STING is an innate immune adaptor, classically activated by cytosolic DNA via cGAS-cGAMP to induce interferon signaling. Recent studies reveal that STING participates in non-canonical signaling pathways and localizes to the nucleus, where its functions remain poorly understood. In Hutchinson-Gilford Progeria Syndrome (HGPS), a premature aging disease caused by expression of the lamin-A mutant protein ‘progerin’, STING accumulates in the nucleus and drives chronic inflammation. Here, we show that replication stress is a trigger of STING nuclear accumulation and chromatin binding. In addition, we uncover that STING binds to nascent DNA and promotes replication stress in progeria and tumor cells. Mechanistically, STING causes replication fork slowing and stalling by limiting dNTPs availability. Upon fork stalling, STING hinders replication fork protection/stability by facilitating MRE11-mediated nascent DNA degradation (NDD). Importantly, STING’s contribution to dNTP depletion and NDD is mediated by SAMHD1. Depletion of SAMHD1 phenocopies STING abrogation in reducing replication stress in progeria cells, and rescues replication fork speed and stability in STING-expressing tumor cells. These findings define a pathological STING-SAMHD1 axis that drives replication stress and genome instability in both progeria cells and tumor cells with elevated STING activity, uncovering a feedforward loop between innate immune signaling and impaired DNA replication.

DNA replication is remarkably accurate with estimates of only a handful of mutations per human genome per cell division cycle. Replication stress caused by DNA lesions, transcription-replication conflicts, and other obstacles to the replication machinery must be efficiently overcome in ways that minimize errors and maximize completion of DNA synthesis. Replication fork reversal is one mechanism that helps cells tolerate replication stress. This process involves reannealing of parental template DNA strands and generation of a nascent-nascent DNA duplex. While fork reversal may be beneficial by facilitating DNA repair or template switching, it must be confined to the appropriate contexts to preserve genome stability. Many enzymes have been implicated in this process including ATP-dependent DNA translocases like SMARCAL1, ZRANB3, HLTF, and the helicase FBH1. In addition, the RAD51 recombinase is required. Many additional factors and regulatory activities also act to ensure reversal is beneficial instead of yielding undesirable outcomes. Finally, reversed forks must also be stabilized and often need to be restarted to complete DNA synthesis. Disruption or deregulation of fork reversal causes a variety of human diseases. In this review we will describe the latest models for reversal and key mechanisms of regulation.

Replication fork reversal is a fundamental process required for resolution of encounters with DNA damage. A key step in the stabilization and eventual resolution of reversed forks is formation of RAD51 nucleoprotein filaments on exposed single strand DNA (ssDNA). To avoid genome instability, RAD51 filaments are tightly controlled by a variety of positive and negative regulators. RADX (RPA-related RAD51-antagonist on the X chromosome) is a recently discovered negative regulator that binds tightly to ssDNA, directly interacts with RAD51, and regulates replication fork reversal and stabilization in a context-dependent manner. Here, we present a structure-based investigation of RADX’s mechanism of action. Mass photometry experiments showed that RADX forms multiple oligomeric states in a concentration-dependent manner, with a predominance of trimers in the presence of ssDNA. The structure of RADX, which has no structurally characterized orthologs, was determined ab initio by cryo-electron microscopy (cryo-EM) from maps in the 2 to 4 Å range. The structure reveals the molecular basis for RADX oligomerization and the coupled multi-valent binding of ssDNA binding. The interaction of RADX with RAD51 filaments was imaged by negative stain EM, which showed a RADX oligomer at the end of filaments. Based on these results, we propose a model in which RADX functions by capping and restricting the end of RAD51 filaments.

The APOBEC cytidine/deoxycytidine deaminase family of enzymes has 11 members in humans. These enzymes carry out essential developmental, metabolic, and immunological functions through the deamination of cytosine to form uracil in RNA or single-stranded DNA. The known physiological functions relate to lipid absorption (APOBEC1), immunoglobulin gene diversification (AID), virus restriction (APOBEC3A-H, excluding E), and muscle differentiation (APOBEC2). The ability to characterize in vitro how APOBEC enzymes interact with and catalyze cytidine/deoxycytidine deamination of their substrate has provided key insights and understanding of their physiological functions. Having the most highly active and soluble enzyme to carry out in vitro experiments is essential. For APOBEC enzymes this requires purification from a mammalian or insect cell system. Since mammalian cell expression is lower than robustly engineered recombinant systems such as the Spodoptera frugiperda 9 (Sf9) and baculovirus systems, we have developed recombinant baculovirus expression and purification methods for APOBEC enzymes from Sf9 cells. The yield for all family members is suitable for biochemical assays, with some enzymes yielding milligram amounts (suitable for structural studies). Here we describe the expression and purification of APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3F, APOBEC3G, APOBEC3H (Haplotypes II, V, VII), and APOBEC1 using existing molecular biology reagents. We also describe how to clone a novel gene into the system for expression and purification. Due to different expression levels and solubility, three purification methods are detailed that enable high, medium, and low expressing APOBECs to be purified.