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Prim-pol is a recently identified DNA primase-polymerase belonging to the archaeao-eukaryotic primase (AEP) superfamily. Here, we characterize a previously unrecognized prim-pol in human cells, which we designate hPrimpol1 (human primase-polymerase 1). hPrimpol1 possesses primase and DNA polymerase activities in vitro, interacts directly with RPA1 and is recruited to sites of DNA damage and stalled replication forks in an RPA1-dependent manner. Cells depleted of hPrimpol1 display increased spontaneous DNA damage and defects in the restart of stalled replication forks. Both RPA1 binding and the primase activity of hPrimpol1 are required for its cellular function during DNA replication. Our results indicate that hPrimpol1 is a novel factor involved in the response to DNA replication stress.

The mammalian DNA polymerase-α–primase (Polα–primase) complex is essential for DNA metabolism, providing the de novo RNA–DNA primer for several DNA replication pathways 1 – 4 such as lagging-strand synthesis and telomere C-strand fill-in. The physical mechanism underlying how Polα–primase, alone or in partnership with accessory proteins, performs its complicated multistep primer synthesis function is unknown. Here we show that CST, a single-stranded DNA-binding accessory protein complex for Polα–primase, physically organizes the enzyme for efficient primer synthesis. Cryogenic electron microscopy structures of the CST-Polα–primase preinitiation complex (PIC) bound to various types of telomere overhang reveal that template-bound CST partitions the DNA and RNA catalytic centres of Polα–primase into two separate domains and effectively arranges them in RNA–DNA synthesis order. The architecture of the PIC provides a single solution for the multiple structural requirements for the synthesis of RNA–DNA primers by Polα–primase. Several insights into the template-binding specificity of CST, template requirement for assembly of the CST-Polα–primase PIC and activation are also revealed in this study. A structural analysis demonstrates how the single-stranded DNA-binding accessory protein complex CST physically organizes the human DNA polymerase-α–primase complex for efficient primer synthesis during telomere replication.

The synthesis of RNA–DNA primer by primosome requires coordination between primase and DNA polymerase α subunits, which is accompanied by unknown architectural rearrangements of multiple domains. Using cryogenic electron microscopy, we solved a 3.6 Å human primosome structure caught at an early stage of RNA primer elongation with deoxynucleotides. The structure confirms a long-standing role of primase large subunit and reveals new insights into how primosome is limited to synthesizing short RNA–DNA primers. Here, the authors solve the cryogenic electron microscopy structure of a human primosome to shed light on the mechanism by which RNA–DNA primers are synthesized for the initiation of DNA replication and the structural basis of the primer length limitation.

The mechanism by which polymerase α–primase (polα–primase) synthesizes chimeric RNA-DNA primers of defined length and composition, necessary for replication fidelity and genome stability, is unknown. Here, we report cryo-EM structures of Xenopus laevis polα–primase in complex with primed templates representing various stages of DNA synthesis. Our data show how interaction of the primase regulatory subunit with the primer 5′ end facilitates handoff of the primer to polα and increases polα processivity, thereby regulating both RNA and DNA composition. The structures detail how flexibility within the heterotetramer enables synthesis across two active sites and provide evidence that termination of DNA synthesis is facilitated by reduction of polα and primase affinities for the varied conformations along the chimeric primer–template duplex. Together, these findings elucidate a critical catalytic step in replication initiation and provide a comprehensive model for primer synthesis by polα–primase. The DNA polymerase α–primase complex undergoes dramatic configurational rearrangements to synthesize chimeric RNA-DNA primers across two separate active sites while maintaining simultaneous interactions at opposite ends of the primer–template duplex.

Stalled replication forks can be restarted and repaired by RAD51-mediated homologous recombination (HR), but HR can also perform post-replicative repair after bypass of the obstacle. Bulky DNA adducts are important replication-blocking lesions, but it is unknown whether they activate HR at stalled forks or behind ongoing forks. Using mainly BPDE-DNA adducts as model lesions, we show that HR induced by bulky adducts in mammalian cells predominantly occurs at post-replicative gaps formed by the DNA/RNA primase PrimPol. RAD51 recruitment under these conditions does not result from fork stalling, but rather occurs at gaps formed by PrimPol re-priming and resection by MRE11 and EXO1. In contrast, RAD51 loading at double-strand breaks does not require PrimPol. At bulky adducts, PrimPol promotes sister chromatid exchange and genetic recombination. Our data support that HR at bulky adducts in mammalian cells involves post-replicative gap repair and define a role for PrimPol in HR-mediated DNA damage tolerance. Bulky DNA adducts are important replication-blocking lesions. Here the authors reveal that homologous recombination at bulky adducts in mammalian cells involves post-replicative gap repair in a PrimPol dependent manner.

Bacteriophages have long been known to use modified bases in their DNA to prevent cleavage by the host’s restriction endonucleases. Among them, cyanophage S-2L is unique because its genome has all its adenines (A) systematically replaced by 2-aminoadenines (Z). Here, we identify a member of the PrimPol family as the sole possible polymerase of S-2L and we find it can incorporate both A and Z in front of a T. Its crystal structure at 1.5 Å resolution confirms that there is no structural element in the active site that could lead to the rejection of A in front of T. To resolve this contradiction, we show that a nearby gene is a triphosphohydolase specific of dATP (DatZ), that leaves intact all other dNTPs, including dZTP. This explains the absence of A in S-2L genome. Crystal structures of DatZ with various ligands, including one at sub-angstrom resolution, allow to describe its mechanism as a typical two-metal-ion mechanism and to set the stage for its engineering. The cyanophage S-2L incorporates 2-aminoadenine (Z) instead of adenine (A) in its genome. Here, the authors provide an explanation for the absence of A in S-2L genome by identifying and characterising functionally and structurally both the HD phosphohydrolase ( datZ ) that specifically cleaves dATP, and the sole DNA primase-polymerase of S-2L, nonspecific of dATP or dZTP.

The BRCA2 tumor suppressor protects genome integrity by promoting homologous recombination-based repair of DNA breaks, stability of stalled DNA replication forks and DNA damage-induced cell cycle checkpoints. BRCA2 deficient cells display the radio-resistant DNA synthesis (RDS) phenotype, however the mechanism has remained elusive. Here we show that cells without BRCA2 are unable to sufficiently restrain DNA replication fork progression after DNA damage, and the underrestrained fork progression is due primarily to Primase-Polymerase (PRIMPOL)-mediated repriming of DNA synthesis downstream of lesions, leaving behind single-stranded DNA gaps. Moreover, we find that BRCA2 associates with the essential DNA replication factor MCM10 and this association suppresses PRIMPOL-mediated repriming and ssDNA gap formation, while having no impact on the stability of stalled replication forks. Our findings establish an important function for BRCA2, provide insights into replication fork control during the DNA damage response, and may have implications in tumor suppression and therapy response. Tumor suppressor BRCA2 is known to stabilize and restart stalled DNA replication forks. Here the authors show that BRCA2 is recruited to the replication fork through its interaction with MCM10 and inhibits Primase-Polymerase-mediated repriming, lesion bypass and single strand DNA gap formation after DNA damage.

Telomeres are the physical ends of linear chromosomes. They are composed of short repeating sequences (such as TTGGGG in the G-strand for Tetrahymena thermophila ) of double-stranded DNA with a single-strand 3′ overhang of the G-strand and, in humans, the six shelterin proteins: TPP1, POT1, TRF1, TRF2, RAP1 and TIN2 1 , 2 . TPP1 and POT1 associate with the 3′ overhang, with POT1 binding the G-strand 3 and TPP1 (in complex with TIN2 4 ) recruiting telomerase via interaction with telomerase reverse transcriptase 5 (TERT). The telomere DNA ends are replicated and maintained by telomerase 6 , for the G-strand, and subsequently DNA polymerase α–primase 7 , 8 (PolαPrim), for the C-strand 9 . PolαPrim activity is stimulated by the heterotrimeric complex CTC1–STN1–TEN1 10 – 12 (CST), but the structural basis of the recruitment of PolαPrim and CST to telomere ends remains unknown. Here we report cryo-electron microscopy (cryo-EM) structures of Tetrahymena CST in the context of the telomerase holoenzyme, in both the absence and the presence of PolαPrim, and of PolαPrim alone. Tetrahymena Ctc1 binds telomerase subunit p50, a TPP1 orthologue, on a flexible Ctc1 binding motif revealed by cryo-EM and NMR spectroscopy. The PolαPrim polymerase subunit POLA1 binds Ctc1 and Stn1, and its interface with Ctc1 forms an entry port for G-strand DNA to the POLA1 active site. We thus provide a snapshot of four key components that are required for telomeric DNA synthesis in a single active complex—telomerase-core ribonucleoprotein, p50, CST and PolαPrim—that provides insights into the recruitment of CST and PolαPrim and the handoff between G-strand and C-strand synthesis. Cryo-electron microscopy structures of Tetrahymena thermophila telomerase-bound Ctc1–Stn1–Ten1 and DNA polymerase α–primase provide insights into the molecular mechanisms underlying telomere replication and maintenance.

DNA can transport electrical charge over long distances and has the potential to act as a signaling system. The iron-sulfur complex [4Fe4S] found in some proteins is known to be involved in redox reactions. The eukaryotic DNA primase is involved in DNA replication and contains a [4Fe4S] cluster that is required for its RNA primer synthesis activity. O'Brien et al. show that the [4Fe4S] cluster in DNA primase can regulate the protein's DNA binding activity through DNA-mediated charge transfer. This in turn plays a role in primer initiation and length determination. Science , this issue p. eaag1789 Charge transfer along DNA can regulate the activity of an enzyme involved in eukaryotic DNA replication. DNA charge transport chemistry offers a means of long-range, rapid redox signaling. We demonstrate that the [4Fe4S] cluster in human DNA primase can make use of this chemistry to coordinate the first steps of DNA synthesis. Using DNA electrochemistry, we found that a change in oxidation state of the [4Fe4S] cluster acts as a switch for DNA binding. Single-atom mutations that inhibit this charge transfer hinder primase initiation without affecting primase structure or polymerization. Generating a single base mismatch in the growing primer duplex, which attenuates DNA charge transport, inhibits primer truncation. Thus, redox signaling by [4Fe4S] clusters using DNA charge transport regulates primase binding to DNA and illustrates chemistry that may efficiently drive substrate handoff between polymerases during DNA replication.

The eukaryotic polymerase α (Pol α) synthesizes an RNA-DNA hybrid primer of 20–30 nucleotides. Pol α is composed of Pol1, Pol12, Primase 1 (Pri1), and Pri2. Pol1 and Pri1 contain the DNA polymerase and RNA primase activities, respectively. It has been unclear how Pol α hands over an RNA primer from Pri1 to Pol1 for DNA primer extension, and how the primer length is defined. Here we report the cryo-EM analysis of yeast Pol α in the apo, primer initiation, primer elongation, RNA primer hand-off from Pri1 to Pol1, and DNA extension states, revealing a series of very large movements. We reveal a critical point at which Pol1-core moves to take over the 3’-end of the RNA from Pri1. DNA extension is limited by a spiral motion of Pol1-core. Since both Pri1 and Pol1-core are flexibly attached to a stable platform, primer growth produces stress that limits the primer length. DNA Polymerase α has separate RNA and DNA Polymerase subunits to form a hybrid RNA-DNA primer of unique length. Cryo-EM structures of each stage in primer synthesis reveal large movements among the four subunits and explain how primer length may be limited.