Impact of DNA _3′ pp _5′ G capping on repair reactions at DNA 3′ ends

Abstract

Many biological scenarios generate "dirty" DNA 3′-PO 4 ends that cannot be sealed by classic DNA ligases or extended by DNA polymerases. The noncanonical ligase RtcB can "cap" these ends via a unique chemical mechanism entailing transfer of GMP from a covalent RtcB-GMP intermediate to a DNA 3′-PO 4 to form DNA 3′ pp 5′ G. Here, we show that capping protects DNA 3′ ends from resection by Escherichia coli exonucleases I and III and from end-healing by T4 polynucleotide 3′ phosphatase. By contrast, the cap is an effective primer for DNA synthesis. E. coli DNA polymerase I and Mycobacterium DinB1 extend the DNAppG primer to form an alkali-labile DNApp(rG)pDNA product. The addition of dNTP depends on pairing of the cap guanine with an opposing cytosine in the template strand. Aprataxin, an enzyme implicated in repair of A 5′ pp 5′ DNA ends formed during abortive ligation by classic ligases, is highly effective as a DNA 3′ decapping enzyme, converting DNAppG to DNA 3′ p and GMP. We conclude that the biochemical impact of DNA capping is to prevent resection and healing of a 3′-PO 4 end, while permitting DNA synthesis, at the price of embedding a ribonucleotide and a pyrophosphate linkage in the repaired strand. Aprataxin affords a means to counter the impact of DNA capping.DNA repair | 3′ exonuclease T he synthesis and repair of DNA are driven by enzymatic reactions at 3′ ends. The terminal 3′-OH is the nucleophile that primes 3′-5′ phosphodiester bond formation by DNA polymerases and DNA ligases. To contend with the many situations in biology when incision of DNA generates a 3′-PO 4 (a so-called "dirty" end, which cannot be extended by polymerases or sealed by classic ligases), nature has evolved an assortment of endhealing enzymes that remove the 3′-PO 4 and/or resect 3′ nucleotides (1). We recently described a different fate for DNA 3′-PO 4 ends at the hands of the unconventional ligase RtcB (2, 3), which adds a guanylate "cap" to form DNA 3′ pp 5′ G (4). DNA capping by RtcB is highly efficient, whether at the 3′-PO 4 ends of ssDNAs or at a nick in duplex DNA (4). DNA 3′ capping has tantalizing implications. For analogy, one need only consider the multifaceted role of the 5′ cap structure, m 7 GpppN-, in RNA metabolism (5). Here, we address the consequences of DNA 3′ capping for the reactions of exemplary exonucleases, phosphatases, and polymerases at 3′ ends. In addition, we illuminate aprataxin as a DNA 3′ decapping enzyme. Results and DiscussionDNA Capping Confers Resistance to End Resection by 3′ Exonucleases and 3′ Phosphatase. Escherichia coli exonucleases I and III are DNA repair enzymes that hydrolyze 3′-OH DNA ends in a stepwise fashion to liberate 5′ deoxyribonucleoside monophosphate (dNMP) mononucleotide products. ExoI acts processively on ssDNA with a 3′-OH end but has minimal activity at a 3′-PO 4 end (6, 7). By contrast, ExoIII distributively resects 3′-OH and 3′-PO 4 DNA ends, the latter by virtue of its intrinsic DNA 3′ phosphatase activity (7-9). Here, we gauged the effect of DNA 3′ capping on end r...

Molecular underpinnings of Aprataxin RNA/DNA deadenylase function and dysfunction in neurological disease

“…Intriguingly, reversibility of the serine hydrogen bond donor/acceptor would also enable the recognition of guanine in the lesion-binding pocket. Consistent with this, SpAptx has recently been reported to catalyze removal of GMP from 3’–GMP–capped DNA ends (Das et al, 2014). We speculate that GMP binds in an analogous manner to AMP during a deguanylation reaction.…”

Characterization of 3′-Phosphate RNA Ligase Paralogs RtcB1, RtcB2, and RtcB3 from Myxococcus xanthus Highlights DNA and RNA 5′-Phosphate Capping Activity of RtcB3

“…Initially described as an adenylate decapping enzyme acting on abortive AppDNA intermediates formed by ATP-dependent DNA ligases, aprataxin was subsequently shown to have 3= decapping activity on a DNAppG end formed by E. coli RtcB (12,24) and on DNAppA ends generated by an ATP-dependent thermophilic RNA ligase (38). Here, we extend the aprataxin repertoire by showing that it can remove a 5= guanylate cap from GppDNA formed by M. xanthus RtcB3.…”

“…The DNA repair enzyme aprataxin has a DNA 3= decapping activity whereby it converts DNAppG (synthesized by E. coli RtcB) to DNAp and GMP (12,24). Aprataxin is a member of the histidine triad family of nucleotidyltransferases that act via a covalent enzyme-(histidinyl)-NMP intermediate.…”

“…Biochemical studies of the impact of DNA 3= capping on repair reactions highlighted two key points: (i) the cap protects DNA ends from 3= exonucleases, and (ii) the cap guanosine 3=-OH can serve as a primer for templated DNA synthesis by exemplary members of five different DNA polymerase families (12,13).…”

Polynucleotide 3′-terminal Phosphate Modifications by RNA and DNA Ligases

“…It is first activated by the transfer of GMP from the RtcB-GMP complex to form the 3Ј-guanylated intermediate RNA 3Ј pp 5Ј G. This intermediate is then ligated to the 5Ј-hydroxyl of an RNA acceptor to form a phosphodiester bond or to the adjacent 2Ј-OH to form a cyclic phosphate. Moreover, recent studies showed potential for this activity to be involved in DNA repair (15,16).…”