Kinetic mechanism of nick sealing by T4 RNA ligase 2 and effects of 3′-OH base mispairs and damaged base lesions

Chauleau, Mathieu; Shuman, Stewart

doi:10.1261/rna.041731.113

Cited by 17 publications

(16 citation statements)

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“…The physiology and biochemistry of the Rnl families suggest a division of labor in RNA repair, whereby Rnl1 and Rnl4 ligases are tailored to seal single-strand breaks in the loop of RNA stem-loops (Amitsur et al 1987;Wang et al 2007;Nandakumar et al 2008;Zhang et al 2012), whereas Rnl2 ligases are designed to seal 3 ′ -OH/5 ′ -PO 4 nicks in duplex RNAs and/ or RNA:DNA hybrids (Blanc et al 1999;Chauleau and Shuman 2013).…”

Section: Introductionmentioning

confidence: 99%

Characterization of a novel eukaryal nick-sealing RNA ligase from Naegleria gruberi

Unciuleac

Shuman

2015

RNA

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The proteome of the amoebo-flagellate protozoan Naegleria gruberi is rich in candidate RNA repair enzymes, including 15 putative RNA ligases, one of which, NgrRnl, is a eukaryal homolog of Deinococcus radiodurans RNA ligase, DraRnl. Here we report that purified recombinant NgrRnl seals nicked 3 ′ -OH/5 ′ -PO 4 duplexes in which the 3 ′ -OH strand is RNA. It does so via the "classic" ligase pathway, entailing reaction with ATP to form a covalent NgrRnl-AMP intermediate, transfer of AMP to the nick 5 ′ -PO 4 , and attack of the RNA 3 ′ -OH on the adenylylated nick to form a 3 ′ -5 ′ phosphodiester. Unlike members of the four known families of ATP-dependent RNA ligases, NgrRnl lacks a carboxy-terminal appendage to its nucleotidyltransferase domain. Instead, it contains a defining amino-terminal domain that we show is important for 3 ′ -OH/5 ′ -PO 4 nick-sealing and ligase adenylylation, but dispensable for phosphodiester synthesis at a preadenylylated nick. We propose that NgrRnl, DraRnl, and their homologs from diverse bacteria, viruses, and unicellular eukarya comprise a new "Rnl5 family" of nick-sealing ligases with a signature domain organization.

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Section: Introductionmentioning

confidence: 99%

Characterization of a novel eukaryal nick-sealing RNA ligase from Naegleria gruberi

Unciuleac

Shuman

2015

RNA

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“…First, DraRnl accepts the A-oxoG pair as correct, while it discriminates against 3= U-oxoG and GoxoG mispairs. Bacteriophage T4 Rnl2 displays similar properties in this regard (41). Second, DraRnl obeys the A rule in its preference for sealing when a 3=-OH A is opposite an abasic template site.…”

Section: Discussionmentioning

confidence: 93%

“…With the 3=-OH mispairs (which we suspect are more commonly encountered by ligases during gap repair than 5=-PO 4 mispairs), the DraRnl sealing rate varied widely in a hierarchical fashion, with G-T and A-C mispairs being the best substrates and the G-G, G-A, and A-A mispairs being the worst. It is noteworthy that when the 3=-OH mispaired nicks were recently used to interrogate the fidelity of bacteriophage T4 Rnl2, the 3= A-C and G-T configurations were the ones that were best tolerated by Rnl2 (41). Bacteriophage T4 Rnl2 and DraRnl exemplify distinct families of nick-sealing enzymes that require a 3=-OH RNA strand (34,65).…”

Section: Discussionmentioning

confidence: 99%

“…Oxidized gRNAs could engender mutagenic RNA editing if nucleotides are misincorporated opposite 8-oxopurines in the gRNA during gap filling and the mispaired 3=-OH N-oxopurine nicks are sealed by RELs. Recent studies of bacteriophage T4 Rnl2, a homolog of the RELs, highlight its ability to seal, in high yield, nicks with mispaired 3=-OH ends, including 3= A-oxoG ends (41).…”

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confidence: 99%

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Effects of 3′-OH and 5′-PO ₄ Base Mispairs and Damaged Base Lesions on the Fidelity of Nick Sealing by Deinococcus radiodurans RNA Ligase

Schmier

Shuman

2014

J Bacteriol

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Deinococcus radiodurans RNA ligase (DraRnl) is the founding member of a family of end-joining enzymes encoded by diverse microbes and viruses. DraRnl ligates 3=-OH, 5=-PO 4 nicks in double-stranded nucleic acids in which the nick 3=-OH end is RNA. Here we gauge the effects of 3=-OH and 5=-PO 4 base mispairs and damaged base lesions on the rate of nick sealing. DraRnl is indifferent to the identity of the 3=-OH nucleobase, provided that it is correctly paired. With 3=-OH mispairs, the DraRnl sealing rate varies widely, with G-T and A-C mispairs being the best substrates and G-G, G-A, and A-A mispairs being the worst. DraRnl accepts 3= A-8-oxoguanine (oxoG) to be correctly paired, while it discriminates against U-oxoG and G-oxoG mispairs. DraRnl displays high activity and low fidelity in sealing 3=-OH ends opposite an 8-oxoadenine lesion. It prefers 3=-OH adenosine when sealing opposite an abasic template site. With 5=-PO 4 mispairs, DraRnl seals a 5= T-G mispair as well as it does a 5= C-G pair; in most other respects, the ligation fidelity at 5= mispairs is similar to that at 3= mispairs. DraRnl accepts a 5= A-oxoG end to be correctly paired, yet it is more tolerant of 5= T-oxoG and 5= G-oxoG mispairs than the equivalent configurations on the 3= side of the nick. At 5= nucleobase-abasic site nicks, DraRnl prefers to ligate when the nucleobase is a purine. The biochemical properties of DraRnl are compatible with its participation in the templated repair of RNA damage or in the sealing of filled DNA gaps that have a 3= ribopatch. Ionizing radiation (IR) damages nucleic acids via reactive hydroxyl radicals, generated by the radiolysis of water, that chemically alter the nucleobases and sugar-phosphate backbone. Closely spaced lesions on opposing strands of duplex DNA can lead to lethal double-strand breaks. Cells and viruses deploy multiple enzymatic pathways to recognize and repair radiation-damaged DNA. The final step in the repair cascade is the restoration of the phosphodiester backbone by DNA ligase, an enzyme that seals 3=-OH and 5=-PO 4 ends (1, 2).RNA is also susceptible to base and backbone damage by IR and oxidative stress. Hydroxyl radicals and the bleomycin class of anticancer drugs cleave RNA in vitro and in vivo (3-6). Oxidative damage triggered by exposure of cells to hydrogen peroxide generates 8-oxopurine nucleobases in RNA, principally, 8-oxoguanine (oxoG) (7). Accumulation of oxoG in cellular RNA is a feature of human neurodegenerative syndromes, such as Alzheimer's disease, parkinsonism, and amyotrophic lateral sclerosis (8, 9). It is envisioned that damaged RNAs are degraded (9-11). However, it may be advantageous at times for cells and viruses to repair broken RNAs, rather than simply resynthesize them, in order to rapidly restore the necessary templates and machinery for protein synthesis. Enzymes capable of healing and sealing broken RNA ends are widely prevalent in bacterial, archaeal, and eukaryal taxa and are also encoded by bacterial and eukaryal viruses (12-25). It has been suggested th...

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“…Secondarily, both the rates of adenylation (Step 2) and the strand connection (Step 3) in RNA ligation are signicantly reduced, when mismatches are introduced into the 3 0 -OH terminal region. 30,31 Apparently, strict freezing of molecular dynamics in the stems is important for efficient catalysis. Thirdly, the 3 0 -OH at the joining site must derive from ssRNA, although the 5 0 -phosphate can come from either ssRNA or ssDNA.…”

Section: Discussionmentioning

confidence: 99%

RNA ligation of very small pseudo nick structures by T4 RNA ligase 2, leading to efficient production of versatile RNA rings

Cheng

Cui

et al. 2019

RSC Adv.

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Kinetic mechanism of nick sealing by T4 RNA ligase 2 and effects of 3′-OH base mispairs and damaged base lesions

Cited by 17 publications

References 45 publications

Characterization of a novel eukaryal nick-sealing RNA ligase from Naegleria gruberi

Characterization of a novel eukaryal nick-sealing RNA ligase from Naegleria gruberi

Effects of 3′-OH and 5′-PO ₄ Base Mispairs and Damaged Base Lesions on the Fidelity of Nick Sealing by Deinococcus radiodurans RNA Ligase

RNA ligation of very small pseudo nick structures by T4 RNA ligase 2, leading to efficient production of versatile RNA rings

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Kinetic mechanism of nick sealing by T4 RNA ligase 2 and effects of 3′-OH base mispairs and damaged base lesions

Cited by 17 publications

References 45 publications

Characterization of a novel eukaryal nick-sealing RNA ligase from Naegleria gruberi

Characterization of a novel eukaryal nick-sealing RNA ligase from Naegleria gruberi

Effects of 3′-OH and 5′-PO 4 Base Mispairs and Damaged Base Lesions on the Fidelity of Nick Sealing by Deinococcus radiodurans RNA Ligase

RNA ligation of very small pseudo nick structures by T4 RNA ligase 2, leading to efficient production of versatile RNA rings

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Effects of 3′-OH and 5′-PO ₄ Base Mispairs and Damaged Base Lesions on the Fidelity of Nick Sealing by Deinococcus radiodurans RNA Ligase