Both R-loop removal and ribonucleotide excision repair activities of RNase H2 contribute substantially to chromosome stability

Abstract Mono-ribonucleotides are building blocks for polynucleotide RNA chains (e.g., messenger RNA), but if mis-incorporated into duplex DNA can cause mutagenesis and chromosomal instability. During DNA synthesis by Pol γ, remnants of unremoved RNA primers contribute to elevated mono-ribonucleotide triphosphates resulting in nucleotide pool imbalance, ultimately favoring mis-incorporated ribonucleotides during replication. Moreover, although polymerases generally replicate DNA with high fidelity, the steric gate occasionally allows a mis-incorporated ribonucleotide. Thus, a mono-ribonucleotide is one of the most abundant lesions in genomic DNA of eukaryotes. If unremoved from double-stranded DNA, the ribonucleotide exerts negative effects on replication, transcription, and genomic maintenance, with lasting effects on cellular homeostasis. Even a single ribonucleotide in telomeric DNA comprises shelterin binding and telomere capping causing vulnerability to spontaneous hydrolysis which potentiates telomere shortening. Consistent with this, a ribonucleotide positioned in double-helical DNA alters its structure by torsinally distorting the sugar-phosphate backbone. Fortunately, cellular response and repair pathways exist to help cells cope with mis-incorporated mono-ribonucleotides. The Ribonucleotide Excision Repair (RER) or a Topoisomerase 1 (Top1)-mediated pathway remove embedded ribonucleotides. For RER, RNase H2 incises 5’ of a mono-ribonucleotide, creating an access point for its removal. If cells are deficient in RNase H2, Top1 initiates removal of the ribonucleotide. However, Top1 is less accurate than RNase H2, which can lead to mutagenesis. Studying the mechanisms in which ribonucleotides are incorporated into DNA or further metabolized should provide insight to their negative consequences for chromosomal integrity, cancer, and auto-immune disease attributed to a genetic deficiency of RNase H2.

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RNase H1 and H2 are differentially regulated to eliminate RNA-DNA hybrids

10.1101/593152 ◽

2019 ◽

Cited By ~ 1

Author(s):

Arianna Lockhart ◽

Vanessa Borges Pires ◽

Fabio Bento ◽

Vanessa Kellner ◽

Sarah Luke-Glaser ◽

...

Keyword(s):

Cell Cycle ◽

Excision Repair ◽

Model System ◽

Rnase H ◽

Genome Integrity ◽

Dependent Manner ◽

Complete Understanding ◽

Hybrid Dynamics ◽

Cycle Dependent ◽

Rnase H2

SUMMARYRNA-DNA hybrids are tightly regulated to ensure genome integrity. The RNase H enzymes, RNase H1 and H2, contribute to chromosomal stability through the removal of RNA-DNA hybrids. Loss of RNase H2 function is implicated in human diseases of the nervous system and cancer. To better understand RNA-DNA hybrid dynamics, we have focused on elucidating the regulation of the RNase H enzymes themselves. Using yeast as a model system, we demonstrate that RNase H1 and H2 are controlled in different manners. RNase H2 is regulated in a strict cell cycle dependent manner, both in terms of its R-loop removal, and ribonucleotide excision repair functions. RNase H1, however, can function independent of cell cycle stage to remove R-loops, but appears to become activated in response to high R-loop loads. These results provide us with a more complete understanding of how and when RNA-DNA hybrids are acted upon by the RNase H enzymes.

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High density of unrepaired genomic ribonucleotides leads to Topoisomerase 1-mediated severe growth defects in absence of ribonucleotide reductase

Nucleic Acids Research ◽

10.1093/nar/gkaa103 ◽

2020 ◽

Vol 48 (8) ◽

pp. 4274-4297 ◽

Cited By ~ 1

Author(s):

Susana M Cerritelli ◽

Jaime Iranzo ◽

Sushma Sharma ◽

Andrei Chabes ◽

Robert J Crouch ◽

...

Keyword(s):

Ribonucleotide Reductase ◽

Genomic Dna ◽

Genome Instability ◽

Excision Repair ◽

Topoisomerase 1 ◽

Growth Defects ◽

Rnase H2 ◽

Cell Lethality ◽

Severe Growth ◽

Excessive Accumulation

Abstract Cellular levels of ribonucleoside triphosphates (rNTPs) are much higher than those of deoxyribonucleoside triphosphates (dNTPs), thereby influencing the frequency of incorporation of ribonucleoside monophosphates (rNMPs) by DNA polymerases (Pol) into DNA. RNase H2-initiated ribonucleotide excision repair (RER) efficiently removes single rNMPs in genomic DNA. However, processing of rNMPs by Topoisomerase 1 (Top1) in absence of RER induces mutations and genome instability. Here, we greatly increased the abundance of genomic rNMPs in Saccharomyces cerevisiae by depleting Rnr1, the major subunit of ribonucleotide reductase, which converts ribonucleotides to deoxyribonucleotides. We found that in strains that are depleted of Rnr1, RER-deficient, and harbor an rNTP-permissive replicative Pol mutant, excessive accumulation of single genomic rNMPs severely compromised growth, but this was reversed in absence of Top1. Thus, under Rnr1 depletion, limited dNTP pools slow DNA synthesis by replicative Pols and provoke the incorporation of high levels of rNMPs in genomic DNA. If a threshold of single genomic rNMPs is exceeded in absence of RER and presence of limited dNTP pools, Top1-mediated genome instability leads to severe growth defects. Finally, we provide evidence showing that accumulation of RNA/DNA hybrids in absence of RNase H1 and RNase H2 leads to cell lethality under Rnr1 depletion.

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High Flexibility of RNaseH2 Catalytic Activity with Respect to Non-Canonical DNA Structures

International Journal of Molecular Sciences ◽

10.3390/ijms22105201 ◽

2021 ◽

Vol 22 (10) ◽

pp. 5201

Author(s):

Maria Dede ◽

Silvia Napolitano ◽

Anna Melati ◽

Valentina Pirota ◽

Giovanni Maga ◽

...

Keyword(s):

Dna Sequences ◽

Genome Instability ◽

Excision Repair ◽

Dna Lesions ◽

Hydroxyl Group ◽

Dna Structures ◽

Geometrical Features ◽

Fork Stalling ◽

High Flexibility ◽

Rnase H2

Ribonucleotides misincorporated in the human genome are the most abundant DNA lesions. The 2′-hydroxyl group makes them prone to spontaneous hydrolysis, potentially resulting in strand breaks. Moreover, their presence may decrease the rate of DNA replication causing replicative fork stalling and collapse. Ribonucleotide removal is initiated by Ribonuclease H2 (RNase H2), the key player in Ribonucleotide Excision Repair (RER). Its absence leads to embryonic lethality in mice, while mutations decreasing its activity cause Aicardi–Goutières syndrome. DNA geometry can be altered by DNA lesions or by peculiar sequences forming secondary structures, like G-quadruplex (G4) and trinucleotide repeats (TNR) hairpins, which significantly differ from canonical B-form. Ribonucleotides pairing to lesioned nucleotides, or incorporated within non-B DNA structures could avoid RNase H2 recognition, potentially contributing to genome instability. In this work, we investigate the ability of RNase H2 to process misincorporated ribonucleotides in a panel of DNA substrates showing different geometrical features. RNase H2 proved to be a flexible enzyme, recognizing as a substrate the majority of the constructs we generated. However, some geometrical features and non-canonical DNA structures severely impaired its activity, suggesting a relevant role of misincorporated ribonucleotides in the physiological instability of specific DNA sequences.

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RNase H2-Initiated Ribonucleotide Excision Repair

Molecular Cell ◽

10.1016/j.molcel.2012.06.035 ◽

2012 ◽

Vol 47 (6) ◽

pp. 980-986 ◽

Cited By ~ 205

Author(s):

Justin L. Sparks ◽

Hyongi Chon ◽

Susana M. Cerritelli ◽

Thomas A. Kunkel ◽

Erik Johansson ◽

...

Keyword(s):

Excision Repair ◽

Rnase H2

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A role for Rad5 in ribonucleoside monophosphate (rNMP) tolerance

Life Science Alliance ◽

10.26508/lsa.202000966 ◽

2021 ◽

Vol 4 (10) ◽

pp. e202000966

Author(s):

Menattallah Elserafy ◽

Iman El-Shiekh ◽

Dalia Fleifel ◽

Reham Atteya ◽

Abdelrahman AlOkda ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Excision Repair ◽

Replication Stress ◽

Repair Pathway ◽

Tolerance Mechanism ◽

Tolerance Mechanisms ◽

Dna Damage Tolerance ◽

Bona Fide ◽

Rnase H2

Ribonucleoside monophosphate (rNMP) incorporation in genomic DNA poses a significant threat to genomic integrity. In addition to repair, DNA damage tolerance mechanisms ensure replication progression upon encountering unrepaired lesions. One player in the tolerance mechanism is Rad5, which is an E3 ubiquitin ligase and helicase. Here, we report a new role for yeast Rad5 in tolerating rNMP incorporation, in the absence of the bona fide ribonucleotide excision repair pathway via RNase H2. This role of Rad5 is further highlighted after replication stress induced by hydroxyurea or by increasing rNMP genomic burden using a mutant DNA polymerase (Pol ε - Pol2-M644G). We further demonstrate the importance of the ATPase and ubiquitin ligase domains of Rad5 in rNMP tolerance. These findings suggest a similar role for the human Rad5 homologues helicase-like transcription factor (HLTF) and SNF2 Histone Linker PHD RING Helicase (SHPRH) in rNMP tolerance, which may impact the response of cancer cells to replication stress-inducing therapeutics.

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SAC3B is a target of CML19, the centrin 2 of Arabidopsis thaliana

Biochemical Journal ◽

10.1042/bcj20190674 ◽

2020 ◽

Vol 477 (1) ◽

pp. 173-189 ◽

Cited By ~ 1

Author(s):

Marco Pedretti ◽

Carolina Conter ◽

Paola Dominici ◽

Alessandra Astegno

Keyword(s):

Excision Repair ◽

Complex Structure ◽

Binding Motif ◽

C Terminus ◽

Repair Protein ◽

Nucleotide Excision ◽

Ef Hand ◽

Molecular Determinants ◽

Structure In Solution

Arabidopsis centrin 2, also known as calmodulin-like protein 19 (CML19), is a member of the EF-hand superfamily of calcium (Ca2+)-binding proteins. In addition to the notion that CML19 interacts with the nucleotide excision repair protein RAD4, CML19 was suggested to be a component of the transcription export complex 2 (TREX-2) by interacting with SAC3B. However, the molecular determinants of this interaction have remained largely unknown. Herein, we identified a CML19-binding site within the C-terminus of SAC3B and characterized the binding properties of the corresponding 26-residue peptide (SAC3Bp), which exhibits the hydrophobic triad centrin-binding motif in a reversed orientation (I8W4W1). Using a combination of spectroscopic and calorimetric experiments, we shed light on the SAC3Bp–CML19 complex structure in solution. We demonstrated that the peptide interacts not only with Ca2+-saturated CML19, but also with apo-CML19 to form a protein–peptide complex with a 1 : 1 stoichiometry. Both interactions involve hydrophobic and electrostatic contributions and include the burial of Trp residues of SAC3Bp. However, the peptide likely assumes different conformations upon binding to apo-CML19 or Ca2+-CML19. Importantly, the peptide dramatically increases the affinity for Ca2+ of CML19, especially of the C-lobe, suggesting that in vivo the protein would be Ca2+-saturated and bound to SAC3B even at resting Ca2+-levels. Our results, providing direct evidence that Arabidopsis SAC3B is a CML19 target and proposing that CML19 can bind to SAC3B through its C-lobe independent of a Ca2+ stimulus, support a functional role for these proteins in TREX-2 complex and mRNA export.

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