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Isabel X Wang, Christopher Grunseich, Jennifer Fox, Joshua Burdick, Zhengwei Zhu, Niema Ravazian, Markus Hafner, Vivian G Cheung
RNA/DNA hybrids form when RNA hybridizes with its template DNA generating a three-stranded structure known as the R-loop. Knowledge of how they form and resolve, as well as their functional roles, is limited. Here, by pull-down assays followed by mass spectrometry, we identified 803 proteins that bind to RNA/DNA hybrids. Because these proteins were identified using in vitro assays, we confirmed that they bind to R-loops in vivo. They include proteins that are involved in a variety of functions, including most steps of RNA processing...
August 14, 2018: Genome Research
Tom Killelea, Michelle Hawkins, Jamieson L Howard, Peter McGlynn, Edward L Bolt
Cascade complexes underpin E. coli CRISPR-Cas immunity systems by stimulating 'adaptation' reactions that update immunity and by initiating 'interference' reactions that destroy invader DNA. Recognition of invader DNA in Cascade catalysed R-loops provokes DNA capture and its subsequent integration into CRISPR loci by Cas1 and Cas2. DNA capture processes are unclear but may involve RecG helicase, which stimulates adaptation during its role responding to genome instability. We show that Cascade is a potential source of genome instability because it blocks DNA replication and that RecG helicase alleviates this by dissociating Cascade...
August 10, 2018: RNA Biology
Isabel Strohkendl, Fatema A Saifuddin, James R Rybarski, Ilya J Finkelstein, Rick Russell
Class 2 CRISPR-Cas nucleases are programmable genome editing tools with promising applications in human health and disease. However, DNA cleavage at off-target sites that resemble the target sequence is a pervasive problem that remains poorly understood mechanistically. Here, we use quantitative kinetics to dissect the reaction steps of DNA targeting by Acidaminococcus sp Cas12a (also known as Cpf1). We show that Cas12a binds DNA tightly in two kinetically separable steps. Protospacer-adjacent motif (PAM) recognition is followed by rate-limiting R-loop propagation, leading to inevitable DNA cleavage of both strands...
July 24, 2018: Molecular Cell
Lorenzo Costantino, Douglas Koshland
DNA-RNA hybrids associated with R-loops promote DNA damage and genomic instability. The capacity of hybrids at different genomic sites to cause DNA damage was not known, and the mechanisms leading from hybrid to damage were poorly understood. Here, we adopt a new strategy to map and characterize the sites of hybrid-induced damage genome-wide in budding yeast. We show that hybrid removal is essential for life because persistent hybrids cause irreparable DNA damage and cell death. We identify that a subset of hybrids is prone to cause damage, and the chromosomal context of hybrids dramatically impacts their ability to induce damage...
July 27, 2018: Molecular Cell
Stefano G Manzo, Stella R Hartono, Lionel A Sanz, Jessica Marinello, Sara De Biasi, Andrea Cossarizza, Giovanni Capranico, Frederic Chedin
BACKGROUND: Co-transcriptional R-loops are abundant non-B DNA structures in mammalian genomes. DNA Topoisomerase I (Top1) is often thought to regulate R-loop formation owing to its ability to resolve both positive and negative supercoils. How Top1 regulates R-loop structures at a global level is unknown. RESULTS: Here, we perform high-resolution strand-specific R-loop mapping in human cells depleted for Top1 and find that Top1 depletion results in both R-loop gains and losses at thousands of transcribed loci, delineating two distinct gene classes...
July 30, 2018: Genome Biology
Hai Dang Nguyen, Wan Yee Leong, Weiling Li, Pavankumar N G Reddy, Jack D Sullivan, Matthew J Walter, Lee Zou, Timothy A Graubert
Heterozygous somatic mutations in spliceosome genes (U2AF1, SF3B1, ZRSR2, or SRSF2) occur in >50% of myelodysplastic syndrome (MDS) patients. These mutations occur early in disease development, suggesting that they contribute to MDS pathogenesis and may represent a unique genetic vulnerability for targeted therapy. Here we show that RNA splicing perturbation by expression of the U2AF1(S34F) mutant causes accumulation of R loops, a transcription intermediate containing RNA:DNA hybrids and displaced single-stranded DNA, and elicits an ATR response...
July 27, 2018: Cancer Research
Vladimir A Kuznetsov, Vladyslav Bondarenko, Thidathip Wongsurawat, Surya P Yenamandra, Piroon Jenjaroenpun
No abstract text is available yet for this article.
July 24, 2018: Nucleic Acids Research
Jani B Mohammad, Marcus Wallgren, Nasim Sabouri
Pfh1, the sole member of the Pif1 helicases in Schizosaccharomyces pombe, is multifunctional and essential for maintenance of both the nuclear and mitochondrial genomes. However, we lack mechanistic insights into the functions of Pfh1 and its different motifs. This paper is specifically concerned with the importance of the Pif1 signature motif (SM), a 23 amino acids motif unique to Pif1 helicases, because a single amino acid substitution in this motif is associated with increased risk of breast cancer in humans and inviability in S...
July 24, 2018: Nucleic Acids Research
Martina G L Perego, Michela Taiana, Nereo Bresolin, Giacomo P Comi, Stefania Corti
R loops are transient three-stranded nucleic acid structures that form physiologically during transcription when a nascent RNA transcript hybridizes with the DNA template strand, leaving a single strand of displaced nontemplate DNA. However, aberrant persistence of R-loops can cause DNA damage by inducing genomic instability. Indeed, evidence has emerged that R-loops might represent a key element in the pathogenesis of human diseases, including cancer, neurodegeneration, and motor neuron disorders. Mutations in genes directly involved in R-loop biology, such as SETX (senataxin), or unstable DNA expansion eliciting R-loop generation, such as C9ORF72 HRE, can cause DNA damage and ultimately result in motor neuron cell death...
July 25, 2018: Molecular Neurobiology
Yongmoon Jeon, You Hee Choi, Yunsu Jang, Jihyeon Yu, Jiyoung Goo, Gyejun Lee, You Kyeong Jeong, Seung Hwan Lee, In-San Kim, Jin-Soo Kim, Cherlhyun Jeong, Sanghwa Lee, Sangsu Bae
Cas12a (also called Cpf1) is a representative type V-A CRISPR effector RNA-guided DNA endonuclease, which provides an alternative to type II CRISPR-Cas9 for genome editing. Previous studies have revealed that Cas12a has unique features distinct from Cas9, but the detailed mechanisms of target searching and DNA cleavage by Cas12a are still unclear. Here, we directly observe this entire process by using single-molecule fluorescence assays to study Cas12a from Acidaminococcus sp. (AsCas12a). We determine that AsCas12a ribonucleoproteins search for their on-target site by a one-dimensional diffusion along elongated DNA molecules and induce cleavage in the two DNA strands in a well-defined order, beginning with the non-target strand...
July 17, 2018: Nature Communications
Annapoorna Kannan, Kanchan Bhatia, Dana Branzei, Laxman Gangwani
Chronic low levels of survival motor neuron (SMN) protein cause spinal muscular atrophy (SMA). SMN is ubiquitously expressed, but the mechanisms underlying predominant neuron degeneration in SMA are poorly understood. We report that chronic low levels of SMN cause Senataxin (SETX)-deficiency, which results in increased RNA-DNA hybrids (R-loops) and DNA double-strand breaks (DSBs), and deficiency of DNA-activated protein kinase-catalytic subunit (DNA-PKcs), which impairs DSB repair. Consequently, DNA damage accumulates in patient cells, SMA mice neurons and patient spinal cord tissues...
July 16, 2018: Nucleic Acids Research
Kenji Kojima, Misato Baba, Motoki Tsukiashi, Takuto Nishimura, Kiyoshi Yasukawa
Ribonuclease H (RNase H) [EC] is an enzyme that specifically degrades RNA from RNA/DNA hybrids. Since its discovery in 1969, the enzyme has been extensively studied for its catalytic mechanism and physiological role. RNase H has been classified into two major families, Type 1 and Type 2. Type 1 enzymes are designated RNase HI in prokaryotes and RNase H1 in eukaryotes, while Type 2 enzymes are designated RNase HII in prokaryotes and RNase H2 in eukaryotes. Type 2 enzymes are able to cleave the 5'-phosphodiester bond of one ribonucleotide embedded in a DNA double strand...
July 12, 2018: Briefings in Functional Genomics
Hemanth Tummala, Arran D Dokal, Amanda Walne, Alicia Ellison, Shirleny Cardoso, Saranha Amirthasigamanipillai, Michael Kirwan, Isobel Browne, Jasmin K Sidhu, Vinothini Rajeeve, Ana Rio-Machin, Ahad Al Seraihi, Andrew S Duncombe, Matthew Jenner, Owen P Smith, Helen Enright, Alice Norton, Tekin Aksu, Namık Yaşar Özbek, Nikolas Pontikos, Pedro Cutillas, Inderjeet Dokal, Tom Vulliamy
Biallelic variants in the ERCC excision repair 6 like 2 gene ( ERCC6L2 ) are known to cause bone marrow failure (BMF) due to defects in DNA repair and mitochondrial function. Here, we report on eight cases of BMF from five families harboring biallelic variants in ERCC6L2 , two of whom present with myelodysplasia. We confirm that ERCC6L2 patients' lymphoblastoid cell lines (LCLs) are hypersensitive to DNA-damaging agents that specifically activate the transcription coupled nucleotide excision repair (TCNER) pathway...
July 24, 2018: Proceedings of the National Academy of Sciences of the United States of America
Alex M Andrews, Heather J McCartney, Tim M Errington, Alan D D'Andrea, Ian G Macara
Interstrand DNA cross-links (ICLs) block both replication and transcription, and are commonly repaired by the Fanconi anemia (FA) pathway. However, FA-independent repair mechanisms of ICLs remain poorly understood. Here we report a previously uncharacterized protein, SAN1, as a 5' exonuclease that acts independently of the FA pathway in response to ICLs. Deletion of SAN1 in HeLa cells and mouse embryonic fibroblasts causes sensitivity to ICLs, which is prevented by re-expression of wild type but not nuclease-dead SAN1...
July 3, 2018: Nature Communications
Divyesh Patel, Manthan Patel, Bengt Westermark, Umashankar Singh
OBJECTIVES: Although CpG methylation is well studied, mechanisms of non-CpG methylation in mammals remains elusive. Studying proteins with non-CpG cytosine methylation-sensitive DNA-binding, such as human CGGBP1, can unveil cytosine methylation regulatory mechanisms. Here we have resequenced a published genome-wide bisulfite sequencing library and analyzed it at base level resolution. CpG, CHG and CHH (where H is any nucleotide other than G) methylation states in non-targeting or CGGBP1-targeting shmiR lentivirus-transduced cells have been analyzed to identify how CGGBP1 regulates CpG and non-CpG methylation...
July 2, 2018: BMC Research Notes
María García-Rubio, Paula Aguilera, Juan Lafuente-Barquero, José F Ruiz, Marie-Noelle Simon, Vincent Geli, Ana G Rondón, Andrés Aguilera
R loops are an important source of genome instability, largely due to their negative impact on replication progression. Yra1/ALY is an abundant RNA-binding factor conserved from yeast to humans and required for mRNA export, but its excess causes lethality and genome instability. Here, we show that, in addition to ssDNA and ssRNA, Yra1 binds RNA-DNA hybrids in vitro and, when artificially overexpressed, can be recruited to chromatin in an RNA-DNA hybrid-dependent manner, stabilizing R loops and converting them into replication obstacles in vivo...
July 1, 2018: Genes & Development
Aparna Gorthi, July Carolina Romero, Eva Loranc, Lin Cao, Liesl A Lawrence, Elicia Goodale, Amanda Balboni Iniguez, Xavier Bernard, V Pragathi Masamsetti, Sydney Roston, Elizabeth R Lawlor, Jeffrey A Toretsky, Kimberly Stegmaier, Stephen L Lessnick, Yidong Chen, Alexander J R Bishop
In this Letter, the sentence beginning "This work was funded…." in the Acknowledgements should have read "CPRIT (RP140105) to J.C.R." rather than "CPRIT (RP150445) to J.C.R." This error has been corrected online.
July 2018: Nature
Vladimir A Kuznetsov, Vladyslav Bondarenko, Thidathip Wongsurawat, Surya P Yenamandra, Piroon Jenjaroenpun, Author Name
R-loops are three-stranded RNA:DNA hybrid structures essential for many normal and pathobiological processes. Previously, we generated a quantitative R-loop forming sequence (RLFS) model, quantitative model of R-loop-forming sequences (QmRLFS) and predicted ∼660 000 RLFSs; most of them located in genes and gene-flanking regions, G-rich regions and disease-associated genomic loci in the human genome. Here, we conducted a comprehensive comparative analysis of these RLFSs using experimental data and demonstrated the high performance of QmRLFS predictions on the nucleotide and genome scales...
June 26, 2018: Nucleic Acids Research
Brian P Weiser, Gaddiel Rodriguez, Philip A Cole, James T Stivers
The N-terminal domain (NTD) of nuclear human uracil DNA glycosylase (hUNG2) assists in targeting hUNG2 to replication forks through specific interactions with replication protein A (RPA). Here, we explored hUNG2 activity in the presence and absence of RPA using substrates with ssDNA-dsDNA junctions that mimic structural features of the replication fork and transcriptional R-loops. We find that when RPA is tightly bound to the ssDNA overhang of junction DNA substrates, base excision by hUNG2 is strongly biased toward uracils located 21 bp or less from the ssDNA-dsDNA junction...
June 18, 2018: Nucleic Acids Research
Craig L Bennett, Albert R La Spada
Senataxin (SETX) is a DNA-RNA helicase whose C-terminal region shows homology to the helicase domain of the yeast protein Sen1p. Genetic discoveries have established the importance of SETX for neural function, as recessive mutations in the SETX gene cause Ataxia with Oculomotor Apraxia type 2 (AOA2) (OMIM: 606002), which is the third most common form of recessive ataxia, after Friedreich's ataxia and Ataxia-Telangiectasia. In addition, rare, dominant SETX mutations cause a juvenile-onset form of Amyotrophic Lateral Sclerosis (ALS), known as ALS4...
2018: Advances in Neurobiology
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