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1.
Mol Cell ; 82(19): 3538-3552.e5, 2022 10 06.
Article in English | MEDLINE | ID: mdl-36075220

ABSTRACT

DNA becomes single stranded (ssDNA) during replication, transcription, and repair. Transiently formed ssDNA segments can adopt alternative conformations, including cruciforms, triplexes, and quadruplexes. To determine whether there are stable regions of ssDNA in the human genome, we utilized S1-END-seq to convert ssDNA regions to DNA double-strand breaks, which were then processed for high-throughput sequencing. This approach revealed two predominant non-B DNA structures: cruciform DNA formed by expanded (TA)n repeats that accumulate in microsatellite unstable human cancer cell lines and DNA triplexes (H-DNA) formed by homopurine/homopyrimidine mirror repeats common across a variety of cell lines. We show that H-DNA is enriched during replication, that its genomic location is highly conserved, and that H-DNA formed by (GAA)n repeats can be disrupted by treatment with a (GAA)n-binding polyamide. Finally, we show that triplex-forming repeats are hotspots for mutagenesis. Our results identify dynamic DNA secondary structures in vivo that contribute to elevated genome instability.


Subject(s)
DNA, Cruciform , Nylons , DNA/metabolism , DNA Breaks, Double-Stranded , DNA Replication , Humans , Nucleic Acid Conformation
2.
Cell Rep ; 39(9): 110871, 2022 05 31.
Article in English | MEDLINE | ID: mdl-35649380

ABSTRACT

The maintenance of genome stability relies on coordinated control of origin activation and replication fork progression. How the interplay between these processes influences human genetic disease and cancer remains incompletely characterized. Here we show that mouse cells featuring Polε instability exhibit impaired genome-wide activation of DNA replication origins, in an origin-location-independent manner. Strikingly, Trp53 ablation in primary Polε hypomorphic cells increased Polε levels and origin activation and reduced DNA damage in a transcription-dependent manner. Transcriptome analysis of primary Trp53 knockout cells revealed that the TRP53-CDKN1A/P21 axis maintains appropriate levels of replication factors and CDK activity during unchallenged S phase. Loss of this control mechanism deregulates origin activation and perturbs genome-wide replication fork progression. Thus, while our data support an impaired origin activation model for genetic diseases affecting CMG formation, we propose that loss of the TRP53-CDKN1A/P21 tumor suppressor axis induces inappropriate origin activation and deregulates genome-wide fork progression.


Subject(s)
Cyclin-Dependent Kinase Inhibitor p21 , DNA Polymerase II , DNA Replication , Poly-ADP-Ribose Binding Proteins , Replication Origin , Tumor Suppressor Protein p53 , Animals , Cell Cycle Proteins/metabolism , Cyclin-Dependent Kinase Inhibitor p21/genetics , DNA Damage/genetics , DNA Polymerase II/genetics , DNA Replication/genetics , Mice , Poly-ADP-Ribose Binding Proteins/genetics , S Phase , Tumor Suppressor Protein p53/genetics
3.
Curr Opin Genet Dev ; 71: 34-38, 2021 12.
Article in English | MEDLINE | ID: mdl-34284257

ABSTRACT

One of the goals of precision medicine is to uncover selective vulnerabilities in various cancers. A notable success has been the development of PARP inhibitors for the treatment of breast and ovarian cancers with mutations in BRCA genes. Only two years ago, it was discovered that cancers with microsatellite instability (MSI) were selectively dependent on the RecQ DNA helicase WRN. Subsequently, the molecular mechanism underlying WRN dependency in MSI cancers was uncovered. Here, we review how these developments have led to a promising new drug target in MSI cancers.


Subject(s)
Microsatellite Instability , Neoplasms , Exodeoxyribonucleases/genetics , Humans , Microsatellite Repeats , Neoplasms/drug therapy , Neoplasms/genetics , RecQ Helicases/genetics , RecQ Helicases/metabolism , Werner Syndrome Helicase/genetics
4.
Nature ; 593(7859): 440-444, 2021 05.
Article in English | MEDLINE | ID: mdl-33767446

ABSTRACT

Defects in DNA repair frequently lead to neurodevelopmental and neurodegenerative diseases, underscoring the particular importance of DNA repair in long-lived post-mitotic neurons1,2. The cellular genome is subjected to a constant barrage of endogenous DNA damage, but surprisingly little is known about the identity of the lesion(s) that accumulate in neurons and whether they accrue throughout the genome or at specific loci. Here we show that post-mitotic neurons accumulate unexpectedly high levels of DNA single-strand breaks (SSBs) at specific sites within the genome. Genome-wide mapping reveals that SSBs are located within enhancers at or near CpG dinucleotides and sites of DNA demethylation. These SSBs are repaired by PARP1 and XRCC1-dependent mechanisms. Notably, deficiencies in XRCC1-dependent short-patch repair increase DNA repair synthesis at neuronal enhancers, whereas defects in long-patch repair reduce synthesis. The high levels of SSB repair in neuronal enhancers are therefore likely to be sustained by both short-patch and long-patch processes. These data provide the first evidence of site- and cell-type-specific SSB repair, revealing unexpected levels of localized and continuous DNA breakage in neurons. In addition, they suggest an explanation for the neurodegenerative phenotypes that occur in patients with defective SSB repair.


Subject(s)
DNA Breaks, Single-Stranded , DNA Repair , Enhancer Elements, Genetic/genetics , Neurons/metabolism , 5-Methylcytosine/metabolism , Cell Line , DNA/biosynthesis , DNA Replication , Humans , Male , Methylation , Poly(ADP-ribose) Polymerases/metabolism , Sequence Analysis, DNA
5.
Nature ; 586(7828): 292-298, 2020 10.
Article in English | MEDLINE | ID: mdl-32999459

ABSTRACT

The RecQ DNA helicase WRN is a synthetic lethal target for cancer cells with microsatellite instability (MSI), a form of genetic hypermutability that arises from impaired mismatch repair1-4. Depletion of WRN induces widespread DNA double-strand breaks in MSI cells, leading to cell cycle arrest and/or apoptosis. However, the mechanism by which WRN protects MSI-associated cancers from double-strand breaks remains unclear. Here we show that TA-dinucleotide repeats are highly unstable in MSI cells and undergo large-scale expansions, distinct from previously described insertion or deletion mutations of a few nucleotides5. Expanded TA repeats form non-B DNA secondary structures that stall replication forks, activate the ATR checkpoint kinase, and require unwinding by the WRN helicase. In the absence of WRN, the expanded TA-dinucleotide repeats are susceptible to cleavage by the MUS81 nuclease, leading to massive chromosome shattering. These findings identify a distinct biomarker that underlies the synthetic lethal dependence on WRN, and support the development of therapeutic agents that target WRN for MSI-associated cancers.


Subject(s)
DNA Breaks, Double-Stranded , DNA Repeat Expansion/genetics , Dinucleotide Repeats/genetics , Neoplasms/genetics , Werner Syndrome Helicase/metabolism , Ataxia Telangiectasia Mutated Proteins/metabolism , Cell Line, Tumor , Chromosomes, Human/genetics , Chromosomes, Human/metabolism , Chromothripsis , DNA Cleavage , DNA Replication , DNA-Binding Proteins/metabolism , Endodeoxyribonucleases/metabolism , Endonucleases/metabolism , Genomic Instability , Humans , Recombinases/metabolism
6.
Genes (Basel) ; 10(11)2019 10 31.
Article in English | MEDLINE | ID: mdl-31683575

ABSTRACT

Guanine quadruplex (G4) structures are among the most stable secondary DNA structures that can form in vitro, and evidence for their existence in vivo has been steadily accumulating. Originally described mainly for their deleterious effects on genome stability, more recent research has focused on (potential) functions of G4 structures in telomere maintenance, gene expression, and other cellular processes. The combined research on G4 structures has revealed that properly regulating G4 DNA structures in cells is important to prevent genome instability and disruption of normal cell function. In this short review we provide some background and historical context of our work resulting in the identification of FANCJ, RTEL1 and BLM as helicases that act on G4 structures in vivo. Taken together these studies highlight important roles of different G4 DNA structures and specific G4 helicases at selected genomic locations and telomeres in regulating gene expression and maintaining genome stability.


Subject(s)
G-Quadruplexes , RNA Helicases/metabolism , RecQ Helicases/metabolism , Telomere Homeostasis , Animals , Genomic Instability , Humans
7.
Mol Cell ; 71(6): 877-878, 2018 09 20.
Article in English | MEDLINE | ID: mdl-30241603

ABSTRACT

Two recent studies implicate PARP as sensors of incompletely processed Okazaki fragments, changing our view about how single-strand breaks arise in unperturbed cells. Unligated Okazaki fragments may trigger homologous recombination-mediated repair and underpin genome instability in BRCA1/BRCA2-deficient cancers.


Subject(s)
Breast Neoplasms , Poly(ADP-ribose) Polymerases , BRCA1 Protein/genetics , BRCA2 Protein/genetics , DNA , DNA Repair , DNA Replication , Humans , Poly(ADP-ribose) Polymerase Inhibitors , Synthetic Lethal Mutations
8.
Cell ; 174(5): 1127-1142.e19, 2018 08 23.
Article in English | MEDLINE | ID: mdl-30078706

ABSTRACT

Replication origins, fragile sites, and rDNA have been implicated as sources of chromosomal instability. However, the defining genomic features of replication origins and fragile sites are among the least understood elements of eukaryote genomes. Here, we map sites of replication initiation and breakage in primary cells at high resolution. We find that replication initiates between transcribed genes within nucleosome-depleted structures established by long asymmetrical poly(dA:dT) tracts flanking the initiation site. Paradoxically, long (>20 bp) (dA:dT) tracts are also preferential sites of polar replication fork stalling and collapse within early-replicating fragile sites (ERFSs) and late-replicating common fragile sites (CFSs) and at the rDNA replication fork barrier. Poly(dA:dT) sequences are fragile because long single-strand poly(dA) stretches at the replication fork are unprotected by the replication protein A (RPA). We propose that the evolutionary expansion of poly(dA:dT) tracts in eukaryotic genomes promotes replication initiation, but at the cost of chromosome fragility.


Subject(s)
DNA Replication , DNA, Ribosomal/chemistry , Nucleosomes/metabolism , Poly dA-dT/chemistry , Replication Origin , Amino Acid Motifs , Animals , Cell Line , Chromatin Immunoprecipitation , Chromosomal Instability , Chromosome Fragile Sites , Chromosome Fragility , Female , Male , Mice , Mice, Inbred C57BL , Saccharomyces cerevisiae , Schizosaccharomyces , Transcription Initiation Site , Transcription, Genetic
9.
Nat Commun ; 9(1): 271, 2018 01 18.
Article in English | MEDLINE | ID: mdl-29348659

ABSTRACT

Bloom syndrome is a cancer predisposition disorder caused by mutations in the BLM helicase gene. Cells from persons with Bloom syndrome exhibit striking genomic instability characterized by excessive sister chromatid exchange events (SCEs). We applied single-cell DNA template strand sequencing (Strand-seq) to map the genomic locations of SCEs. Our results show that in the absence of BLM, SCEs in human and murine cells do not occur randomly throughout the genome but are strikingly enriched at coding regions, specifically at sites of guanine quadruplex (G4) motifs in transcribed genes. We propose that BLM protects against genome instability by suppressing recombination at sites of G4 structures, particularly in transcribed regions of the genome.


Subject(s)
Bloom Syndrome/genetics , G-Quadruplexes , Neoplasms/etiology , RecQ Helicases/metabolism , Sister Chromatid Exchange , Animals , Bloom Syndrome/complications , Cell Line , Genomic Instability , Humans , Loss of Heterozygosity , Mice
10.
Genome Res ; 26(11): 1565-1574, 2016 11.
Article in English | MEDLINE | ID: mdl-27646535

ABSTRACT

Haplotypes are fundamental to fully characterize the diploid genome of an individual, yet methods to directly chart the unique genetic makeup of each parental chromosome are lacking. Here we introduce single-cell DNA template strand sequencing (Strand-seq) as a novel approach to phasing diploid genomes along the entire length of all chromosomes. We demonstrate this by building a complete haplotype for a HapMap individual (NA12878) at high accuracy (concordance 99.3%), without using generational information or statistical inference. By use of this approach, we mapped all meiotic recombination events in a family trio with high resolution (median range ∼14 kb) and phased larger structural variants like deletions, indels, and balanced rearrangements like inversions. Lastly, the single-cell resolution of Strand-seq allowed us to observe loss of heterozygosity regions in a small number of cells, a significant advantage for studies of heterogeneous cell populations, such as cancer cells. We conclude that Strand-seq is a unique and powerful approach to completely phase individual genomes and map inheritance patterns in families, while preserving haplotype differences between single cells.


Subject(s)
Chromosome Mapping/methods , Chromosomes, Human/genetics , Haplotypes , Single-Cell Analysis/methods , Cell Line , HapMap Project , Homologous Recombination , Humans , Lymphocytes/cytology , Lymphocytes/metabolism , Mutation
11.
Nucleic Acids Res ; 44(14): 6787-93, 2016 08 19.
Article in English | MEDLINE | ID: mdl-27185886

ABSTRACT

Sister chromatid exchanges (SCEs) are considered sensitive indicators of genome instability. Detection of SCEs typically requires cells to incorporate bromodeoxyuridine (BrdU) during two rounds of DNA synthesis. Previous studies have suggested that SCEs are induced by DNA replication over BrdU-substituted DNA and that BrdU incorporation alone could be responsible for the high number of SCE events observed in cells from patients with Bloom syndrome (BS), a rare genetic disorder characterized by marked genome instability and high SCE frequency. Here we show using Strand-seq, a single cell DNA template strand sequencing technique, that the presence of variable BrdU concentrations in the cell culture medium and in DNA template strands has no effect on SCE frequency in either normal or BS cells. We conclude that BrdU does not induce SCEs and that SCEs detected in either normal or BS cells reflect DNA repair events that occur spontaneously.


Subject(s)
Bloom Syndrome/metabolism , Bloom Syndrome/pathology , Bromodeoxyuridine/pharmacology , Sister Chromatid Exchange/drug effects , Cell Division/drug effects , DNA/metabolism , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibroblasts/pathology , Humans , Lymphocytes/drug effects , Lymphocytes/metabolism , Lymphocytes/pathology , Templates, Genetic
12.
Cell Stem Cell ; 17(5): 543-56, 2015 Nov 05.
Article in English | MEDLINE | ID: mdl-26412562

ABSTRACT

Induced pluripotent stem cells (iPSCs) undergo extensive nuclear reprogramming and are generally indistinguishable from embryonic stem cells (ESCs) in their functional capacity and transcriptome and DNA methylation profiles. However, direct conversion of cells from one lineage to another often yields incompletely reprogrammed, functionally compromised cells, raising the question of whether pluripotency is required to achieve a high degree of nuclear reprogramming. Here, we show that transient expression of Gata3, Eomes, and Tfap2c in mouse fibroblasts induces stable, transgene-independent trophoblast stem-like cells (iTSCs). iTSCs possess transcriptional profiles highly similar to blastocyst-derived TSCs, with comparable methylation and H3K27ac patterns and genome-wide H2A.X deposition. iTSCs generate trophoectodermal lineages upon differentiation, form hemorrhagic lesions, and contribute to developing placentas in chimera assays, indicating a high degree of nuclear reprogramming, with no evidence of passage through a transient pluripotent state. Together, these data demonstrate that extensive nuclear reprogramming can be achieved independently of pluripotency.


Subject(s)
Cell Lineage , Cell Nucleus/metabolism , Cellular Reprogramming , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Trophoblasts/cytology , Animals , Cells, Cultured , Mice , Mice, Transgenic , Trophoblasts/metabolism
13.
Cell Stem Cell ; 15(3): 295-309, 2014 Sep 04.
Article in English | MEDLINE | ID: mdl-25192464

ABSTRACT

Induced pluripotent stem cells (iPSCs) are commonly generated by transduction of Oct4, Sox2, Klf4, and Myc (OSKM) into cells. Although iPSCs are pluripotent, they frequently exhibit high variation in terms of quality, as measured in mice by chimera contribution and tetraploid complementation. Reliably high-quality iPSCs will be needed for future therapeutic applications. Here, we show that one major determinant of iPSC quality is the combination of reprogramming factors used. Based on tetraploid complementation, we found that ectopic expression of Sall4, Nanog, Esrrb, and Lin28 (SNEL) in mouse embryonic fibroblasts (MEFs) generated high-quality iPSCs more efficiently than other combinations of factors including OSKM. Although differentially methylated regions, transcript number of master regulators, establishment of specific superenhancers, and global aneuploidy were comparable between high- and low-quality lines, aberrant gene expression, trisomy of chromosome 8, and abnormal H2A.X deposition were distinguishing features that could potentially also be applicable to human.


Subject(s)
Cellular Reprogramming , Induced Pluripotent Stem Cells/metabolism , Transcription Factors/metabolism , Animals , Cell Line , Chimera , Chromosomes, Human, Pair 8/genetics , DNA Methylation/genetics , Embryonic Stem Cells/metabolism , Enhancer Elements, Genetic/genetics , Gene Expression Profiling , Genome/genetics , Histones/metabolism , Humans , Kruppel-Like Factor 4 , Mice, Inbred C57BL , Mice, Inbred DBA , RNA, Messenger/genetics , RNA, Messenger/metabolism , Trisomy/genetics
14.
J Biol Chem ; 287(52): 43351-8, 2012 Dec 21.
Article in English | MEDLINE | ID: mdl-23150669

ABSTRACT

Iron-sulfur (Fe-S) clusters are essential cofactors of proteins with a wide range of biological functions. A dedicated cytosolic Fe-S cluster assembly (CIA) system is required to assemble Fe-S clusters into cytosolic and nuclear proteins. Here, we show that the mammalian nucleotide excision repair protein homolog MMS19 can simultaneously bind probable cytosolic iron-sulfur protein assembly protein CIAO1 and Fe-S proteins, confirming that MMS19 is a central protein of the CIA machinery that brings Fe-S cluster donor proteins and the receiving apoproteins into proximity. In addition, we show that mitotic spindle-associated MMXD complex subunit MIP18 also interacts with both CIAO1 and Fe-S proteins. Specifically, it binds the Fe-S cluster coordinating regions in Fe-S proteins. Furthermore, we show that ADP/ATP translocase 2 (ANT2) interacts with Fe-S apoproteins and MMS19 in the CIA complex but not with the individual proteins. Together, these results elucidate the composition and interactions within the late CIA complex.


Subject(s)
Adenine Nucleotide Translocator 2/metabolism , Carrier Proteins/metabolism , Cytoplasm/metabolism , Multiprotein Complexes/metabolism , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Adenine Nucleotide Translocator 2/genetics , Animals , Carrier Proteins/genetics , Cytoplasm/genetics , HEK293 Cells , Humans , Metallochaperones/genetics , Metallochaperones/metabolism , Metalloproteins , Mice , Multiprotein Complexes/genetics , Nuclear Proteins/genetics , Protein Binding/physiology , Spindle Apparatus/genetics , Spindle Apparatus/metabolism , Transcription Factors/genetics
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