Topoisomerase 1-Mediated Removal of Ribonucleotides from Nascent Leading-Strand DNA

Williams, Jessica S.; Smith, Dana J.; Marjavaara, Lisette; Lujan, Scott A.; Chabes, Andrei; Kunkel, Thomas A.

doi:10.1016/j.molcel.2012.12.021

Cited by 129 publications

(175 citation statements)

References 34 publications

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“…Although the metabolism of 2′,3′-cyclic-PO 4 DNA breaks is entirely uncharted, such ends are potential fodder for the cyclic phosphodiesterase activity of RtcB and subsequent end joining to restore the DNA backbone. If RtcB seals the duplex nick without intervening processing steps, the effect of such a faithful ligation could be to redirect repair of the embedded ribonucleotide to an apparently "safer" pathway driven by RNase H2 (18,19). Alternatively, RtcB might splice the 2′,3′-cyclic-PO 4 DNA end to a different 5′-OH acceptor, resulting in a mutagenic repair outcome.…”

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confidence: 99%

Rewriting the rules for end joining via enzymatic splicing of DNA 3′-PO ₄ and 5′-OH ends

Das

Chakravarty

Remus

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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There are many biological contexts in which DNA damage generates "dirty" breaks with 3′-PO 4 (or cyclic-PO 4 ) and 5′-OH ends that cannot be sealed by DNA ligases. Here we show that the Escherichia coli RNA ligase RtcB can splice these dirty DNA ends via a unique chemical mechanism. RtcB transfers GMP from a covalent RtcB-GMP intermediate to a DNA 3′-PO 4 to form a "capped" 3′ end structure, DNA 3′ pp 5′ G. When a suitable DNA 5′-OH end is available, RtcB catalyzes attack of the 5′-OH on DNA 3′ pp 5′ G to form a 3′-5′ phosphodiester splice junction. Our findings unveil an enzymatic capacity for DNA 3′ capping and the sealing of DNA breaks with 3′-PO 4 and 5′-OH termini, with implications for DNA repair and DNA rearrangements.T he Escherichia coli RtcB is a founding member of a recently discovered family of RNA repair/splicing enzymes that join RNA 2′,3′-cyclic-PO 4 or 3′-PO 4 ends to RNA 5′-OH ends (1-4). RtcB executes a four-step pathway that requires GTP as an energy source and Mn 2+ as a cofactor (5-7). RtcB first reacts with GTP to form a covalent RtcB-(histidinyl 337 -N)-GMP intermediate. It then hydrolyzes the RNA 2′,3′-cyclic-PO 4 end to a 3′-PO 4 and transfers guanylate from His337 to the RNA 3′-PO 4 to form an RNA 3′ pp 5′ G intermediate. Finally, RtcB catalyzes the attack of an RNA 5′-OH on the RNA 3′ pp 5′ G end to form the 3′-5′ phosphodiester splice junction and liberate GMP.The unique chemical mechanism of RtcB overturned a longstanding tenet of nucleic acid enzymology, which held that synthesis of polynucleotide 3′-5′ phosphodiesters proceeds via the attack of a 3′-OH on a high-energy 5′-phosphoanhydride: either a nucleoside 5′-triphosphate in the case of RNA/DNA polymerases or an adenylylated intermediate A 5′ pp 5′ N, in the case of classic RNA/DNA ligases. In light of the wide distribution of RtcB proteins in bacteria, archaea, and metazoa, we raised the prospect of an alternative enzymology based on covalently activated 3′-PO 4 ends (6).In principle, the chemistry of RNA 3′-PO 4 /5′-OH end joining by RtcB might be portable to DNA transactions and pertinent to DNA repair. A variety of hydrolytic nucleases incise the DNA phosphodiester backbone to yield 3′-PO 4 and 5′-OH termini that cannot be joined by DNA ligases. Nonligatable 3′-PO 4 ends are also generated during base excision repair catalyzed by DNA glycosylase/lyase enzymes, during the repair of trapped covalent topoisomerase IB-DNA adducts by tyrosyl-DNA phosphodiesterase 1, and during DNA damage inflicted by ionizing radiation. One way nature solves this "dirty end" problem is by deploying a variety of "end healing" enzymes (8-14). These include 3′-phosphoesterases that convert a 3′-PO 4 to a 3′-OH and 5′-kinases that transform a 5′-OH to a 5′-PO 4 , thereby enabling break sealing by the classic ligase pathway. Given what we now know about RtcB, would it not make sense for nature to also endow a pathway for direct joining of DNA 3′-PO 4 and 5′-OH ends, be it via RtcB or another ligase yet to be discovered?We can extend this thought to DN...

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confidence: 99%

Rewriting the rules for end joining via enzymatic splicing of DNA 3′-PO ₄ and 5′-OH ends

Das

Chakravarty

Remus

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…9 There is good evidence that Top1-mediated nicks at rNMP residues act as the initiating DNA lesion in slippage mutation induction. 1,2,10,11 To determine the linkage of the ligation step to mutations, we used the dinucleotide slippage reporter to examine the effect of the top1-T722A allele on mutation rates. As shown in Figure 2A, top1-T722A behaves identically to the null allele, thus showing that both rNMP cleavage and DNA ligation are needed to generate the necessary lesion for mutation induction.…”

Section: Roles Of Top1 and Tdp1 In Cleavage And Processing At An Rnmpmentioning

confidence: 99%

Roles of DNA helicases and Exo1 in the avoidance of mutations induced by Top1-mediated cleavage at ribonucleotides in DNA

et al. 2016

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The replicative DNA polymerases insert ribonucleotides into DNA at a frequency of approximately 1/6500 nucleotides replicated. The rNMP residues make the DNA backbone more susceptible to hydrolysis and can also distort the helix, impeding the transcription and replication machineries. rNMPs in DNA are efficiently removed by RNaseH2 by a process called ribonucleotides excision repair (RER). In the absence of functional RNaseH2, rNMPs are subject to cleavage by Topoisomerase I, followed by further processing to result in deletion mutations due to slippage in simple DNA repeats. The topoisomerase I-mediated cleavage at rNMPs results in DNA ends that cannot be ligated by DNA ligase I, a 5 0 OH end and a 2 0 -3 0 cyclic phosphate end. In the budding yeast, the mutation level in RNaseH2 deficient cells is kept low via the action of the Srs2 helicase and the Exo1 nuclease, which collaborate to process the Top1-induced nick with subsequent non-mutagenic gap filling. We have surveyed other helicases and nucleases for a possible role in reducing mutagenesis at Top1 nicks at rNMPs and have uncovered a novel role for the RecQ family helicase Sgs1 in this process.

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“…Ribonucleotides are also incorporated into DNA by DNA polymerases (Pol) α, δ, and e, because they discriminate against ribonucleoside triphosphates (rNTPs) efficiently but imperfectly (4) and because cellular rNTP concentrations are much higher than dNTP concentrations (4). As a consequence, large numbers of ribonucleotides are incorporated during replication, and are present in the genomes of cells defective in the repair enzymes that initiate their removal, RNase H2 (5-9) and topoisomerase 1 (10).…”

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confidence: 99%

“…Yeast strains defective in RNase H2-dependent ribonucleotide excision repair (RER) (5,14) exhibit several characteristics of replicative stress, including strongly elevated rates for deleting 2-5 bp from repetitive DNA sequences (5,15), events that are initiated by topoisomerase 1 cleavage of a ribonucleotide in DNA (10,16). Yeast strains defective in RNase H2 and RNase H1 progress slowly through S phase, accumulate ubiquitylated proliferating cell nuclear antigen (PCNA), and are sensitive to treatment with hydroxyurea (17).…”

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confidence: 99%

“…Moreover, their survival in the presence of hydroxyurea partly depends on MMS2-dependent template switching and on REV3, which encodes the catalytic subunit of the translesion synthesis (TLS) enzyme Pol ζ. When ribonucleotide incorporation during leading strand replication is increased by a M644G substitution in the Pol e active site, a defect in RNase H2 results in elevated deletion mutagenesis, elevated dNTP pools, slow growth and activation of the S-phase checkpoint (5,10,18), and concomitant deletion of the RNH1 gene encoding RNases H1 is lethal (17). In mice, knocking out any of the genes encoding the three subunits of RNase H2 is embryonic lethal (7,19).…”

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Structure–function analysis of ribonucleotide bypass by B family DNA replicases

Clausen

Murray

Passer

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Ribonucleotides are frequently incorporated into DNA during replication, they are normally removed, and failure to remove them results in replication stress. This stress correlates with DNA polymerase (Pol) stalling during bypass of ribonucleotides in DNA templates. Here we demonstrate that stalling by yeast replicative Pols δ and e increases as the number of consecutive template ribonucleotides increases from one to four. The homologous bacteriophage RB69 Pol also stalls during ribonucleotide bypass, with a pattern most similar to that of Pol e. Crystal structures of an exonuclease-deficient variant of RB69 Pol corresponding to multiple steps in single ribonucleotide bypass reveal that increased stalling is associated with displacement of Tyr391 and an unpreferred C2´-endo conformation for the ribose. Even less efficient bypass of two consecutive ribonucleotides in DNA correlates with similar movements of Tyr391 and displacement of one of the ribonucleotides along with the primer-strand DNA backbone. These structurefunction studies have implications for cellular signaling by ribonucleotides, and they may be relevant to replication stress in cells defective in ribonucleotide excision repair, including humans suffering from autoimmune disease associated with RNase H2 defects.translesion synthesis | replication stalling | DNA replication

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Topoisomerase 1-Mediated Removal of Ribonucleotides from Nascent Leading-Strand DNA

Cited by 129 publications

References 34 publications

Rewriting the rules for end joining via enzymatic splicing of DNA 3′-PO ₄ and 5′-OH ends

Rewriting the rules for end joining via enzymatic splicing of DNA 3′-PO ₄ and 5′-OH ends

Roles of DNA helicases and Exo1 in the avoidance of mutations induced by Top1-mediated cleavage at ribonucleotides in DNA

Structure–function analysis of ribonucleotide bypass by B family DNA replicases

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Topoisomerase 1-Mediated Removal of Ribonucleotides from Nascent Leading-Strand DNA

Cited by 129 publications

References 34 publications

Rewriting the rules for end joining via enzymatic splicing of DNA 3′-PO 4 and 5′-OH ends

Rewriting the rules for end joining via enzymatic splicing of DNA 3′-PO 4 and 5′-OH ends

Roles of DNA helicases and Exo1 in the avoidance of mutations induced by Top1-mediated cleavage at ribonucleotides in DNA

Structure–function analysis of ribonucleotide bypass by B family DNA replicases

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Rewriting the rules for end joining via enzymatic splicing of DNA 3′-PO ₄ and 5′-OH ends

Rewriting the rules for end joining via enzymatic splicing of DNA 3′-PO ₄ and 5′-OH ends