R-loop-mediated genomic instability is caused by impairment of replication fork progression

doi:10.1101/GAD.17010011

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R-loop-mediated genomic instability is caused by impairment of replication fork progression

Journal Article•DOI•

R-loop-mediated genomic instability is caused by impairment of replication fork progression

Wenjian Gan¹, Zhishuang Guan, Jie Liu, Ting Gui, Keng Shen, James L. Manley, Xialu Li - Show less +3 more•Institutions (1)

Peking University¹

01 Oct 2011-Genes & Development (Cold Spring Harbor Lab)-Vol. 25, Iss: 19, pp 2041-2056

TL;DR: It is shown that R-loop formation induces chromosomal DNA rearrangements and recombination in Escherichia coli, just as it does in eukaryotes, and that this underlies the effects of R loops on genomic stability.

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Abstract: Transcriptional R loops are anomalous RNA:DNA hybrids that have been detected in organisms from bacteria to humans. These structures have been shown in eukaryotes to result in DNA damage and rearrangements; however, the mechanisms underlying these effects have remained largely unknown. To investigate this, we first show that R-loop formation induces chromosomal DNA rearrangements and recombination in Escherichia coli, just as it does in eukaryotes. More importantly, we then show that R-loop formation causes DNA replication fork stalling, and that this in fact underlies the effects of R loops on genomic stability. Strikingly, we found that attenuation of replication strongly suppresses R-loop-mediated DNA rearrangements in both E. coli and HeLa cells. Our findings thus provide a direct demonstration that R-loop formation impairs DNA replication and that this is responsible for the deleterious effects of R loops on genome stability from bacteria to humans.

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Journal Article•DOI•

R Loops: From Transcription Byproducts to Threats to Genome Stability

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Andrés Aguilera¹, Tatiana García-Muse¹•Institutions (1)

University of Seville¹

27 Apr 2012-Molecular Cell

TL;DR: The factors and cellular processes that control R loop formation and the mechanisms by which R loops may influence gene expression and the integrity of the genome are discussed.

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825 citations

Cites background from "R-loop-mediated genomic instability..."

...Finally, R loops accumulate naturally at the G-rich 50-UTR regions immediately downstream of the CpG-non-methylated promoters in humans (Ginno et al., 2012)....
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...6 hybridoma (El Hage et al., 2010; Pohjoismäki et al., 2010; Mischo et al., 2011; Wahba et al., 2011; Stirling et al., 2012; Ginno et al., 2012) or an inactivated RNase H (Ginno et al....
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...6 hybridoma (El Hage et al., 2010; Pohjoismäki et al., 2010; Mischo et al., 2011; Wahba et al., 2011; Stirling et al., 2012; Ginno et al., 2012) or an inactivated RNase H (Ginno et al., 2012)....
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..., 2012) or an inactivated RNase H (Ginno et al., 2012)....
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...It has been proposed that the displaced ssDNA in the R loop acts as a signal to recruit either the protective H3K4 trimethyl mark or the DNA demethylases complex (Ginno et al., 2012) (Figure 1F)....
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Journal Article•DOI•

R loops: new modulators of genome dynamics and function

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José M. Santos-Pereira¹, Andrés Aguilera¹•Institutions (1)

Spanish National Research Council¹

01 Oct 2015-Nature Reviews Genetics

TL;DR: The current knowledge of the mechanisms controlling R loops and their putative relationship with disease is reviewed and several DNA and RNA metabolism factors prevent R-loop formation in cells.

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Abstract: R loops are nucleic acid structures composed of an RNA-DNA hybrid and a displaced single-stranded DNA. Recently, evidence has emerged that R loops occur more often in the genome and have greater physiological relevance, including roles in transcription and chromatin structure, than was previously predicted. Importantly, however, R loops are also a major threat to genome stability. For this reason, several DNA and RNA metabolism factors prevent R-loop formation in cells. Dysfunction of these factors causes R-loop accumulation, which leads to replication stress, genome instability, chromatin alterations or gene silencing, phenomena that are frequently associated with cancer and a number of genetic diseases. We review the current knowledge of the mechanisms controlling R loops and their putative relationship with disease.

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595 citations

Journal Article•DOI•

A double-edged sword: R loops as threats to genome integrity and powerful regulators of gene expression

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Konstantina Skourti-Stathaki¹, Nick J. Proudfoot¹•Institutions (1)

University of Oxford¹

01 Jul 2014-Genes & Development

TL;DR: Recent findings about R loops, three-stranded nucleic acid structures that comprise nascent RNA hybridized with the DNA template, leaving the nontemplate DNA single-STRanded, are reviewed.

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Abstract: R loops are three-stranded nucleic acid structures that comprise nascent RNA hybridized with the DNA template, leaving the nontemplate DNA single-stranded. R loops form naturally during transcription even though their persistent formation can be a risky outcome with deleterious effects on genome integrity. On the other hand, over the last few years, an increasingly strong case has been built for R loops as potential regulators of gene expression. Therefore, understanding their function and regulation under these opposite situations is essential to fully characterize the mechanisms that control genome integrity and gene expression. Here we review recent findings about these interesting structures that highlight their opposite roles in cellular fitness.

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442 citations

Cites background from "R-loop-mediated genomic instability..."

...An additional possible scenario suggests that transcriptional R loops induce genomic instability by interfering with DNA replication (Aguilera 2002; Gan et al. 2011; Houlard et al. 2011; Aguilera and Garcia-Muse 2012)....
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Journal Article•DOI•

Transcription-Coupled Nucleotide Excision Repair Factors Promote R-Loop-Induced Genome Instability

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Julie Sollier¹, Caroline Townsend Stork¹, María L. García-Rubio², Renee D. Paulsen¹, Andrés Aguilera², Karlene A. Cimprich¹ - Show less +2 more•Institutions (2)

Stanford University¹, Spanish National Research Council²

18 Dec 2014-Molecular Cell

TL;DR: Surprisingly, DSB formation requires the transcription-coupled nucleotide excision repair (TC-NER) factor Cockayne syndrome group B (CSB), but not the global genome repair protein XPC, which reveals an unexpected and potentially deleterious role for TC-NER factors in driving R-loop-induced DNA damage and genome instability.

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434 citations

Cites background from "R-loop-mediated genomic instability..."

...Several studies in bacteria, yeast, and human cells suggest that R-loop-induced DNA damage is associated with defects in replication fork progression (Alzu et al., 2012; Gan et al., 2011; Wellinger et al., 2006; Yüce and West, 2013; Tuduri et al., 2009)....
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...Several studies in bacteria, yeast, and human cells suggest that R-loop-induced DNA damage is associated with defects in replication fork progression (Alzu et al., 2012; Gan et al., 2011; Wellinger et al., 2006; Yüce and West, 2013; Tuduri et al., 2009)....
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...…genome instability, and the activation of ATR suggested by the phosphorylation of RPA and CHK1 indicates there may be effects of these structures or their processed intermediates in S phase (Alzu et al., 2012; Gan et al., 2011; Tuduri et al., 2009; Wellinger et al., 2006; Yüce and West, 2013)....
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...Indeed, diverse studies suggest that DNA replication is required for Rloop-induced genome instability, and the activation of ATR suggested by the phosphorylation of RPA and CHK1 indicates there may be effects of these structures or their processed intermediates in S phase (Alzu et al., 2012; Gan et al., 2011; Tuduri et al., 2009; Wellinger et al., 2006; Yüce and West, 2013)....
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Journal Article•DOI•

R-Loops as Cellular Regulators and Genomic Threats.

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Madzia P Crossley¹, Michael J. Bocek¹, Karlene A. Cimprich¹•Institutions (1)

Stanford University¹

07 Feb 2019-Molecular Cell

TL;DR: This review highlights recent work suggesting that R-loops can be problematic to cells as blocks to efficient transcription and replication that trigger the DNA damage response and compares the available next-generation sequencing-based approaches to map R-loop genome wide.

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411 citations

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References

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Book•

Molecular Cloning: A Laboratory Manual

[...]

Joseph Sambrook, Edward F. Fritsch, Tom Maniatis

15 Jan 2001

TL;DR: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years as mentioned in this paper and has been so popular, or so influential, that no other manual has been more widely used and influential.

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Abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol

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215,169 citations

Journal Article•DOI•

One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products

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Kirill A. Datsenko¹, Barry L. Wanner•Institutions (1)

Purdue University¹

06 Jun 2000-Proceedings of the National Academy of Sciences of the United States of America

TL;DR: A simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s), which should be widely useful, especially in genome analysis of E. coli and other bacteria.

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Abstract: We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage lambda Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells.

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14,389 citations

"R-loop-mediated genomic instability..." refers methods in this paper

...Mutations were introduced by P1 transduction (Miller 2052 GENES & DEVELOPMENT 1992) or by using the l red recombinase-mediated gene targeting method (Datsenko and Wanner 2000) essentially as described....
[...]

Journal Article•DOI•

DNA Double-stranded Breaks Induce Histone H2AX Phosphorylation on Serine 139

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Emmy P. Rogakou¹, Duane R. Pilch¹, Ann Orr¹, Vessela S. Ivanova¹, William M. Bonner¹ - Show less +1 more•Institutions (1)

National Institutes of Health¹

06 Mar 1998-Journal of Biological Chemistry

TL;DR: In this paper, a histone H2AX species that has been phosphorylated specifically at serine 139 was found to be a major component of DNA double-stranded break.

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5,132 citations

"R-loop-mediated genomic instability..." refers background in this paper

...As our previous studies have established that depletion of splicing factor SRSF1 results in R-loop-mediated genomic instability, reflected by the appearance of DSBs, in both chicken DT40 cells and human HeLa cells (Li and Manley 2005), we first wished to determine whether SRSF1 depletion results in R-loop-mediated replication fork stalling in HeLa cells....
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...We next wished to determine whether replication impairment accounts for the formation of DSBs in HeLa cells upon SRSF1 depletion....
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...Remarkably, compared with control DMSOtreated cells, PHA767491 treatment significantly decreased the percentage of g-H2AX foci-positive cells (from 34.7% to 17.6%, P 0.0001) upon SRSF1 depletion, indicating that active replication is required for the appearance of DSBs in SRSF1-depleted cells (Fig....
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...It is thus likely that R-loop formation and interference with replication fork progression, and thus DNA DSBs and rearrangements, can occur at numerous transcribed regions....
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...6C). g-H2AX foci are one of the earliest DNA damage repair foci to occur at DSBs (Rogakou et al. 1998)....
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Journal Article•DOI•

Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter.

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Luz-Maria Guzman¹, Dominique Belin¹, M. J. Carson¹, Jon Beckwith¹•Institutions (1)

Harvard University¹

01 Jul 1995-Journal of Bacteriology

TL;DR: The tight regulation of the PBAD promoter is exploited to study the phenotypes of null mutations of essential genes and the use of pBAD vectors as an expression system is explored.

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Abstract: We have constructed a series of plasmid vectors (pBAD vectors) containing the PBAD promoter of the araBAD (arabinose) operon and the gene encoding the positive and negative regulator of this promoter, araC. Using the phoA gene and phoA fusions to monitor expression in these vectors, we show that the ratio of induction/repression can be 1,200-fold, compared with 50-fold for PTAC-based vectors. phoA expression can be modulated over a wide range of inducer (arabinose) concentrations and reduced to extremely low levels by the presence of glucose, which represses expression. Also, the kinetics of induction and repression are very rapid and significantly affected by the ara allele in the host strain. Thus, the use of this system which can be efficiently and rapidly turned on and off allows the study of important aspects of bacterial physiology in a very simple manner and without changes of temperature. We have exploited the tight regulation of the PBAD promoter to study the phenotypes of null mutations of essential genes and explored the use of pBAD vectors as an expression system.

...read moreread less

4,997 citations

Additional excerpts

...To construct the pPtac vector, a fragment containing the lacI gene and Ptac promoter replaced the araC gene and the PBAD promoter in the pBAD18 vector (Guzman et al. 1995)....
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Journal Article•DOI•

Class Switch Recombination and Hypermutation Require Activation-Induced Cytidine Deaminase (AID), a Potential RNA Editing Enzyme

[...]

Masamichi Muramatsu¹, Kazuo Kinoshita¹, Sidonia Fagarasan¹, Shuichi Yamada¹, Yoichi Shinkai¹, Tasuku Honjo¹ - Show less +2 more•Institutions (1)

Kyoto University¹

01 Sep 2000-Cell

TL;DR: Results suggest that AID may be involved in regulation or catalysis of the DNA modification step of both class switching and somatic hypermutation in CH12F3-2 B lymphoma.

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3,288 citations

"R-loop-mediated genomic instability..." refers background in this paper

...AID, which is specifically expressed in active B cells, is also essential for the initiation of CSR in IgH (Muramatsu et al. 2000; Okazaki et al. 2002; Longerich et al. 2006)....
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...Second, it has been well documented that transcription through the S region is necessary but not sufficient to confer genetic alteration to the IgH constant region (CH)....
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