Impact of DNA3′pp5′G capping on repair reactions at DNA 3′ ends

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.