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This study shows how the yeast Ctf4 protein couples the DNA helicase, Cdc45–MCM–GINS, to DNA polymerase α — the GINS subunit of the helicase and the polymerase use a similar interaction to bind Ctf4, suggesting that, as Ctf4 is a trimer, two polymerases could be simultaneously coupled to a single helicase during lagging-strand synthesis. Helicase–polymerase coordination During DNA replication, each polymerase is preceded by a helicase that disrupts the two strands, funnelling them into the leading- and lagging-strand machineries. This study, a collaboration between the laboratories of Luca Pellegrini, Alessandro Costa and Karim Labib, examines the structural basis for the action of the yeast Ctf4 protein that links DNA helicase and DNA polymerase components of the replisome. The authors delineate how Ctf4 couples the DNA helicase, Cdc45–MCM–GINS, to polymerase α. The GINS subunit of the helicase and the polymerase use a similar interaction to bind Ctf4, suggesting that, as Ctf4 is a trimer, two polymerases can be simultaneously coupled to a single helicase during lagging-strand synthesis. Efficient duplication of the genome requires the concerted action of helicase and DNA polymerases at replication forks 1 to avoid stalling of the replication machinery and consequent genomic instability 2 , 3 , 4 . In eukaryotes, the physical coupling between helicase and DNA polymerases remains poorly understood. Here we define the molecular mechanism by which the yeast Ctf4 protein links the Cdc45–MCM–GINS (CMG) DNA helicase to DNA polymerase α (Pol α) within the replisome. We use X-ray crystallography and electron microscopy to show that Ctf4 self-associates in a constitutive disk-shaped trimer. Trimerization depends on a β-propeller domain in the carboxy-terminal half of the protein, which is fused to a helical extension that protrudes from one face of the trimeric disk. Critically, Pol α and the CMG helicase share a common mechanism of interaction with Ctf4. We show that the amino-terminal tails of the catalytic subunit of Pol α and the Sld5 subunit of GINS contain a conserved Ctf4-binding motif that docks onto the exposed helical extension of a Ctf4 protomer within the trimer. Accordingly, we demonstrate that one Ctf4 trimer can support binding of up to three partner proteins, including the simultaneous association with both Pol α and GINS. Our findings indicate that Ctf4 can couple two molecules of Pol α to one CMG helicase within the replisome, providing a new model for lagging-strand synthesis in eukaryotes that resembles the emerging model for the simpler replisome of Escherichia coli 5 , 6 , 7 , 8 . The ability of Ctf4 to act as a platform for multivalent interactions illustrates a mechanism for the concurrent recruitment of factors that act together at the fork.

Horizontal gene transfer (HGT), or the transfer of genes between species, has been recognized recently as more pervasive than previously suspected. Here, we report evidence for an unprecedented degree of HGT into an animal genome, based on a draft genome of a tardigrade,Hypsibius dujardini. Tardigrades are microscopic eight-legged animals that are famous for their ability to survive extreme conditions. Genome sequencing, direct confirmation of physical linkage, and phylogenetic analysis revealed that a large fraction of theH. dujardinigenome is derived from diverse bacteria as well as plants, fungi, and Archaea. We estimate that approximately one-sixth of tardigrade genes entered by HGT, nearly double the fraction found in the most extreme cases of HGT into animals known to date. Foreign genes have supplemented, expanded, and even replaced some metazoan gene families within the tardigrade genome. Our results demonstrate that an unexpectedly large fraction of an animal genome can be derived from foreign sources. We speculate that animals that can survive extremes may be particularly prone to acquiring foreign genes.

Genetic information storage and processing rely on just two polymers, DNA and RNA, yet whether their role reflects evolutionary history or fundamental functional constraints is currently unknown. With the use of polymerase evolution and design, we show that genetic information can be stored in and recovered from six alternative genetic polymers based on simple nucleic acid architectures not found in nature [xeno-nucleic acids (XNAs)]. We also select XNA aptamers, which bind their targets with high affinity and specificity, demonstrating that beyond heredity, specific XNAs have the capacity for Darwinian evolution and folding into defined structures. Thus, heredity and evolution, two hallmarks of life, are not limited to DNA and RNA but are likely to be emergent properties of polymers capable of information storage.

The replicative helicase-catalyzed unwinding of the DNA double helix is the initiation of DNA replication. Helicases and primases are functionally related enzymes that have even been expressed as fusion proteins in some organisms and viruses. However, the mechanism underlying DNA unwinding initiation by these helicase-primase fusion enzymes and the functional association between domains have not been elucidated. Herein, we report the cryo-EM structures of mpox virus E5, the founding member of these helicase-primase enzymes, in various enzymatic stages. Notably, E5 forms a head-to-head double hexamer encircling dsDNA, disrupted by the conformational rearrangement of primase domains upon nucleotide incorporation. Five E5-ssDNA-ATP structures further support an ATP cycle-driven non-classical escort model for E5 translocation. Finally, the helicase domain is found to enhance the primase function as a DNA scaffold. Together, our data shed light on the E5-mediated DNA unwinding model including dsDNA loading, DNA melting, ssDNA translocation, and provide a reasonable interpretation for evolutionary preservation of helicase-primase fusion from a functional perspective.

The DNA replisome performs concerted parental-strand separation and DNA synthesis on both strands. Gao et al. report the cryo–electron microscopy structures of the minimum set of bacteriophage T7 proteins that can carry out leading- and lagging-strand synthesis at the replication fork (see the Perspective by Li and O'Donnell). Three key enzymes involved in DNA replication—DNA polymerase, helicase, and primase—were visualized in complex with substrate DNA, demonstrating their highly dynamic organizations on both strands. Comparison of prokaryotic and eukaryotic replisomes reveals evolutionarily conserved operating principles and provides a structural basis for understanding coordination among DNA replication, recombination, and repair. Science , this issue p. eaav7003 ; see also p. 814 Cryo-EM structures of the bacteriophage T7 replisome carry out concerted leading- and lagging-strand DNA synthesis. Visualization in atomic detail of the replisome that performs concerted leading– and lagging–DNA strand synthesis at a replication fork has not been reported. Using bacteriophage T7 as a model system, we determined cryo–electron microscopy structures up to 3.2-angstroms resolution of helicase translocating along DNA and of helicase-polymerase-primase complexes engaging in synthesis of both DNA strands. Each domain of the spiral-shaped hexameric helicase translocates sequentially hand-over-hand along a single-stranded DNA coil, akin to the way AAA+ ATPases (adenosine triphosphatases) unfold peptides. Two lagging-strand polymerases are attached to the primase, ready for Okazaki fragment synthesis in tandem. A β hairpin from the leading-strand polymerase separates two parental DNA strands into a T-shaped fork, thus enabling the closely coupled helicase to advance perpendicular to the downstream DNA duplex. These structures reveal the molecular organization and operating principles of a replisome.

Rev1–Polζ-dependent translesion synthesis (TLS) of DNA is crucial for maintaining genome integrity. To elucidate the mechanism by which the two polymerases cooperate in TLS, we determined the cryogenic electron microscopic structure of the Saccharomyces cerevisiae Rev1–Polζ holocomplex in the act of DNA synthesis (3.53 Å). We discovered that a composite N-helix-BRCT module in Rev1 is the keystone of Rev1–Polζ cooperativity, interacting directly with the DNA template–primer and with the Rev3 catalytic subunit of Polζ. The module is positioned akin to the polymerase-associated domain in Y-family TLS polymerases and is set ideally to interact with PCNA. We delineate the full extent of interactions that the carboxy-terminal domain of Rev1 makes with Polζ and identify potential new druggable sites to suppress chemoresistance from first-line chemotherapeutics. Collectively, our results provide fundamental new insights into the mechanism of cooperativity between Rev1 and Polζ in TLS. The authors elucidate by cryo-EM the mechanism by which DNA polymerases Rev1 and Polζ cooperate in translesion DNA synthesis.

The physicochemical properties of nucleic acids are dominated by their highly charged phosphodiester backbone chemistry. This polyelectrolyte structure decouples information content (base sequence) from bulk properties, such as solubility, and has been proposed as a defining trait of all informational polymers. However, this conjecture has not been tested experimentally. Here, we describe the encoded synthesis of a genetic polymer with an uncharged backbone chemistry: alkyl phosphonate nucleic acids (phNAs) in which the canonical, negatively charged phosphodiester is replaced by an uncharged P-alkyl phosphonodiester backbone. Using synthetic chemistry and polymerase engineering, we describe the enzymatic, DNA-templated synthesis of P-methyl and P-ethyl phNAs, and the directed evolution of specific streptavidin-binding phNA aptamer ligands directly from random-sequence mixed P-methyl/P-ethyl phNA repertoires. Our results establish an example of the DNA-templated enzymatic synthesis and evolution of an uncharged genetic polymer and provide a foundational methodology for their exploration as a source of novel functional molecules. The highly charged phosphodiester chemistry of the natural nucleic acids DNA and RNA has been widely considered to be indispensable for their function as informational molecules. Now, synthetic genetic polymers with an uncharged alkyl phosphonate backbone chemistry have been shown to enable genetic information transfer and evolution.

ADP-ribosyltransferases use NAD + to catalyse substrate ADP-ribosylation 1 , and thereby regulate cellular pathways or contribute to toxin-mediated pathogenicity of bacteria 2 – 4 . Reversible ADP-ribosylation has traditionally been considered a protein-specific modification 5 , but recent in vitro studies have suggested nucleic acids as targets 6 – 9 . Here we present evidence that specific, reversible ADP-ribosylation of DNA on thymidine bases occurs in cellulo through the DarT–DarG toxin–antitoxin system, which is found in a variety of bacteria (including global pathogens such as Mycobacterium tuberculosis , enteropathogenic Escherichia coli and Pseudomonas aeruginosa ) 10 . We report the structure of DarT, which identifies this protein as a diverged member of the PARP family. We provide a set of high-resolution structures of this enzyme in ligand-free and pre- and post-reaction states, which reveals a specialized mechanism of catalysis that includes a key active-site arginine that extends the canonical ADP-ribosyltransferase toolkit. Comparison with PARP–HPF1, a well-established DNA repair protein ADP-ribosylation complex, offers insights into how the DarT class of ADP-ribosyltransferases evolved into specific DNA-modifying enzymes. Together, our structural and mechanistic data provide details of this PARP family member and contribute to a fundamental understanding of the ADP-ribosylation of nucleic acids. We also show that thymine-linked ADP-ribose DNA adducts reversed by DarG antitoxin (functioning as a noncanonical DNA repair factor) are used not only for targeted DNA damage to induce toxicity, but also as a signalling strategy for cellular processes. Using M. tuberculosis as an exemplar, we show that DarT–DarG regulates growth by ADP-ribosylation of DNA at the origin of chromosome replication. Structural and mechanistic data of the ADP-ribosyltransferase DarT demonstrate the role of ADP-ribosylation of DNA by this enzyme in generating toxicity and regulating cellular signalling processes in bacteria.

In eukaryotic transcription initiation, a large multi-subunit pre-initiation complex (PIC) that assembles at the core promoter is required for the opening of the duplex DNA and identification of the start site for transcription by RNA polymerase II. Here we use cryo-electron microscropy (cryo-EM) to determine near-atomic resolution structures of the human PIC in a closed state (engaged with duplex DNA), an open state (engaged with a transcription bubble), and an initially transcribing complex (containing six base pairs of DNA–RNA hybrid). Our studies provide structures for previously uncharacterized components of the PIC, such as TFIIE and TFIIH, and segments of TFIIA, TFIIB and TFIIF. Comparison of the different structures reveals the sequential conformational changes that accompany the transition from each state to the next throughout the transcription initiation process. This analysis illustrates the key role of TFIIB in transcription bubble stabilization and provides strong structural support for a translocase activity of XPB. Cryo-electron microscopy structural models of the human pre-initiation complex at all major steps of transcription initiation at near atomic-level resolution are presented, providing new mechanistic insights into the processes of promoter melting and transcription-bubble formation, as well as an almost complete proposed structural model of all of the pre-initiation complex components and their interactions with DNA. The structural basis of gene initiation The initiation of gene transcription in eukaryotes is tightly controlled at the promoter of each gene through the actions of the pre-initiation complex (PIC), a large multi-subunit composed of general transcription factors, and RNA polymerase II (Pol II) assembles at the promoter to ensure correct loading of Pol II and opening of the duplex DNA for transcription into RNA. Two papers published in this issue report detailed cryo-electron microscopy structures of the Pol II machinery at near-atomic resolution. Eva Nogales and colleagues present structural models of the human PIC at all major steps during transcription initiation at near-atomic resolution. They provide new mechanistic insights into the processes of promoter melting and transcription bubble stabilization, as well as proposing an almost complete structural model of all of the PIC components bound to duplex DNA. Patrick Cramer and colleagues report structures of yeast initiation complexes containing all of the basal transcription factors except TFIIH, and containing either closed or open promoter DNA. They show that DNA opening can occur in the absence of TFIIH, and provide mechanistic insights into DNA opening and template-strand loading. The structures reveal the high structural conservation between yeast and human transcription initiation systems.