytidine deaminase, for DNA stem-loops. Conversely, recurrent APOBEC-signature mutations not in stem-loops are enriched in well-characterized driver genes and may predict new drivers. This demonstrates that mesoscale genomic features need to be integrated into computational models aimed at identifying mutations linked to diseases.
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