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

Abstract

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...

Distinct Contributions of Enzymic Functional Groups to the 2′,3′-Cyclic Phosphodiesterase, 3′-Phosphate Guanylylation, and 3′-ppG/5′-OH Ligation Steps of the Escherichia coli RtcB Nucleic Acid Splicing Pathway

“…Since then, our laboratory has improved the purification of recombinant RtcB protein, such that it is fully dependent on exogenous GTP for sealing activity (5). Moreover, we have developed new substrates to assay the cyclic phosphodiesterase (CPDase), 3=-PO 4 /5=-OH ligation, 3=-PO 4 guanylylation, and 3=-ppG/5=-OH ligation reactions under single-turnover conditions (5)(6)(7)(8). Here we applied these interval technical advances to reexamine the effects of active-site alanine mutations on E. coli RtcB function in vitro.…”

“…2). The predominance of "soft" metal ligands (histidine nitrogens and cysteine sulfur) in the active site explains the observed metal activity and inhibition spectrum of E. coli RtcB (1,5,8), whereby (i) the overall ligation pathway and the component steps of RtcB guanylylation and phosphodiester synthesis rely on Mn 2ϩ , which is favorably coordinated by soft ligands; (ii) "hard" metals Mg 2ϩ and Ca 2ϩ , which favor oxygen ligands, neither support RtcB activity themselves nor inhibit RtcB function in the presence of Mn 2ϩ ; and (iii) alternative soft metals Zn 2ϩ , Co 2ϩ , Cu 2ϩ , and Ni 2ϩ that do not support activity are potent inhibitors of activity in the presence of Mn 2ϩ , presumably because they compete with Mn 2ϩ for binding to one or both metal sites, whence bound they are unable to sustain catalysis. It is an open question whether both Mn 2ϩ sites in the binuclear cluster are required for each step in the RtcB pathway or if some of the steps rely on just one of the metal ions.…”

“…The utility of this approach was exemplified by an analysis of the H337A mutant of E. coli RtcB, which eliminates the site of covalent attachment of GMP to the enzyme, rendering the H337A mutant protein inert in RtcB guanylylation and, hence, formation of the polynucleotide-ppG intermediate (6). The instructive findings were that the H337A mutant was adept at joining DNAppG and HO DNA ends in a reaction that required Mn 2ϩ but not GTP (8), signifying that GTP and His337 play no essential role in the pathway downstream of the step of 3=-end activation.…”

Distinctive kinetics and substrate specificities of plant and fungal tRNA ligases

“…A simple way to address this issue is to query whether any or all of the nucleotides of a spliceable polynucleotide substrate for AtRNL (exclusive of the 3 ′ nucleotide) can be replaced by deoxynucleotides. This approach has been fruitful in delineating the minimal RNA content required for other RNA repair enzymes, including T4 RNA ligase 2 (Nandakumar and Shuman 2004), the RNA terminal 2 ′ -O-methyltransferase Hen1 (Jain and Shuman 2011), the RNA 3 ′ -terminal cyclase RtcA (Das and Shuman 2013), and the RNA 3 ′ -PO 4 /5 ′ -OH ligase RtcB (Das et al 2013a). Here, we tested whether AtRNL could splice a HO D17R>p substrate, consisting of 17 deoxynucleotides and a single 32 Plabeled 3 ′ -terminal ribonucleoside-2 ′ ,3 ′ -cyclic-PO 4 (Fig.…”

“…We also analyze the kinetics and substrate requirements of an exemplary fungal tRNA ligase, Kluyveromyces lactis Trl1 (KlaTrl1). Fungal Trl1 enzymes are potential therapeutic targets, because their structures and biochemical mechanisms are unique compared to the RtcB-type tRNA repair systems elaborated by metazoans, archaea, and many bacteria (Popow et al 2011;Chakravarty et al 2012;Englert et al 2012;Das et al 2013a;Desai et al 2013). Indeed, mammalian proteomes have no discernable homologs of the sealing domain of fungal tRNA ligase.…”