POT1 recruits and regulates CST-Polα/primase at human telomeres.

Telomere maintenance requires the extension of the G-rich telomeric repeat strand by telomerase and the fill-in synthesis of the C-rich strand by Polα/primase. At telomeres, Polα/primase is bound to Ctc1/Stn1/Ten1 (CST), a single-stranded DNA-binding complex. Like mutations in telomerase, mutations affecting CST-Polα/primase result in pathological telomere shortening and cause a telomere biology disorder, Coats plus (CP). We determined cryogenic electron microscopy structures of human CST bound to the shelterin heterodimer POT1/TPP1 that reveal how CST is recruited to telomeres by POT1. Our findings suggest that POT1 hinge phosphorylation is required for CST recruitment, and the complex is formed through conserved interactions involving several residues mutated in CP. Our structural and biochemical data suggest that phosphorylated POT1 holds CST-Polα/primase in an inactive, autoinhibited state until telomerase has extended the telomere ends. We propose that dephosphorylation of POT1 releases CST-Polα/primase into an active state that completes telomere replication through fill-in synthesis.

Single-molecule imaging reveals the mechanism of bidirectional replication initiation in metazoa.

Metazoan genomes are copied bidirectionally from thousands of replication origins. Replication initiation entails the assembly and activation of two CMG helicases (Cdc45⋅Mcm2-7⋅GINS) at each origin. This requires several replication firing factors (including TopBP1, RecQL4, and DONSON) whose exact roles are still under debate. How two helicases are correctly assembled and activated at each origin is a long-standing question. By visualizing the recruitment of GINS, Cdc45, TopBP1, RecQL4, and DONSON in real time, we uncovered that replication initiation is surprisingly dynamic. First, TopBP1 transiently binds to the origin and dissociates before the start of DNA synthesis. Second, two Cdc45 are recruited together, even though Cdc45 alone cannot dimerize. Next, two copies of DONSON and two GINS simultaneously arrive at the origin, completing the assembly of two CMG helicases. Finally, RecQL4 is recruited to the CMG⋅DONSON⋅DONSON⋅CMG complex and promotes DONSON dissociation and CMG activation via its ATPase activity.

RNA genome packaging and capsid assembly of bluetongue virus visualized in host cells.

Unlike those of double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and ssRNA viruses, the mechanism of genome packaging of dsRNA viruses is poorly understood. Here, we combined the techniques of high-resolution cryoelectron microscopy (cryo-EM), cellular cryoelectron tomography (cryo-ET), and structure-guided mutagenesis to investigate genome packaging and capsid assembly of bluetongue virus (BTV), a member of the Reoviridae family of dsRNA viruses. A total of eleven assembly states of BTV capsid were captured, with resolutions up to 2.8 Å, with most visualized in the host cytoplasm. ATPase VP6 was found underneath the vertices of capsid shell protein VP3 as an RNA-harboring pentamer, facilitating RNA packaging. RNA packaging expands the VP3 shell, which then engages middle- and outer-layer proteins to generate infectious virions. These revealed "duality" characteristics of the BTV assembly mechanism reconcile previous contradictory co-assembly and core-filling models and provide insights into the mysterious RNA packaging and capsid assembly of Reoviridae members and beyond.

A glycolytic metabolite bypasses "two-hit" tumor suppression by BRCA2.

Knudson's "two-hit" paradigm posits that carcinogenesis requires inactivation of both copies of an autosomal tumor suppressor gene. Here, we report that the glycolytic metabolite methylglyoxal (MGO) transiently bypasses Knudson's paradigm by inactivating the breast cancer suppressor protein BRCA2 to elicit a cancer-associated, mutational single-base substitution (SBS) signature in nonmalignant mammary cells or patient-derived organoids. Germline monoallelic BRCA2 mutations predispose to these changes. An analogous SBS signature, again without biallelic BRCA2 inactivation, accompanies MGO accumulation and DNA damage in Kras-driven, Brca2-mutant murine pancreatic cancers and human breast cancers. MGO triggers BRCA2 proteolysis, temporarily disabling BRCA2's tumor suppressive functions in DNA repair and replication, causing functional haploinsufficiency. Intermittent MGO exposure incites episodic SBS mutations without permanent BRCA2 inactivation. Thus, a metabolic mechanism wherein MGO-induced BRCA2 haploinsufficiency transiently bypasses Knudson's two-hit requirement could link glycolysis activation by oncogenes, metabolic disorders, or dietary challenges to mutational signatures implicated in cancer evolution.

The SPATA5-SPATA5L1 ATPase complex directs replisome proteostasis to ensure genome integrity.

Ubiquitin-dependent unfolding of the CMG helicase by VCP/p97 is required to terminate DNA replication. Other replisome components are not processed in the same fashion, suggesting that additional mechanisms underlie replication protein turnover. Here, we identify replisome factor interactions with a protein complex composed of AAA+ ATPases SPATA5-SPATA5L1 together with heterodimeric partners C1orf109-CINP (55LCC). An integrative structural biology approach revealed a molecular architecture of SPATA5-SPATA5L1 N-terminal domains interacting with C1orf109-CINP to form a funnel-like structure above a cylindrically shaped ATPase motor. Deficiency in the 55LCC complex elicited ubiquitin-independent proteotoxicity, replication stress, and severe chromosome instability. 55LCC showed ATPase activity that was specifically enhanced by replication fork DNA and was coupled to cysteine protease-dependent cleavage of replisome substrates in response to replication fork damage. These findings define 55LCC-mediated proteostasis as critical for replication fork progression and genome stability and provide a rationale for pathogenic variants seen in associated human neurodevelopmental disorders.

The human pre-replication complex is an open complex.

In eukaryotes, DNA replication initiation requires assembly and activation of the minichromosome maintenance (MCM) 2-7 double hexamer (DH) to melt origin DNA strands. However, the mechanism for this initial melting is unknown. Here, we report a 2.59-Å cryo-electron microscopy structure of the human MCM-DH (hMCM-DH), also known as the pre-replication complex. In this structure, the hMCM-DH with a constricted central channel untwists and stretches the DNA strands such that almost a half turn of the bound duplex DNA is distorted with 1 base pair completely separated, generating an initial open structure (IOS) at the hexamer junction. Disturbing the IOS inhibits DH formation and replication initiation. Mapping of hMCM-DH footprints indicates that IOSs are distributed across the genome in large clusters aligning well with initiation zones designed for stochastic origin firing. This work unravels an intrinsic mechanism that couples DH formation with initial DNA melting to license replication initiation in human cells.

A replication fork determinant for the establishment of sister chromatid cohesion.

Concomitant with DNA replication, the chromosomal cohesin complex establishes cohesion between newly replicated sister chromatids. Cohesion establishment requires acetylation of conserved cohesin lysine residues by Eco1 acetyltransferase. Here, we explore how cohesin acetylation is linked to DNA replication. Biochemical reconstitution of replication-coupled cohesin acetylation reveals that transient DNA structures, which form during DNA replication, control the acetylation reaction. As polymerases complete lagging strand replication, strand displacement synthesis produces DNA flaps that are trimmed to result in nicked double-stranded DNA. Both flaps and nicks stimulate cohesin acetylation, while subsequent nick ligation to complete Okazaki fragment maturation terminates the acetylation reaction. A flapped or nicked DNA substrate constitutes a transient molecular clue that directs cohesin acetylation to a window behind the replication fork, next to where cohesin likely entraps both sister chromatids. Our results provide an explanation for how DNA replication is linked to sister chromatid cohesion establishment.

Cyclin E-induced replicative stress drives p53-dependent whole-genome duplication.

Whole-genome duplication (WGD) is a frequent event in cancer evolution and an important driver of aneuploidy. The role of the p53 tumor suppressor in WGD has been enigmatic: p53 can block the proliferation of tetraploid cells, acting as a barrier to WGD, but can also promote mitotic bypass, a key step in WGD via endoreduplication. In wild-type (WT) p53 tumors, WGD is frequently associated with activation of the E2F pathway, especially amplification of CCNE1, encoding cyclin E1. Here, we show that elevated cyclin E1 expression causes replicative stress, which activates ATR- and Chk1-dependent G2 phase arrest. p53, via its downstream target p21, together with Wee1, then inhibits mitotic cyclin-dependent kinase activity sufficiently to activate APC/CCdh1 and promote mitotic bypass. Cyclin E expression suppresses p53-dependent senescence after mitotic bypass, allowing cells to complete endoreduplication. Our results indicate that p53 can contribute to cancer evolution through the promotion of WGD.

Antagonistic roles of canonical and Alternative-RPA in disease-associated tandem CAG repeat instability.

Expansions of repeat DNA tracts cause >70 diseases, and ongoing expansions in brains exacerbate disease. During expansion mutations, single-stranded DNAs (ssDNAs) form slipped-DNAs. We find the ssDNA-binding complexes canonical replication protein A (RPA1, RPA2, and RPA3) and Alternative-RPA (RPA1, RPA3, and primate-specific RPA4) are upregulated in Huntington disease and spinocerebellar ataxia type 1 (SCA1) patient brains. Protein interactomes of RPA and Alt-RPA reveal unique and shared partners, including modifiers of CAG instability and disease presentation. RPA enhances in vitro melting, FAN1 excision, and repair of slipped-CAGs and protects against CAG expansions in human cells. RPA overexpression in SCA1 mouse brains ablates expansions, coincident with decreased ATXN1 aggregation, reduced brain DNA damage, improved neuron morphology, and rescued motor phenotypes. In contrast, Alt-RPA inhibits melting, FAN1 excision, and repair of slipped-CAGs and promotes CAG expansions. These findings suggest a functional interplay between the two RPAs where Alt-RPA may antagonistically offset RPA's suppression of disease-associated repeat expansions, which may extend to other DNA processes.

Recycling of modified H2A-H2B provides short-term memory of chromatin states.

Chromatin landscapes are disrupted during DNA replication and must be restored faithfully to maintain genome regulation and cell identity. The histone H3-H4 modification landscape is restored by parental histone recycling and modification of new histones. How DNA replication impacts on histone H2A-H2B is currently unknown. Here, we measure H2A-H2B modifications and H2A.Z during DNA replication and across the cell cycle using quantitative genomics. We show that H2AK119ub1, H2BK120ub1, and H2A.Z are recycled accurately during DNA replication. Modified H2A-H2B are segregated symmetrically to daughter strands via POLA1 on the lagging strand, but independent of H3-H4 recycling. Post-replication, H2A-H2B modification and variant landscapes are quickly restored, and H2AK119ub1 guides accurate restoration of H3K27me3. This work reveals epigenetic transmission of parental H2A-H2B during DNA replication and identifies cross talk between H3-H4 and H2A-H2B modifications in epigenome propagation. We propose that rapid short-term memory of recycled H2A-H2B modifications facilitates restoration of stable H3-H4 chromatin states.

Chromatin remodeling of histone H3 variants by DDM1 underlies epigenetic inheritance of DNA methylation.

Nucleosomes block access to DNA methyltransferase, unless they are remodeled by DECREASE in DNA METHYLATION 1 (DDM1LSH/HELLS), a Snf2-like master regulator of epigenetic inheritance. We show that DDM1 promotes replacement of histone variant H3.3 by H3.1. In ddm1 mutants, DNA methylation is partly restored by loss of the H3.3 chaperone HIRA, while the H3.1 chaperone CAF-1 becomes essential. The single-particle cryo-EM structure at 3.2 Å of DDM1 with a variant nucleosome reveals engagement with histone H3.3 near residues required for assembly and with the unmodified H4 tail. An N-terminal autoinhibitory domain inhibits activity, while a disulfide bond in the helicase domain supports activity. DDM1 co-localizes with H3.1 and H3.3 during the cell cycle, and with the DNA methyltransferase MET1Dnmt1, but is blocked by H4K16 acetylation. The male germline H3.3 variant MGH3/HTR10 is resistant to remodeling by DDM1 and acts as a placeholder nucleosome in sperm cells for epigenetic inheritance.

Molecular mechanisms of stress-induced reactivation in mumps virus condensates.

Negative-stranded RNA viruses can establish long-term persistent infection in the form of large intracellular inclusions in the human host and cause chronic diseases. Here, we uncover how cellular stress disrupts the metastable host-virus equilibrium in persistent infection and induces viral replication in a culture model of mumps virus. Using a combination of cell biology, whole-cell proteomics, and cryo-electron tomography, we show that persistent viral replication factories are dynamic condensates and identify the largely disordered viral phosphoprotein as a driver of their assembly. Upon stress, increased phosphorylation of the phosphoprotein at its interaction interface with the viral polymerase coincides with the formation of a stable replication complex. By obtaining atomic models for the authentic mumps virus nucleocapsid, we elucidate a concomitant conformational change that exposes the viral genome to its replication machinery. These events constitute a stress-mediated switch within viral condensates that provide an environment to support upregulation of viral replication.

The Initiation of Eukaryotic DNA Replication.

DNA replication in eukaryotic cells initiates from large numbers of sites called replication origins. Initiation of replication from these origins must be tightly controlled to ensure the entire genome is precisely duplicated in each cell cycle. This is accomplished through the regulation of the first two steps in replication: loading and activation of the replicative DNA helicase. Here we describe what is known about the mechanism and regulation of these two reactions from a genetic, biochemical, and structural perspective, focusing on recent progress using proteins from budding yeast.

The Life of SARS-CoV-2 Inside Cells: Replication-Transcription Complex Assembly and Function.

The persistence of the coronavirus disease 2019 (COVID-19) pandemic has resulted in increasingly disruptive impacts, and it has become the most devastating challenge to global health in a century. The rapid emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants challenges the currently available therapeutics for clinical application. Nonstructural proteins (also known as replicase proteins) with versatile biological functions play central roles in viral replication and transcription inside the host cells, and they are the most conserved target proteins among the SARS-CoV-2 variants. Specifically, they constitute the replication-transcription complexes (RTCs) dominating the synthesis of viral RNA. Knowledge of themolecular mechanisms of nonstructural proteins and their assembly into RTCs will benefit the development of antivirals targeting them against existing or potentially emerging variants. In this review, we summarize current knowledge of the structures and functions of coronavirus nonstructural proteins as well as the assembly and functions of RTCs in the life cycle of the virus.

Replication stress impairs chromosome segregation and preimplantation development in human embryos.

Human cleavage-stage embryos frequently acquire chromosomal aneuploidies during mitosis due to unknown mechanisms. Here, we show that S phase at the 1-cell stage shows replication fork stalling, low fork speed, and DNA synthesis extending into G2 phase. DNA damage foci consistent with collapsed replication forks, DSBs, and incomplete replication form in G2 in an ATR- and MRE11-dependent manner, followed by spontaneous chromosome breakage and segmental aneuploidies. Entry into mitosis with incomplete replication results in chromosome breakage, whole and segmental chromosome errors, micronucleation, chromosome fragmentation, and poor embryo quality. Sites of spontaneous chromosome breakage are concordant with sites of DNA synthesis in G2 phase, locating to gene-poor regions with long neural genes, which are transcriptionally silent at this stage of development. Thus, DNA replication stress in mammalian preimplantation embryos predisposes gene-poor regions to fragility, and in particular in the human embryo, to the formation of aneuploidies, impairing developmental potential.

From Bioorganic Models to Cells.

I endeavor to share how various choices-some deliberate, some unconscious-and the unmistakable influence of many others shaped my scientific pursuits. I am fascinated by how two long-term, major streams of my research, DNA replication and purine biosynthesis, have merged with unexpected interconnections. If I have imparted to many of the talented individuals who have passed through my lab a degree of my passion for uncloaking the mysteries hidden in scientific research and an understanding of the honesty and rigor it demands and its impact on the world community, then my mentorship has been successful.

Structural Mechanisms for Replicating DNA in Eukaryotes.

The faithful and timely copying of DNA by molecular machines known as replisomes depends on a disparate suite of enzymes and scaffolding factors working together in a highly orchestrated manner. Large, dynamic protein-nucleic acid assemblies that selectively morph between distinct conformations and compositional states underpin this critical cellular process. In this article, we discuss recent progress outlining the physical basis of replisome construction and progression in eukaryotes.

Repair of DNA Breaks by Break-Induced Replication.

Double-strand DNA breaks (DSBs) are the most lethal type of DNA damage, making DSB repair critical for cell survival. However, some DSB repair pathways are mutagenic and promote genome rearrangements, leading to genome destabilization. One such pathway is break-induced replication (BIR), which repairs primarily one-ended DSBs, similar to those formed by collapsed replication forks or telomere erosion. BIR is initiated by the invasion of a broken DNA end into a homologous template, synthesizes new DNA within the context of a migrating bubble, and is associated with conservative inheritance of new genetic material. This mode of synthesis is responsible for a high level of genetic instability associated with BIR. Eukaryotic BIR was initially investigated in yeast, but now it is also actively studied in mammalian systems. Additionally, a significant breakthrough has been made regarding the role of microhomology-mediated BIR in the formation of complex genomic rearrangements that underly various human pathologies.

Coupling of N7-methyltransferase and 3'-5' exoribonuclease with SARS-CoV-2 polymerase reveals mechanisms for capping and proofreading.

The capping of mRNA and the proofreading play essential roles in SARS-CoV-2 replication and transcription. Here, we present the cryo-EM structure of the SARS-CoV-2 replication-transcription complex (RTC) in a form identified as Cap(0)-RTC, which couples a co-transcriptional capping complex (CCC) composed of nsp12 NiRAN, nsp9, the bifunctional nsp14 possessing an N-terminal exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), and nsp10 as a cofactor of nsp14. Nsp9 and nsp12 NiRAN recruit nsp10/nsp14 into the Cap(0)-RTC, forming the N7-CCC to yield cap(0) (7MeGpppA) at 5' end of pre-mRNA. A dimeric form of Cap(0)-RTC observed by cryo-EM suggests an in trans backtracking mechanism for nsp14 ExoN to facilitate proofreading of the RNA in concert with polymerase nsp12. These results not only provide a structural basis for understanding co-transcriptional modification of SARS-CoV-2 mRNA but also shed light on how replication fidelity in SARS-CoV-2 is maintained.

TMEM41B Is a Pan-flavivirus Host Factor.

Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection, we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results, we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms present at nearly 20% in East Asian populations reduce flavivirus infection. Based on our mechanistic studies, we propose that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication.