Helicases in R-loop Formation and Resolution

Yang, Shizhuo; Winstone, Lacey; Mondal, Sohaumn; Wu, Yuliang

doi:10.1016/j.jbc.2023.105307

Cited by 12 publications

(3 citation statements)

References 154 publications

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“…Moreover, different previously identified non-histone substrates of NSD2 have important roles in DNA damage repair such as PTEN 27 and Aurora kinase A (AURKA) 26 , and other studies showed that NSD2 enhances DNA damage repair leading to an increase in resistance to chemotherapeutic agents 53 . This connection between NSD2 and DNA damage repair is further enhanced by our finding that ATRX and FANCM are direct targets of NSD2, because these two proteins are helicases with important roles in DNA repair and R-loop metabolism 54 .…”

Section: Discussionmentioning

confidence: 85%

Discovery of NSD2 non-histone substrates and design of a super-substrate

Weirich,

Kusevic,

Schnee

et al. 2024

Commun Biol

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The human protein lysine methyltransferase NSD2 catalyzes dimethylation at H3K36. It has very important roles in development and disease but many mechanistic features and its full spectrum of substrate proteins are unclear. Using peptide SPOT array methylation assays, we investigate the substrate sequence specificity of NSD2 and discover strong readout of residues between G33 (-3) and P38 (+2) on H3K36. Unexpectedly, we observe that amino acid residues different from natural ones in H3K36 are preferred at some positions. Combining four preferred residues led to the development of a super-substrate which is methylated much faster by NSD2 at peptide and protein level. Molecular dynamics simulations demonstrate that this activity increase is caused by distinct hyperactive conformations of the enzyme-peptide complex. To investigate the substrate spectrum of NSD2, we conducted a proteome wide search for nuclear proteins matching the specificity profile and discovered 22 peptide substrates of NSD2. In protein methylation studies, we identify K1033 of ATRX and K819 of FANCM as NSD2 methylation sites and also demonstrate their methylation in human cells. Both these proteins have important roles in DNA repair strengthening the connection of NSD2 and H3K36 methylation to DNA repair.

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Section: Discussionmentioning

confidence: 85%

Discovery of NSD2 non-histone substrates and design of a super-substrate

Weirich,

Kusevic,

Schnee

et al. 2024

Commun Biol

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“…R-loops can be resolved by RNA unwinding by the helicases senataxin, aquarius (AQR), Werner syndrome protein (WRN), Bloom syndrome protein (BLM), regulator of telomere elongation helicase 1 (RTEL1), petite integration factor (PIF1), Fanconi anemia complementation group M (FANCM), alphathalassemia/mental retardation, X-linked (ATRX), and CRISPR-associated DinG protein (CasDinG) [247][248][249][250]. Several DEAD/H-box proteins including DDX1, DDX17, and DHX9 (RNA helicase A) are involved in both R-loop formation and resolution [251][252][253]. Proteins involved in HR and interstrand crosslink repair (FANCA, FANCD2) also function in R-loop resolution [254][255][256][257], and BRCA1 recruits senataxin to R-loops [219,[258][259][260][261].…”

Section: Other Nucleases Involved In the Replication Stress Responsementioning

confidence: 99%

Cellular Responses to Widespread DNA Replication Stress

Nickoloff,

Jaiswal,

Sharma

et al. 2023

IJMS

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Replicative DNA polymerases are blocked by nearly all types of DNA damage. The resulting DNA replication stress threatens genome stability. DNA replication stress is also caused by depletion of nucleotide pools, DNA polymerase inhibitors, and DNA sequences or structures that are difficult to replicate. Replication stress triggers complex cellular responses that include cell cycle arrest, replication fork collapse to one-ended DNA double-strand breaks, induction of DNA repair, and programmed cell death after excessive damage. Replication stress caused by specific structures (e.g., G-rich sequences that form G-quadruplexes) is localized but occurs during the S phase of every cell division. This review focuses on cellular responses to widespread stress such as that caused by random DNA damage, DNA polymerase inhibition/nucleotide pool depletion, and R-loops. Another form of global replication stress is seen in cancer cells and is termed oncogenic stress, reflecting dysregulated replication origin firing and/or replication fork progression. Replication stress responses are often dysregulated in cancer cells, and this too contributes to ongoing genome instability that can drive cancer progression. Nucleases play critical roles in replication stress responses, including MUS81, EEPD1, Metnase, CtIP, MRE11, EXO1, DNA2-BLM, SLX1-SLX4, XPF-ERCC1-SLX4, Artemis, XPG, FEN1, and TATDN2. Several of these nucleases cleave branched DNA structures at stressed replication forks to promote repair and restart of these forks. We recently defined roles for EEPD1 in restarting stressed replication forks after oxidative DNA damage, and for TATDN2 in mitigating replication stress caused by R-loop accumulation in BRCA1-defective cells. We also discuss how insights into biological responses to genome-wide replication stress can inform novel cancer treatment strategies that exploit synthetic lethal relationships among replication stress response factors.

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“…Specifically, loss of FANCM increased the efficiency of AAV-HR in human cells 16 . FANCM is also known to regulate the resolution of RNA-DNA hybrid-containing structures called R-loops 17 . R-loops form on the genome during transcription when the newly synthesized RNA molecule anneals to its template DNA and in turn displaces the other DNA strand.…”

Section: Introductionmentioning

confidence: 99%

AAV-mediated genome editing is influenced by the formation of R-loops

Puzzo,

Crossley,

Goswami

et al. 2024

Preprint

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Recombinant adeno-associated viral vectors (rAAV) hold an intrinsic ability to stimulate homologous recombination (AAV-HR) and are the most used in clinical settings for in vivo gene therapy. However, rAAVs also integrate throughout the genome. Here, we describe DNA-RNA immunoprecipitation sequencing (DRIP-seq) in murine HEPA1-6 hepatoma cells and whole murine liver to establish the similarities and differences in genomic R-loop formation in a transformed cell line and intact tissue. We show enhanced AAV-HR in mice upon genetic and pharmacological upregulation of R-loops. Selecting the highly expressed Albumin gene as a model locus for genome editing in both in vitro and in vivo experiments showed that the R-loop prone, 3 prime end of Albumin was efficiently edited by AAV-HR, whereas the upstream R-loop-deficient region did not result in detectable vector integration. In addition, we found a positive correlation between previously reported off-target rAAV integration sites and R-loop enriched genomic regions. Thus, we conclude that high levels of R-loops, present in highly transcribed genes, promote rAAV vector genome integration. These findings may shed light on potential mechanisms for improving the safety and efficacy of genome editing by modulating R-loops and may enhance our ability to predict regions most susceptible to off-target insertional mutagenesis by rAAV vectors.

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Helicases in R-loop Formation and Resolution

Cited by 12 publications

References 154 publications

Discovery of NSD2 non-histone substrates and design of a super-substrate

Discovery of NSD2 non-histone substrates and design of a super-substrate

Cellular Responses to Widespread DNA Replication Stress

AAV-mediated genome editing is influenced by the formation of R-loops

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