Catalytically inactive, purified RNase H1: A specific and sensitive probe for RNA–DNA hybrid imaging

Crossley, Magdalena P.; Brickner, Joshua R.; Song, Chenlin; Zar, Su Mon Thin; Maw, Su S.; Chédin, Frédéric; Tsai, Miaw Sheue; Cimprich, Karlene A.

doi:10.1083/jcb.202101092

Cited by 48 publications

(29 citation statements)

References 44 publications

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“…To detect R-loops, we used a recombinant, V5-epitope-tagged version of a catalytically inactive, mutant (D210N) RNASEH1 protein (rRNASEH1) 41 – 43 ( Fig. 2a ) in preference to the S9.6 antibody against RNA:DNA hybrids 44 which gave high background staining.…”

Section: Resultsmentioning

confidence: 99%

TET deficiency perturbs mature B cell homeostasis and promotes oncogenesis associated with accumulation of G-quadruplex and R-loop structures

et al. 2021

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Enzymes of the TET family are methylcytosine dioxygenases that undergo frequent mutational or functional inactivation in human cancers. Recurrent loss-of-function mutations in TET proteins are frequent in human Diffuse Large B-Cell Lymphoma (DLBCL). Here we investigate the role of TET proteins in B-cell homeostasis and development of B cell lymphomas with features of DLBCL. We show that deletion of Tet2 and Tet3 genes in mature B cells in mice perturbs B-cell homeostasis and results in spontaneous development of germinal center-derived B cell lymphomas with increased G-quadruplexes and R-loops. At a genome-wide level, G-quadruplexes and R-loops were associated with increased DNA double strand breaks at immunoglobulin switch regions. Deletion of the DNA methyltransferase DNMT1 in TET-deficient B cells prevented expansion of germinal center B cells, diminished the accumulation of G-quadruplexes and R-loops, and delayed B lymphoma development, consistent with the opposing functions of DNMT and TET enzymes in DNA methylation and demethylation. CRISPR-mediated depletion of nucleases and helicases that regulate G-quadruplexes and R-loops decreased the viability of TET-deficient B cells. Our studies suggest a molecular mechanism by which TET loss-of-function might predispose to development of B cell malignancies.

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Section: Resultsmentioning

confidence: 99%

TET deficiency perturbs mature B cell homeostasis and promotes oncogenesis associated with accumulation of G-quadruplex and R-loop structures

et al. 2021

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“…Moreover, the emergence of S9.6 alternatives that can detect R-loops more specifically will improve researchers’ ability to assess R-loop binding and modulatory functions in the future. For example, the recent use of a purified recombinant catalytically inactive human RNase H1 tagged with GFP shows strong specificity against R-loops ( Crossley et al 2021 ). Refinements to methods and curation of functionally validated RLBPs will help to improve this resource going forward.…”

Section: Discussionmentioning

confidence: 99%

Integrative analysis and prediction of human R-loop binding proteins

Kumar

Fournier

Stirling

2022

G3 Genes|Genomes|Genetics

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In the past decade there has been a growing appreciation for R-loop structures as important regulators of the epigenome, telomere maintenance, DNA repair and replication. Given these numerous functions, dozens, or potentially hundreds, of proteins could serve as direct or indirect regulators of R-loop writing, reading, and erasing. In order to understand common properties shared amongst potential R-loop binding proteins (RLBPs) we mined published proteomic studies and distilled 10 features that were enriched in RLBPs compared to the rest of the proteome. Applying an easy-ensemble machine learning approach, we used these RLBP-specific features along with their amino acid composition to create random forest classifiers that predict the likelihood of a protein to bind to R-loops. Known R-loop regulating pathways such as splicing, DNA damage repair and chromatin remodeling are highly enriched in our datasets, and we validate two new R-loop binding proteins LIG1 and FXR1 in human cells. Together these datasets provide a reference to pursue analyses of novel R-loop regulatory proteins.

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“…Some sequence bias was also reported 32 . Recently, S9.6-independent R-loop detection methods have been developed, which employ either a non-cleaving RNase H domain to recognize the hybrids (e.g., DRIVE-seq and R-ChIP) 33 – 35 or bisulfite treatment or activation-induced cytidine deaminase (AID) (e.g., SMRF-seq 36 ) to chemically or enzymatically modify and mark the ssDNA strands of R-loops 37 . However, these alternative methods have their own shortcomings such as inefficient R-loop detection and cannot wholly replace S9.6-based methods 27 , 30 , 35 , 38 .…”

Section: Introductionmentioning

confidence: 99%

Structural basis of R-loop recognition by the S9.6 monoclonal antibody

et al. 2022

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R-loops are ubiquitous, dynamic nucleic-acid structures that play fundamental roles in DNA replication and repair, chromatin and transcription regulation, as well as telomere maintenance. The DNA-RNA hybrid–specific S9.6 monoclonal antibody is widely used to map R-loops. Here, we report crystal structures of a S9.6 antigen-binding fragment (Fab) free and bound to a 13-bp hybrid duplex. We demonstrate that S9.6 exhibits robust selectivity in binding hybrids over double-stranded (ds) RNA and in categorically rejecting dsDNA. S9.6 asymmetrically recognizes a compact epitope of two consecutive RNA nucleotides via their 2′-hydroxyl groups and six consecutive DNA nucleotides via their backbone phosphate and deoxyribose groups. Recognition is mediated principally by aromatic and basic residues of the S9.6 heavy chain, which closely track the curvature of the hybrid minor groove. These findings reveal the molecular basis for S9.6 recognition of R-loops, detail its binding specificity, identify a new hybrid-recognition strategy, and provide a framework for S9.6 protein engineering.

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Catalytically inactive, purified RNase H1: A specific and sensitive probe for RNA–DNA hybrid imaging

Cited by 48 publications

References 44 publications

TET deficiency perturbs mature B cell homeostasis and promotes oncogenesis associated with accumulation of G-quadruplex and R-loop structures

TET deficiency perturbs mature B cell homeostasis and promotes oncogenesis associated with accumulation of G-quadruplex and R-loop structures

Integrative analysis and prediction of human R-loop binding proteins

Structural basis of R-loop recognition by the S9.6 monoclonal antibody

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