DNA and Chromosomes
Hyperextended telomeres promote formation of C-circle DNA in telomerase positive human cells
Telomere length maintenance is crucial to cancer cell immortality. Up to 15% of cancers utilize a telomerase-independent, recombination-based mechanism termed alternative lengthening of telomeres (ALT). Currently, the primary ALT biomarker is the C-circle, a type of circular DNA with extrachromosomal telomere repeats (cECTRs). How C-circles form is not well characterized. We investigated C-circle formation in the human cen3tel cell line, a long-telomere, telomerase+ (LTT+) cell line with progressively hyper-elongated telomeres (up to ∼100 kb).Termination of DNA replication at Tus-ter barriers results in under-replication of template DNA
The complete and accurate duplication of genomic information is vital to maintain genome stability in all domains of life. In Escherichia coli, replication termination, the final stage of the duplication process, is confined to the “replication fork trap” region by multiple unidirectional fork barriers formed by the binding of Tus protein to genomic ter sites. Termination typically occurs away from Tus-ter complexes, but they become part of the fork fusion process when a delay to one replisome allows the second replisome to travel more than halfway around the chromosome.An automated, high-throughput methodology optimized for quantitative cell-free mitochondrial and nuclear DNA isolation from plasma
Progress in the study of circulating, cell-free nuclear DNA (ccf-nDNA) in cancer detection has led to the development of noninvasive clinical diagnostic tests and has accelerated the evaluation of ccf-nDNA abundance as a disease biomarker. Likewise, circulating, cell-free mitochondrial DNA (ccf-mtDNA) is under similar investigation. However, optimal ccf-mtDNA isolation parameters have not been established, and inconsistent protocols for ccf-nDNA collection, storage, and analysis have hindered its clinical utility.Processing and integration of functionally oriented prespacers in the Escherichia coli CRISPR system depends on bacterial host exonucleases
CRISPR-Cas systems provide bacteria with adaptive immunity against viruses. During spacer adaptation, the Cas1-Cas2 complex selects fragments of foreign DNA, called prespacers, and integrates them into CRISPR arrays in an orientation that provides functional immunity. Cas4 is involved in both the trimming of prespacers and the cleavage of protospacer adjacent motif (PAM) in several type I CRISPR-Cas systems, but how the prespacers are processed in systems lacking Cas4, such as the type I-E and I-F systems, is not understood.Target sequence requirements of a type III-B CRISPR-Cas immune system
CRISPR-Cas systems are RNA-based immune systems that protect many prokaryotes from invasion by viruses and plasmids. Type III CRISPR systems are unique, as their targeting mechanism requires target transcription. Upon transcript binding, DNA cleavage by type III effector complexes is activated. Type III systems must differentiate between invader and native transcripts to prevent autoimmunity. Transcript origin is dictated by the sequence that flanks the 3′ end of the RNA target site (called the PFS).The abundant DNA adduct N7-methyl deoxyguanosine contributes to miscoding during replication by human DNA polymerase η
Aside from abasic sites and ribonucleotides, the DNA adduct N7-methyl deoxyguanosine (N7-CH3 dG) is one of the most abundant lesions in mammalian DNA. Because N7-CH3 dG is unstable, leading to deglycosylation and ring-opening, its miscoding potential is not well-understood. Here, we employed a 2′-fluoro isostere approach to synthesize an oligonucleotide containing an analog of this lesion (N7-CH3 2′-F dG) and examined its miscoding potential with four Y-family translesion synthesis DNA polymerases (pols): human pol (hpol) η, hpol κ, and hpol ι and Dpo4 from the archaeal thermophile Sulfolobus solfataricus.Dynamic interactions of the homologous pairing 2 (Hop2)–meiotic nuclear divisions 1 (Mnd1) protein complex with meiotic presynaptic filaments in budding yeast
Homologous recombination (HR) is a universally conserved DNA repair pathway that can result in the exchange of genetic material. In eukaryotes, HR has evolved into an essential step in meiosis. During meiosis many eukaryotes utilize a two-recombinase pathway. This system consists of Rad51 and the meiosis-specific recombinase Dmc1. Both recombinases have distinct activities during meiotic HR, despite being highly similar in sequence and having closely related biochemical activities, raising the question of how these two proteins can perform separate functions.Introduction to the Thematic Minireview Series: DNA double-strand break repair and pathway choice
Environmental agents and reactive metabolites induce myriad chromosomal lesions that challenge the integrity of our genome. In particular, the DNA double-strand break (DSB) has the highest potential to cause the types of chromosome aberrations and rearrangements found in transformed and cancer cells. Several conserved pathways of DSB repair exist in eukaryotes, and these have been the subject of intense studies in recent years. In this Thematic Minireview Series, four leading research groups review recent progress in deciphering DSB repair mechanisms and the intricate regulatory network that helps determine the preferential engagement of one pathway over others.Long repeating (TTAGGG)n single-stranded DNA self-condenses into compact beaded filaments stabilized by G-quadruplex formation
Conformations adopted by long stretches of single-stranded DNA (ssDNA) are of central interest in understanding the architecture of replication forks, R loops, and other structures generated during DNA metabolism in vivo. This is particularly so if the ssDNA consists of short nucleotide repeats. Such studies have been hampered by the lack of defined substrates greater than ∼150 nt and the absence of high-resolution biophysical approaches. Here we describe the generation of very long ssDNA consisting of the mammalian telomeric repeat (5′-TTAGGG-3′)n, as well as the interrogation of its structure by EM and single-molecule magnetic tweezers (smMT).The bacterial condensin MukB compacts DNA by sequestering supercoils and stabilizing topologically isolated loops
MukB is a structural maintenance of chromosome-like protein required for DNA condensation. The complete condensin is a large tripartite complex of MukB, the kleisin, MukF, and an accessory protein, MukE. As found previously, MukB DNA condensation is a stepwise process. We have defined these steps topologically. They proceed first via the formation of negative supercoils that are sequestered by the protein followed by hinge–hinge interactions between MukB dimers that stabilize topologically isolated loops in the DNA.The MukB–topoisomerase IV interaction is required for proper chromosome compaction
The bacterial condensin MukB and the cellular decatenating enzyme topoisomerase IV interact. This interaction stimulates intramolecular reactions catalyzed by topoisomerase IV, supercoiled DNA relaxation, and DNA knotting but not intermolecular reactions such as decatenation of linked DNAs. We have demonstrated previously that MukB condenses DNA by sequestering negative supercoils and stabilizing topologically isolated loops in the DNA. We show here that the MukB–topoisomerase IV interaction stabilizes MukB on DNA, increasing the extent of DNA condensation without increasing the amount of MukB bound to the DNA.Replisome-mediated translesion synthesis by a cellular replicase
Genome integrity relies on the ability of the replisome to navigate ubiquitous DNA damage during DNA replication. The Escherichia coli replisome transiently stalls at leading-strand template lesions and can either reinitiate replication downstream of the lesion or recruit specialized DNA polymerases that can bypass the lesion via translesion synthesis. Previous results had suggested that the E. coli replicase might play a role in lesion bypass, but this possibility has not been tested in reconstituted DNA replication systems.The DEAD-box protein DDX43 (HAGE) is a dual RNA-DNA helicase and has a K-homology domain required for full nucleic acid unwinding activity
The K-homology (KH) domain is a nucleic acid-binding domain present in many proteins but has not been reported in helicases. DDX43, also known as HAGE (helicase antigen gene), is a member of the DEAD-box protein family. It contains a helicase core domain in its C terminus and a potential KH domain in its N terminus. DDX43 is highly expressed in many tumors and is, therefore, considered a potential target for immunotherapy. Despite its potential as a therapeutic target, little is known about its activities.Sap1 is a replication-initiation factor essential for the assembly of pre-replicative complex in the fission yeast Schizosaccharomyces pombe
A central step in the initiation of chromosomal DNA replication in eukaryotes is the assembly of pre-replicative complex (pre-RC) at late M and early G1 phase of the cell cycles. Since 1973, four proteins or protein complexes, including cell division control protein 6 (Cdc6)/Cdc18, minichromosome maintenance protein complex, origin recognition complex (ORC), and Cdt1, are known components of the pre-RC. Previously, we reported that a non-ORC protein binds to the essential element Δ9 of the Schizosaccharomyces pombe DNA-replication origin ARS3001.Structural Basis for the Lesion-scanning Mechanism of the MutY DNA Glycosylase
The highly mutagenic A:8-oxoguanine (oxoG) base pair is generated mainly by misreplication of the C:oxoG base pair, the oxidation product of the C:G base pair. The A:oxoG base pair is particularly insidious because neither base in it carries faithful information to direct the repair of the other. The bacterial MutY (MUTYH in humans) adenine DNA glycosylase is able to initiate the repair of A:oxoG by selectively cleaving the A base from the A:oxoG base pair. The difference between faithful repair and wreaking mutagenic havoc on the genome lies in the accurate discrimination between two structurally similar base pairs: A:oxoG and A:T.Shared Subunits of Tetrahymena Telomerase Holoenzyme and Replication Protein A Have Different Functions in Different Cellular Complexes
In most eukaryotes, telomere maintenance relies on telomeric repeat synthesis by a reverse transcriptase named telomerase. To synthesize telomeric repeats, the catalytic subunit telomerase reverse transcriptase (TERT) uses the RNA subunit (TER) as a template. In the ciliate Tetrahymena thermophila, the telomerase holoenzyme consists of TER, TERT, and eight additional proteins, including the telomeric repeat single-stranded DNA-binding protein Teb1 and its heterotrimer partners Teb2 and Teb3. Teb1 is paralogous to the large subunit of the general single-stranded DNA binding heterotrimer replication protein A (RPA).MukB-mediated Catenation of DNA Is ATP and MukEF Independent
Properly condensed chromosomes are necessary for accurate segregation of the sisters after DNA replication. The Escherichia coli condesin is MukB, a structural maintenance of chromosomes (SMC)-like protein, which forms a complex with MukE and the kleisin MukF. MukB is known to be able to mediate knotting of a DNA ring, an intramolecular reaction. In our investigations of how MukB condenses DNA we discovered that it can also mediate catenation of two DNA rings, an intermolecular reaction. This activity of MukB requires DNA binding by the head domains of the protein but does not require either ATP or its partner proteins MukE or MukF.Identification of a Substrate Recognition Domain in the Replication Stress Response Protein Zinc Finger Ran-binding Domain-containing Protein 3 (ZRANB3)
DNA damage and other forms of replication stress can cause replication forks to stall. Replication stress response proteins stabilize and resolve stalled forks by mechanisms that include fork remodeling to facilitate repair or bypass of damaged templates. Several enzymes including SMARCAL1, HLTF, and ZRANB3 catalyze these reactions. SMARCAL1 and HLTF utilize structurally distinct accessory domains attached to an ATPase motor domain to facilitate DNA binding and catalysis of fork remodeling reactions.Genetic Control of Replication through N1-methyladenine in Human Cells
N1-methyl adenine (1-MeA) is formed in DNA by reaction with alkylating agents and naturally occurring methyl halides. The 1-MeA lesion impairs Watson-Crick base pairing and blocks normal DNA replication. Here we identify the translesion synthesis (TLS) DNA polymerases (Pols) required for replicating through 1-MeA in human cells and show that TLS through this lesion is mediated via three different pathways in which Pols ι and θ function in one pathway and Pols η and ζ, respectively, function in the other two pathways.DNA Replication Dynamics of the GGGGCC Repeat of the C9orf72 Gene
Background: The (GGGGCC)n hexanucleotide repeat expansion of C9orf72 is the most common genetic cause of ALS-FTD.Results: C9orf72 repeat expansion increases instability and decreases replication efficiency by disrupting replication fork progression.Conclusion: C9orf72 repeat length and replication direction contribute to repeat instability in human cells.Significance: DNA replication-induced instability at the C9orf72 GGGGCC repeat can lead to further expansion and more severe disease.Unifying the DNA End-processing Roles of the Artemis Nuclease: KU-DEPENDENT ARTEMIS RESECTION AT BLUNT DNA ENDS
Background: Artemis is a nuclease that is necessary for hairpin opening in V(D)J recombination.Results: Artemis action on blunt DNA ends is dependent on DNA sequence (breathing) and Ku.Conclusion: Breathing of blunt DNA ends into a transient ss/dsDNA boundary is needed for Artemis action.Significance: Unification of Artemis nuclease action explains the features of NHEJ and V(D)J recombination.Hexapeptides That Inhibit Processing of Branched DNA Structures Induce a Dynamic Ensemble of Holliday Junction Conformations
Background: Anti-microbial hexapeptides trap Holliday junctions and inhibit junction-processing enzymes.Results: Hexapeptides induce multiple conformations and dynamic fluctuations of two Holliday junctions that differ in core sequence.Conclusion: Destabilization of the functional junction conformation likely contributes to inhibition of enzymes that process Holliday junctions.Significance: Ligand-induced conformational dynamics may contribute generally to the action of anti-microbial agents that target specialized DNA structures.Human DNA Exonuclease TREX1 Is Also an Exoribonuclease That Acts on Single-stranded RNA
3′ repair exonuclease 1 (TREX1) is a known DNA exonuclease involved in autoimmune disorders and the antiviral response. In this work, we show that TREX1 is also a RNA exonuclease. Purified TREX1 displays robust exoribonuclease activity that degrades single-stranded, but not double-stranded, RNA. TREX1-D200N, an Aicardi-Goutieres syndrome disease-causing mutant, is defective in degrading RNA. TREX1 activity is strongly inhibited by a stretch of pyrimidine residues as is a bacterial homolog, RNase T.The Roles of Family B and D DNA Polymerases in Thermococcus Species 9°N Okazaki Fragment Maturation
Background:During replication, the lagging strand is synthesized discontinuously from a series of Okazaki fragments.Results:Okazaki fragment maturation was reconstituted using purified proteins from Thermococcus.Conclusion:In Thermococcus, efficient Okazaki fragment processing requires DNA polymerase B, flap endonuclease, and DNA ligase.Significance:Okazaki fragment maturation in Thermococcus shares similarities to both bacterial and eukaryotic systems.Dpb11 Protein Helps Control Assembly of the Cdc45·Mcm2-7·GINS Replication Fork Helicase
Background: Dpb11 is required for the initiation of DNA replication. The replication fork helicase is composed of Cdc45, Mcm2-7, and GINS.Results: Dpb11 recruits Cdc45 to Mcm2-7, and Dpb11 blocks GINS interaction with Mcm2-7. Dpb11 also binds to ssDNA, and this interaction releases Dpb11 from Mcm2-7.Conclusion: Dpb11 helps control assembly of the replication fork helicase.Significance: A mechanism for Dpb11 function is described.
