Cation trafficking propels RNA hydrolysis

Samara, N.L.; Yang, Wei

doi:10.1038/s41594-018-0099-4

Cited by 50 publications

(147 citation statements)

References 32 publications

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“…This slow rate is reflected by a relatively high free energy barrier (~29 kcal/mol) from our calculations for the intrinsic cleavage reaction. Interestingly, recent structural work suggested the potential involvement of additional transiently bound metal ions in DNA polymerase reaction by DNA polymerase η 13 and DNA polymerase β 14 , as well as RNA cleavage conducted by RNase H 22 . To date, direct structural evidences in supporting the involvement of additional transiently-bound metal in transcriptional intrinsic cleavage by Pol II are not available.…”

Section: Discussionmentioning

confidence: 99%

“…To date, direct structural evidences in supporting the involvement of additional transiently-bound metal in transcriptional intrinsic cleavage by Pol II are not available. Nevertheless, we tested the potential role of a third Mg 2+ (Mg C ) by modelling its possible locations based on previously published Mg C conformations 14,22 (Supplementary Fig. 13).…”

Section: Discussionmentioning

confidence: 99%

“…The reaction mechanism of intrinsic cleavage by Pol II remains elusive. Although time-resolved X-ray studies have emerged to be a powerful technique for real-time monitoring the enzymatic reaction progress 12–20,22 , it remains difficult to fully reveal the reaction mechanism using this technique for Pol II due to the resolution limit of current available crystal structures. The currently available crystal structures of one-residue-backtracked RNAP 7,8 are unable to reveal a general base for activating the attacking nucleophile, as well as a general acid for protonating the oxyanion leaving group.…”

Section: Introductionmentioning

confidence: 99%

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Intrinsic cleavage of RNA polymerase II adopts a nucleobase-independent mechanism assisted by transcript phosphate

Tse

et al. 2019

Nat Catal

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RNA polymerase II (Pol II) utilises the same active site for polymerization and intrinsic cleavage. Pol II proofreads the nascent transcript by its intrinsic nuclease activity to maintain high transcriptional fidelity critical for cell growth and viability. The detailed catalytic mechanism of intrinsic cleavage remains unknown. Here, we combined ab initio quantum mechanics/molecular mechanics studies and biochemical cleavage assays to show that Pol II utilises downstream phosphate oxygen to activate the attacking nucleophile in hydrolysis, while the newly formed 3’-end is protonated through active-site water without a defined general acid. Experimentally, alteration of downstream phosphate oxygen either by 2’−5’ sugar linkage or stereo-specific thio-substitution of phosphate oxygen drastically reduced cleavage rate. We showed by N7-modification that guanine nucleobase does not directly involve as acid-base catalyst. Our proposed mechanism provides important insights into the understanding of intrinsic transcriptional cleavage reaction, an essential step of transcriptional fidelity control.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Intrinsic cleavage of RNA polymerase II adopts a nucleobase-independent mechanism assisted by transcript phosphate

Tse

et al. 2019

Nat Catal

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“…RNase H-deficient systems are involved in the quasi-palindromeassociated mutations (32). Toward this end, the effect of cations on RNase H2 activity has been studied extensively (33).…”

mentioning

confidence: 99%

Human DNA polymerase η has reverse transcriptase activity in cellular environments

Ghodke

Egli

et al. 2019

Journal of Biological Chemistry

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Edited by Patrick SungClassical DNA and RNA polymerase (pol) enzymes have defined roles with their respective substrates, but several pols have been found to have multiple functions. We reported previously that purified human DNA pol (hpol ) can incorporate both deoxyribonucleoside triphosphates (dNTPs) and ribonucleoside triphosphates (rNTPs) and can use both DNA and RNA as substrates. X-ray crystal structures revealed that two pol residues, Phe-18 and Tyr-92, behave as steric gates to influence sugar selectivity. However, the physiological relevance of these phenomena has not been established. Here, we show that purified hpol adds rNTPs to DNA primers at physiological rNTP concentrations and in the presence of competing dNTPs. When two rATPs were inserted opposite a cyclobutane pyrimidine dimer, the substrate was less efficiently cleaved by human RNase H2. Human XP-V fibroblast extracts, devoid of hpol , could not add rNTPs to a DNA primer, but the expression of transfected hpol in the cells restored this ability. XP-V cell extracts did not add dNTPs to DNA primers hybridized to RNA, but could when hpol was expressed in the cells. HEK293T cell extracts could add dNTPs to DNA primers hybridized to RNA, but lost this ability if hpol was deleted. Interestingly, a similar phenomenon was not observed when other translesion synthesis (TLS) DNA polymerases-hpol , , or -were individually deleted. These results suggest that hpol is one of the major reverse transcriptases involved in physiological processes in human cells.

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“…The catalytic domain was predicted to adopt a similar fold as previously observed in viruses (47), bacteria (7,8,48,49) and mammals (9), and includes the conserved DEDD motif ( Fig. S1), from which residue D252 was shown to be essential for activity, as in other organisms (16).…”

Section: Functional Characterization Of the Domains Of Dm Rnase H1mentioning

confidence: 58%

A second hybrid-binding domain modulates the activity of Drosophila ribonuclease H1

Cózar

Carretero-Junquera

Ciesielski

et al. 2020

Preprint

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In eukaryotes, ribonuclease H1 (RNase H1) is involved in the processing and removal of RNA/DNA hybrids in both nuclear and mitochondrial DNA. The enzyme comprises a C-terminal catalytic domain and an N-terminal hybrid-binding domain (HBD), separated by a linker of variable length, which in Drosophila melanogaster (Dm) is exceptionally long, 115 amino acids. Molecular modeling predicted this extended linker to fold into a structure similar to the conserved HBD. We measured catalytic activity and substrate binding by EMSA and biolayer interferometry, using a deletion series of protein variants. Both the catalytic domain and the conserved HBD were required for high-affinity binding to heteroduplex substrates, whilst loss of the novel HBD led to a ~90% drop in K[cat] with a decreased K[M], and a large increase in the stability of the RNA/DNA hybrid-enzyme complex. The findings support a bipartite binding model for the enzyme, whereby the second HBD facilitates dissociation of the active site from the product, allowing for processivity. We used shotgun proteomics to identify protein partners of the enzyme involved in mediating these effects. Single-stranded DNA-binding proteins from both the nuclear and mitochondrial compartments, respectively RpA-70 and mtSSB, were prominently detected by this method. However, we were not able to document direct interactions between mtSSB and Dm RNase H1 when co-overexpressed in S2 cells, or functional interactions in vitro. Further studies are needed to determine the exact reaction mechanism of Dm RNase H1, the nature of its interaction with mtSSB and the role of the second HBD in both.

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Cation trafficking propels RNA hydrolysis

Cited by 50 publications

References 32 publications

Intrinsic cleavage of RNA polymerase II adopts a nucleobase-independent mechanism assisted by transcript phosphate

Intrinsic cleavage of RNA polymerase II adopts a nucleobase-independent mechanism assisted by transcript phosphate

Human DNA polymerase η has reverse transcriptase activity in cellular environments

A second hybrid-binding domain modulates the activity of Drosophila ribonuclease H1

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