DNA and Chromosomes
Evolution of Rev7 interactions in eukaryotic TLS DNA polymerase Polζ
Translesion synthesis (TLS) DNA polymerase Polζ is crucial for the bypass replication over sites of DNA damage. The Rev7 subunit of Polζ is a HORMA (Hop1, Rev7, Mad2) protein that facilitates recruitment of Polζ to the replication fork via interactions with the catalytic subunit Rev3 and the translesion synthesis scaffold protein Rev1. Human Rev7 (hRev7) interacts with two Rev7-binding motifs (RBMs) of hRev3 by a mechanism conserved among HORMA proteins whereby the safety-belt loop of hRev7 closes on the top of the ligand.The MRN complex and topoisomerase IIIa–RMI1/2 synchronize DNA resection motor proteins
DNA resection—the nucleolytic processing of broken DNA ends—is the first step of homologous recombination. Resection is catalyzed by the resectosome, a multienzyme complex that includes bloom syndrome helicase (BLM), DNA2 or exonuclease 1 nucleases, and additional DNA-binding proteins. Although the molecular players have been known for over a decade, how the individual proteins work together to regulate DNA resection remains unknown. Using single-molecule imaging, we characterized the roles of the MRE11–RAD50–NBS1 complex (MRN) and topoisomerase IIIa (TOP3A)–RMI1/2 during long-range DNA resection.The N-terminal domain of human mitochondrial helicase Twinkle has DNA-binding activity crucial for supporting processive DNA synthesis by polymerase γ
Twinkle is the ring-shaped replicative helicase within the human mitochondria with high homology to bacteriophage T7 gp4 helicase–primase. Unlike many orthologs of Twinkle, the N-terminal domain (NTD) of human Twinkle has lost its primase activity through evolutionarily acquired mutations. The NTD has no demonstrated activity thus far; its role has remained unclear. Here, we biochemically characterize the isolated NTD and C-terminal domain (CTD) with linker to decipher their contributions to full-length Twinkle activities.Characterization of the Escherichia coli XPD/Rad3 iron-sulfur helicase YoaA in complex with the DNA polymerase III clamp loader subunit chi (χ)
Escherichia coli YoaA aids in the resolution of DNA damage that halts DNA synthesis in vivo in conjunction with χ, an accessory subunit of DNA polymerase III. YoaA and χ form a discrete complex separate from the DNA polymerase III holoenzyme, but little is known about how YoaA and χ work together to help the replication fork overcome damage. Although YoaA is predicted to be an iron-sulfur helicase in the XPD/Rad3 helicase family based on sequence analysis, the biochemical activities of YoaA have not been described.DNA repair protein RAD52 is required for protecting G-quadruplexes in mammalian cells
G-quadruplex (G4)-forming DNA sequences are abundant in the human genome, and they are hot spots for inducing DNA double-strand breaks (DSBs) and genome instability. The mechanisms involved in protecting G4s and maintaining genome stability have not been fully elucidated. Here, we demonstrated that RAD52 plays an important role in suppressing DSB accumulation at G4s, and RAD52-deficient cells are sensitive to G4-stabilizing compounds. Mechanistically, we showed that RAD52 is required for efficient homologous recombination repair at G4s, likely due to its function in recruiting structure-specific endonuclease XPF to remove G4 structures at DSB ends.Rapid excision of oxidized adenine by human thymine DNA glycosylase
Oxidation of DNA bases generates mutagenic and cytotoxic lesions that are implicated in cancer and other diseases. Oxidative base lesions, including 7,8-dihydro-8-oxoguanine, are typically removed through base excision repair. In addition, oxidized deoxynucleotides such as 8-oxo-dGTP are depleted by sanitizing enzymes to preclude DNA incorporation. While pathways that counter threats posed by 7,8-dihydro-8-oxoguanine are well characterized, mechanisms protecting against the major adenine oxidation product, 7,8-dihydro-8-oxoadenine (oxoA), are poorly understood.Deubiquitinase USP2 stabilizes the MRE11–RAD50–NBS1 complex at DNA double-strand break sites by counteracting the ubiquitination of NBS1
The MRE11–RAD50–NBS1 (MRN) complex plays essential roles in the cellular response to DNA double-strand breaks (DSBs), which are the most cytotoxic DNA lesions, and is a target of various modifications and controls. Recently, lysine 48-linked ubiquitination of NBS1, resulting in premature disassembly of the MRN complex from DSB sites, was observed in cells lacking RECQL4 helicase activity. However, the role and control of this ubiquitination during the DSB response in cells with intact RECQL4 remain unknown.Yeast 9-1-1 complex acts as a sliding clamp for DNA synthesis by DNA polymerase ε
Eukaryotic cells harbor two DNA-binding clamps, proliferating cell nuclear antigen (PCNA), and another clamp commonly referred to as 9-1-1 clamp. In contrast to the essential role of PCNA in DNA replication as a sliding clamp for DNA polymerase (Pol) δ, no such role in DNA synthesis has been identified for the human 9-1-1 clamp or the orthologous yeast 17-3-1 clamp. The only role identified for either the 9-1-1 or 17-3-1 clamp is in the recruitment of signal transduction kinases, which affect the activation of cell cycle checkpoints in response to DNA damage.Transcription suppression is mediated by the HDAC1–Sin3 complex in Xenopus nucleoplasmic extract
Modification of histones provides a dynamic mechanism to regulate chromatin structure and access to DNA. Histone acetylation, in particular, plays a prominent role in controlling the interaction between DNA, histones, and other chromatin-associated proteins. Defects in histone acetylation patterns interfere with normal gene expression and underlie a wide range of human diseases. Here, we utilize Xenopus egg extracts to investigate how changes in histone acetylation influence transcription of a defined gene construct.Exploiting the distinctive properties of the bacterial and human MutS homolog sliding clamps on mismatched DNA
MutS homologs (MSHs) are highly conserved core components of DNA mismatch repair. Mismatch recognition provokes ATP-binding by MSH proteins that drives a conformational transition from a short-lived lesion-searching clamp to an extremely stable sliding clamp on the DNA. Here, we have expanded on previous bulk biochemical studies to examine the stability, lifetime, and kinetics of bacterial and human MSH sliding clamps on mismatched DNA using surface plasmon resonance and single-molecule analysis of fluorescently labeled proteins.The SOS response-associated peptidase (SRAP) domain of YedK catalyzes ring opening of abasic sites and reversal of its DNA–protein cross-link
Apurinic/apyrimidinic (AP, or abasic) sites in DNA are one of the most common forms of DNA damage. AP sites are reactive and form cross-links to both proteins and DNA, are prone to strand breakage, and inhibit DNA replication and transcription. The replication-associated AP site repair protein HMCES protects cells from strand breaks, inhibits mutagenic translesion synthesis, and participates in repair of interstrand DNA cross-links derived from AP sites by forming a stable thiazolidine DNA–protein cross-link (DPC) to AP sites in single-stranded DNA (ssDNA).Extended DNA-binding interfaces beyond the canonical SAP domain contribute to the function of replication stress regulator SDE2 at DNA replication forks
Elevated DNA replication stress causes instability of the DNA replication fork and increased DNA mutations, which underlies tumorigenesis. The DNA replication stress regulator silencing-defective 2 (SDE2) is known to bind to TIMELESS (TIM), a protein of the fork protection complex, and enhances its stability, thereby supporting replisome activity at DNA replication forks. However, the DNA-binding activity of SDE2 is not well defined. Here, we structurally and functionally characterize a new conserved DNA-binding motif related to the SAP (SAF-A/B, Acinus, PIAS) domain in human SDE2 and establish its preference for ssDNA.The p97 segregase cofactor Ubxn7 facilitates replisome disassembly during S-phase
Complex cellular processes are driven by the regulated assembly and disassembly of large multiprotein complexes. While we are beginning to understand the molecular mechanism for assembly of the eukaryotic DNA replication machinery (replisome), we still know relatively little about the regulation of its disassembly at replication termination. Recently, the first elements of this process have emerged, revealing that the replicative helicase, at the heart of the replisome, is polyubiquitylated prior to unloading and that this unloading requires p97 segregase activity.Development of a universal antibiotic resistance screening reporter for improving efficiency of cytosine and adenine base editing
Base editing has emerged as a revolutionary technology for single nucleotide modifications. The cytosine and adenine base editors (CBEs and ABEs) have demonstrated great potential in clinical and fundamental research. However, screening and isolating target-edited cells remains challenging. In the current study, we developed a universal Adenine and Cytosine Base-Editing Antibiotic Resistance Screening Reporter (ACBE-ARSR) for improving the editing efficiency. To develop the reporter, the CBE-ARSR was first constructed and shown to be capable of enriching cells for those that had undergone CBE editing activity.Interplay between H3K36me3, methyltransferase SETD2, and mismatch recognition protein MutSα facilitates processing of oxidative DNA damage in human cells
Oxidative DNA damage contributes to aging and the pathogenesis of numerous human diseases including cancer. 8-hydroxyguanine (8-oxoG) is the major product of oxidative DNA lesions. Although OGG1-mediated base excision repair is the primary mechanism for 8-oxoG removal, DNA mismatch repair has also been implicated in processing oxidative DNA damage. However, the mechanism of the latter is not fully understood. Here, we treated human cells defective in various 8-oxoG repair factors with H2O2 and performed biochemical, live cell imaging, and chromatin immunoprecipitation sequencing analyses to determine their response to the treatment.Multifaceted regulation of the sumoylation of the Sgs1 DNA helicase
Homologous recombination repairs DNA breaks and sequence gaps via the production of joint DNA intermediates such as Holliday junctions. Dissolving Holliday junctions into linear DNA repair products requires the activity of the Sgs1 helicase in yeast and of its homologs in other organisms. Recent studies suggest that the functions of these conserved helicases are regulated by sumoylation; however, the mechanisms that promote their sumoylation are not well understood. Here, we employed in vitro sumoylation systems and cellular assays to determine the roles of DNA and the scaffold protein Esc2 in Sgs1 sumoylation.In vitro eradication of abasic site-mediated DNA–peptide/protein cross-links by Escherichia coli long-patch base excision repair
Apurinic/apyrimidinic (AP or abasic) sites are among the most abundant DNA lesions. Numerous proteins within different organisms ranging from bacteria to human have been demonstrated to react with AP sites to form covalent Schiff base DNA–protein cross-links (DPCs). These DPCs are unstable due to their spontaneous hydrolysis, but the half-lives of these cross-links can be as long as several hours. Such long-lived DPCs are extremely toxic due to their large sizes, which physically block DNA replication.Concerted actions of DnaA complexes with DNA-unwinding sequences within and flanking replication origin oriC promote DnaB helicase loading
Unwinding of the replication origin and loading of DNA helicases underlie the initiation of chromosomal replication. In Escherichia coli, the minimal origin oriC contains a duplex unwinding element (DUE) region and three (Left, Middle, and Right) regions that bind the initiator protein DnaA. The Left/Right regions bear a set of DnaA-binding sequences, constituting the Left/Right-DnaA subcomplexes, while the Middle region has a single DnaA-binding site, which stimulates formation of the Left/Right-DnaA subcomplexes.Residues located in the primase domain of the bacteriophage T7 primase-helicase are essential for loading the hexameric complex onto DNA
The T7 primase-helicase plays a pivotal role in the replication of T7 DNA. Using affinity isolation of peptide–nucleic acid crosslinks and mass spectrometry, we identify protein regions in the primase-helicase and T7 DNA polymerase that form contacts with the RNA primer and DNA template. The contacts between nucleic acids and the primase domain of the primase-helicase are centered in the RNA polymerase subdomain of the primase domain, in a cleft between the N-terminal subdomain and the topoisomerase-primase fold.Histone chaperone ASF1 acts with RIF1 to promote DNA end joining in BRCA1-deficient cells
Replication timing regulatory factor 1 (RIF1) acts downstream of p53-binding protein 53BP1 to inhibit the resection of DNA broken ends, which plays critical roles in determining the DNA double-strand break repair pathway choice between nonhomologous end joining and homologous recombination (HR). However, the mechanism by which this choice is made is not yet clear. In this study, we identified that histone chaperone protein ASF1 associates with RIF1 and regulates RIF1-dependent functions in the DNA damage response.Intersubunit and intrasubunit interactions driving the MukBEF ATPase
MukBEF, a structural maintenance of chromosome-like protein complex consisting of an ATPase, MukB, and two interacting subunits, MukE and MukF, functions as the bacterial condensin. It is likely that MukBEF compacts DNA via an ATP hydrolysis–dependent DNA loop–extrusion reaction similar to that demonstrated for the yeast structural maintenance of chromosome proteins condensin and cohesin. MukB also interacts with the ParC subunit of the cellular chromosomal decatenase topoisomerase IV, an interaction that is required for proper chromosome condensation and segregation in Escherichia coli, although it suppresses the MukB ATPase activity.The N-terminal domain of the Agrobacterium tumefaciens telomere resolvase, TelA, regulates its DNA cleavage and rejoining activities
Linear replicons can be found in a minority of prokaryotic organisms, including Borrelia species and Agrobacterium tumefaciens. The problem with replicating the lagging strand end of linear DNAs is circumvented in these organisms by the presence of covalently closed DNA hairpin telomeres at the DNA termini. Telomere resolvases are enzymes responsible for generating these hairpin telomeres from a dimeric replication intermediate through a two-step DNA cleavage and rejoining reaction referred to as telomere resolution.Stable G-quadruplex DNA structures promote replication-dependent genome instability
G-quadruplex (G4)–prone structures are abundant in mammalian genomes, where they have been shown to influence DNA replication, transcription, and genome stability. In this article, we constructed cells with a single ectopic homopurine/homopyrimidine repeat tract derived from the polycystic kidney disease type 1 (PKD1) locus, which is capable of forming triplex (H3) and G4 DNA structures. We show that ligand stabilization of these G4 structures results in deletions of the G4 consensus sequence, as well as kilobase deletions spanning the G4 and ectopic sites.Nej1 interacts with Sae2 at DNA double-stranded breaks to inhibit DNA resection
The two major pathways of DNA double-strand break repair, nonhomologous end-joining and homologous recombination, are highly conserved from yeast to mammals. The regulation of 5′-DNA resection controls repair pathway choice and influences repair outcomes. Nej1 was first identified as a canonical NHEJ factor involved in stimulating the ligation of broken DNA ends, and more recently, it was shown to participate in DNA end-bridging and in the inhibition of 5′-resection mediated by the nuclease/helicase complex Dna2–Sgs1.DNA polymerase η promotes nonhomologous end joining upon etoposide exposure dependent on the scaffolding protein Kap1
DNA polymerase eta (Pol η) is a eukaryotic member of the Y-family of DNA polymerase involved in translesion DNA synthesis and genome mutagenesis. Recently, several translesion DNA synthesis polymerases have been found to function in repair of DNA double-strand breaks (DSBs). However, the role of Pol η in promoting DSB repair remains to be well defined. Here, we demonstrated that Pol η could be targeted to etoposide (ETO)-induced DSBs and that depletion of Pol η in cells causes increased sensitivity to ETO.
