Rewriting the rules for end joining via enzymatic splicing of DNA 3′-PO4 and 5′-OH ends

There are many biological contexts in which DNA damage generates dirty breaks with 3'-PO4 (or cyclic-PO4) 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'-PO4 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'-PO4 and 5'-OH termini, with implications for DNA repair and DNA rearrangements.