ABSTRACT
ATR checkpoint signaling is crucial for cellular responses to DNA replication impediments. Using an optogenetic platform, we show that TopBP1, the main activator of ATR, self-assembles extensively to yield micrometer-sized condensates. These opto-TopBP1 condensates are functional entities organized in tightly packed clusters of spherical nano-particles. TopBP1 condensates are reversible, occasionally fuse, and co-localize with TopBP1 partner proteins. We provide evidence that TopBP1 condensation is a molecular switch that amplifies ATR activity to phosphorylate checkpoint kinase 1 (Chk1) and slow down replication forks. Single amino acid substitutions of key residues in the intrinsically disordered ATR activation domain disrupt TopBP1 condensation and consequently ATR/Chk1 signaling. In physiologic salt concentration and pH, purified TopBP1 undergoes liquid-liquid phase separation in vitro. We propose that the actuation mechanism of ATR signaling is the assembly of TopBP1 condensates driven by highly regulated multivalent and cooperative interactions.
Subject(s)
Ataxia Telangiectasia Mutated Proteins , Carrier Proteins , Cell Nucleus , DNA-Binding Proteins , Mutation, Missense , Nuclear Proteins , Signal Transduction , Amino Acid Substitution , Animals , Ataxia Telangiectasia Mutated Proteins/chemistry , Ataxia Telangiectasia Mutated Proteins/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Nucleus/chemistry , Cell Nucleus/genetics , Cell Nucleus/metabolism , Checkpoint Kinase 1/chemistry , Checkpoint Kinase 1/genetics , Checkpoint Kinase 1/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , HeLa Cells , Humans , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Sf9 Cells , SpodopteraABSTRACT
R-loops have both positive and negative impacts on chromosome functions. To identify toxic R-loops in the human genome, here, we map RNA:DNA hybrids, replication stress markers and DNA double-strand breaks (DSBs) in cells depleted for Topoisomerase I (Top1), an enzyme that relaxes DNA supercoiling and prevents R-loop formation. RNA:DNA hybrids are found at both promoters (TSS) and terminators (TTS) of highly expressed genes. In contrast, the phosphorylation of RPA by ATR is only detected at TTS, which are preferentially replicated in a head-on orientation relative to the direction of transcription. In Top1-depleted cells, DSBs also accumulate at TTS, leading to persistent checkpoint activation, spreading of γ-H2AX on chromatin and global replication fork slowdown. These data indicate that fork pausing at the TTS of highly expressed genes containing R-loops prevents head-on conflicts between replication and transcription and maintains genome integrity in a Top1-dependent manner.
Subject(s)
DNA Replication , DNA Topoisomerases, Type I/metabolism , R-Loop Structures/genetics , Terminator Regions, Genetic/genetics , Transcription, Genetic , Ataxia Telangiectasia Mutated Proteins/metabolism , DNA Breaks, Double-Stranded , DNA Topoisomerases, Type I/genetics , Gene Knockdown Techniques , Genomic Instability , HEK293 Cells , HeLa Cells , Humans , Phosphorylation , Promoter Regions, Genetic , RNA, Small Interfering/metabolismABSTRACT
SAMHD1 was previously characterized as a dNTPase that protects cells from viral infections. Mutations in SAMHD1 are implicated in cancer development and in a severe congenital inflammatory disease known as Aicardi-Goutières syndrome. The mechanism by which SAMHD1 protects against cancer and chronic inflammation is unknown. Here we show that SAMHD1 promotes degradation of nascent DNA at stalled replication forks in human cell lines by stimulating the exonuclease activity of MRE11. This function activates the ATR-CHK1 checkpoint and allows the forks to restart replication. In SAMHD1-depleted cells, single-stranded DNA fragments are released from stalled forks and accumulate in the cytosol, where they activate the cGAS-STING pathway to induce expression of pro-inflammatory type I interferons. SAMHD1 is thus an important player in the replication stress response, which prevents chronic inflammation by limiting the release of single-stranded DNA from stalled replication forks.
