scholarly journals Two mechanisms of chromosome fragility at replication-termination sites in bacteria

2021 ◽  
Vol 7 (25) ◽  
pp. eabe2846
Author(s):  
Qian Mei ◽  
Devon M. Fitzgerald ◽  
Jingjing Liu ◽  
Jun Xia ◽  
John P. Pribis ◽  
...  
Keyword(s):  
Replication Fork ◽  
Genome Instability ◽  
Neurological Diseases ◽  
Strand Break ◽  
Fragile Sites ◽  
Holliday Junctions ◽  
Dna Breaks ◽  
Dna Damage And Repair ◽  
Chromosome Fragility ◽  
Chromosomal Fragile Sites

Chromosomal fragile sites are implicated in promoting genome instability, which drives cancers and neurological diseases. Yet, the causes and mechanisms of chromosome fragility remain speculative. Here, we identify three spontaneous fragile sites in the Escherichia coli genome and define their DNA damage and repair intermediates at high resolution. We find that all three sites, all in the region of replication termination, display recurrent four-way DNA or Holliday junctions (HJs) and recurrent DNA breaks. Homology-directed double-strand break repair generates the recurrent HJs at all of these sites; however, distinct mechanisms of DNA breakage are implicated: replication fork collapse at natural replication barriers and, unexpectedly, frequent shearing of unsegregated sister chromosomes at cell division. We propose that mechanisms such as both of these may occur ubiquitously, including in humans, and may constitute some of the earliest events that underlie somatic cell mosaicism, cancers, and other diseases of genome instability.

scholarly journals Holliday junction trap shows how cells use recombination and a junction-guardian role of RecQ helicase

2016 ◽  
Vol 2 (11) ◽  
pp. e1601605 ◽  
Author(s):  
Jun Xia ◽  
Li-Tzu Chen ◽  
Qian Mei ◽  
Chien-Hui Ma ◽  
Jennifer A. Halliday ◽  
...  
Keyword(s):  
Dna Damage ◽  
Genome Instability ◽  
Human Cancer ◽  
Single Cells ◽  
Strand Break ◽  
Holliday Junctions ◽  
Dna Breaks ◽  
E Coli ◽  
Dna Replication And Repair ◽  
The Many

DNA repair by homologous recombination (HR) underpins cell survival and fuels genome instability, cancer, and evolution. However, the main kinds and sources of DNA damage repaired by HR in somatic cells and the roles of important HR proteins remain elusive. We present engineered proteins that trap, map, and quantify Holliday junctions (HJs), a central DNA intermediate in HR, based on catalytically deficient mutant RuvC protein ofEscherichia coli. We use RuvCDefGFP (RDG) to map genomic footprints of HR at defined DNA breaks inE. coliand demonstrate genome-scale directionality of double-strand break (DSB) repair along the chromosome. Unexpectedly, most spontaneous HR-HJ foci are instigated, not by DSBs, but rather by single-stranded DNA damage generated by replication. We show that RecQ, theE. coliortholog of five human cancer proteins, nonredundantly promotes HR-HJ formation in single cells and, in a novel junction-guardian role, also prevents apparent non-HR–HJs promoted by RecA overproduction. We propose that one or more human RecQ orthologs may act similarly in human cancers overexpressing the RecA orthologRAD51and find that cancer genome expression data implicate the orthologs BLM and RECQL4 in conjunction with EME1 and GEN1 as probable HJ reducers in such cancers. Our results support RecA-overproducingE. colias a model of the many human tumors with up-regulatedRAD51and provide the first glimpses of important, previously elusive reaction intermediates in DNA replication and repair in single living cells.

scholarly journals Biology before the SOS Response—DNA Damage Mechanisms at Chromosome Fragile Sites

Cells ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2275
Author(s):  
Devon M. Fitzgerald ◽  
Susan M Rosenberg
Keyword(s):  
Dna Damage ◽  
Genome Instability ◽  
Human Cancer ◽  
Sos Response ◽  
Fragile Sites ◽  
Holliday Junctions ◽  
Cancer Evolution ◽  
Dna Damage Tolerance ◽  
Chromosome Fragility ◽  
Species Evolution

The Escherichia coli SOS response to DNA damage, discovered and conceptualized by Evelyn Witkin and Miroslav Radman, is the prototypic DNA-damage stress response that upregulates proteins of DNA protection and repair, a radical idea when formulated in the late 1960s and early 1970s. SOS-like responses are now described across the tree of life, and similar mechanisms of DNA-damage tolerance and repair underlie the genome instability that drives human cancer and aging. The DNA damage that precedes damage responses constitutes upstream threats to genome integrity and arises mostly from endogenous biology. Radman’s vision and work on SOS, mismatch repair, and their regulation of genome and species evolution, were extrapolated directly from bacteria to humans, at a conceptual level, by Radman, then many others. We follow his lead in exploring bacterial molecular genomic mechanisms to illuminate universal biology, including in human disease, and focus here on some events upstream of SOS: the origins of DNA damage, specifically at chromosome fragile sites, and the engineered proteins that allow us to identify mechanisms. Two fragility mechanisms dominate: one at replication barriers and another associated with the decatenation of sister chromosomes following replication. DNA structures in E. coli, additionally, suggest new interpretations of pathways in cancer evolution, and that Holliday junctions may be universal molecular markers of chromosome fragility.

scholarly journals Genome-wide analysis of DNA replication and DNA double-strand breaks using TrAEL-seq

PLoS Biology ◽  
2021 ◽  
Vol 19 (3) ◽  
pp. e3000886
Author(s):  
Neesha Kara ◽  
Felix Krueger ◽  
Peter Rugg-Gunn ◽  
Jonathan Houseley
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Replication Fork ◽  
Strand Break ◽  
Double Strand Break ◽  
Replication Forks ◽  
Dna Breaks ◽  
Genome Wide ◽  
Fork Stalling ◽  
End Resection

Faithful replication of the entire genome requires replication forks to copy large contiguous tracts of DNA, and sites of persistent replication fork stalling present a major threat to genome stability. Understanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods that profile replication fork position and the formation of recombinogenic DNA ends. Here, we describe Transferase-Activated End Ligation sequencing (TrAEL-seq), a method that captures single-stranded DNA 3′ ends genome-wide and with base pair resolution. TrAEL-seq labels both DNA breaks and replication forks, providing genome-wide maps of replication fork progression and fork stalling sites in yeast and mammalian cells. Replication maps are similar to those obtained by Okazaki fragment sequencing; however, TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq far simpler and more widely applicable than existing replication fork direction profiling methods. The specificity of TrAEL-seq for DNA 3′ ends also allows accurate detection of double-strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double-strand break hotspots in a dmc1Δ mutant that is competent for end resection but not strand invasion. Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability.

scholarly journals Genome-wide analysis of DNA replication and DNA double strand breaks by TrAEL-seq

2020 ◽  
Author(s):  
Neesha Kara ◽  
Felix Krueger ◽  
Peter Rugg-Gunn ◽  
Jonathan Houseley
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Replication Fork ◽  
High Sensitivity ◽  
Strand Break ◽  
Double Strand Break ◽  
Replication Forks ◽  
Dna Breaks ◽  
Biochemical Purification ◽  
Genome Wide

AbstractUnderstanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods sensitive to both changes in replication fork structure and the formation of recombinogenic DNA ends. Here we describe Transferase-Activated End Ligation sequencing (TrAEL-seq), a method that captures single stranded DNA 3’ ends genome-wide and with base pair resolution. TrAEL-seq labels DNA breaks, and profiles both stalled and processive replication forks in yeast and mammalian cells. Replication forks stalling at defined barriers and expressed genes are detected by TrAEL-seq with exceptional signal-to-noise, most likely through labelling of DNA 3’ ends exposed during fork reversal. TrAEL-seq also labels unperturbed processive replication forks to yield maps of replication fork direction similar to those obtained by Okazaki fragment sequencing, however TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq simpler and more widely applicable than existing replication fork direction profiling methods. The specificity of TrAEL-seq for DNA 3’ ends also allows accurate detection of double strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double strand break hotspots in a dmc1Δ mutant. Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability.

scholarly journals Impact of G-Quadruplexes on the Regulation of Genome Integrity, DNA Damage and Repair

Biomolecules ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 1284
Author(s):  
Anzhela V. Pavlova ◽  
Elena A. Kubareva ◽  
Mayya V. Monakhova ◽  
Maria I. Zvereva ◽  
Nina G. Dolinnaya
Keyword(s):  
Dna Damage ◽  
Genome Instability ◽  
Critical Role ◽  
Dna Lesions ◽  
Genome Integrity ◽  
Dna Breaks ◽  
Dna Damage And Repair ◽  
Repair Processes

DNA G-quadruplexes (G4s) are known to be an integral part of the complex regulatory systems in both normal and pathological cells. At the same time, the ability of G4s to impede DNA replication plays a critical role in genome integrity. This review summarizes the results of recent studies of G4-mediated genomic and epigenomic instability, together with associated DNA damage and repair processes. Although the underlying mechanisms remain to be elucidated, it is known that, among the proteins that recognize G4 structures, many are linked to DNA repair. We analyzed the possible role of G4s in promoting double-strand DNA breaks, one of the most deleterious DNA lesions, and their repair via error-prone mechanisms. The patterns of G4 damage, with a focus on the introduction of oxidative guanine lesions, as well as their removal from G4 structures by canonical repair pathways, were also discussed together with the effects of G4s on the repair machinery. According to recent findings, there must be a delicate balance between G4-induced genome instability and G4-promoted repair processes. A broad overview of the factors that modulate the stability of G4 structures in vitro and in vivo is also provided here.

scholarly journals Integrated Genome and Transcriptome Analyses Reveal the Mechanism of Genome Instability in Ataxia with Oculomotor Apraxia 2

2021 ◽  
Author(s):  
Radhakrishnan Kanagaraj ◽  
Richard Mitter ◽  
Theodoros Kantidakis ◽  
Matthew Edwards ◽  
Anaid Benitez ◽  
...  
Keyword(s):  
Genome Instability ◽  
Expression Profiles ◽  
Chromosome Instability ◽  
Gene Expression Profiles ◽  
Fragile Sites ◽  
Oculomotor Apraxia ◽  
A Genome ◽  
Chromosome Fragility ◽  
Ataxia With Oculomotor Apraxia ◽  
Gains And Losses

Mutations in the SETX gene, which encodes Senataxin, are associated with the progressive neurodegenerative diseases Ataxia with Oculomotor Apraxia 2 (AOA2) and Amyotrophic Lateral Sclerosis 4 (ALS4). To identify the causal defect in AOA2, patient-derived cells and SETX knockouts (human and mouse) were analyzed using integrated genomic and transcriptomic approaches. We observed a genome-wide increase in chromosome instability (gains and losses) within genes and at chromosome fragile sites, resulting in changes to gene expression profiles. Senataxin loss caused increased transcription stress near promoters that correlated with high GCskew and R-loop accumulation at promoter-proximal regions. Notably, the chromosomal regions with gains and losses overlapped with regions of elevated transcription stress. In the absence of Senataxin, we found that Cockayne Syndrome protein CSB was required for the recruitment of the transcription-coupled repair endonucleases (XPG and XPF) and recombination protein RAD52 to target and resolve transcription bubbles containing R-loops, leading to error prone repair and genomic instability. These results show that transcription stress is an important contributor to SETX mutation-associated chromosome fragility and AOA2.

scholarly journals Chromosomal Instability at Fragile Sites in Blue Foxes, Silver Foxes, and Their Interspecific Hybrids

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1743
Author(s):  
Marta Kuchta-Gładysz ◽  
Ewa Wójcik ◽  
Anna Grzesiakowska ◽  
Katarzyna Rymuza ◽  
Olga Szeleszczuk
Keyword(s):  
Interspecific Hybrids ◽  
Genome Stability ◽  
Diploid Number ◽  
Fragile Sites ◽  
B Chromosomes ◽  
Silver Fox ◽  
Chromosomal Breaks ◽  
Blue Fox ◽  
Chromosomal Fragile Sites ◽  
Karyotypic Variation

A cytogenetic assay based on fragile sites (FS) enables the identification of breaks, chromatid gaps, and deletions. In healthy individuals, the number of these instabilities remains low. Genome stability in these species is affected by Robertsonian translocations in the karyotype of the blue fox and by B chromosomes in the silver fox. The aims of the study were to characterise the karyotype of blue foxes, silver foxes, and their hybrids and to identify chromosomal fragile sites used to evaluate genome stability. The diploid number of A chromosomes in blue foxes ranged from 48 to 50, while the number of B chromosomes in silver foxes varied from one to four, with a constant number of A chromosomes (2n = 34). In interspecific hybrids, both types of karyotypic variation were identified, with the diploid number of A chromosomes ranging from 40 to 44 and the number of B chromosomes varying from 0 to 3. The mean frequency of FS in foxes was 4.06 ± 0.19: 4.61 ± 0.37 in blue foxes, 3.46 ± 0.28 in silver foxes, and 4.12 ± 0.22 in hybrids. A relationship was identified between an increased number of A chromosomes in the karyotype of the hybrids and the frequency of chromosomal breaks. The FS assay was used as a biomarker for the evaluation of genomic stability in the animals in the study.

Sign in / Sign up

Export Citation Format

Share Document