Cross-over of RNA 3′-phosphate ligase into the DNA world

Abstract

The central dogma of DNA enzymology has long held that for two pieces of DNA to be ligated, the respective DNA pieces must bear a hydroxyl on the 3′-end and a phosphate on the 5′-end. All of the other types of DNA ends are considered incorrect or "dirty," as they cannot be joined by classic DNA ligases. However, intrinsic and extrinsic DNA damage often results in broken DNA with dirty ends. Thus, there are a variety of DNA repair enzymes that function to "clean up" or "heal" such dirty ends to promote the restoration of a continuous DNA phosphodiester backbone. Until now, DNA breaks with phosphate at the 3′-end and hydroxyl at the 5′-end also fell into the category of dirty ends. The study in PNAS by Das et al.(1) demonstrates that a recently identified RNAprocessing enzyme, RtcB, can directly ligate a DNA 3′-phosphate end with a DNA 5′-OH end (Fig. 1). Conservation of RtcBRtcB was known to splice tRNA exons with 2′,3′-cyclic-phosphate and 5′-OH ends (2-8) and possibly plays a role in unconventional splicing of mRNA exons with 2′,3′-cyclicphosphate and 5′-OH ends (4). Phylogenetic analysis indicates that RtcB enzymes are widely prevalent in bacteria, archaea, and metazoa (2, 4-6), although Escherichia coli RtcB is not expressed under normal conditions (3, 9). RtcB is absent in fungi and plants, where end-healing enzymes and a classic RNA ligase are used to splice tRNAs (6, 7). Nevertheless, the high degree of conservation of RtcB in bacteria, archaea, and metazoa, as well as the expression profile of human RtcB (variously named C22orf28, HSPC117, or FAAP), in cancer cell lines (http://discover.nci.nih.gov/cellminer/) are consistent with the enzyme playing an essential role in vertebrate cells. Accordingly, knockdown of RtcB/HSPC117 in cloned mouse embryos leads to placental abnormality, and the mortality rate is 2.5-fold higher than the control group (2).Biochemical Mechanisms of Classical DNA and RNA Ligases vs. RtcB 1A) and E. coli RtcB (Fig. 1B). The classic DNA and RNA ligases catalyze a three-step mechanism of adenylate transfer. First, the catalytic lysine residue is charged by adenylylation in an ATP-dependent step. Second, the adenylate is transferred to the 5′-phosphate end of DNA or RNA, generating an activated 5′-end with a pyrophosphate linkage. Third, the 3′-OH carries out an inline attack on the 5′-phosphoanhydride, generating the 3′-5′ phosphodiester bond.Through a series of elegant biochemical experiments, Shuman and coworkers have elucidated the reaction pathway of RtcB (1, 5, 10, 11). They have now shown RtcB can ligate not only RNA but also DNA ends in a scheme entailing activation of a 3′-phosphate end, the reverse of the strategy used by classic DNA and RNA ligases (cf. Fig. 1 A and B). RtcB carries out the joining of DNA or RNA via a three-or four-step mechanism, depending whether the polynucleotide substrate contains a 3′-phosphate or a ribonucleotide 2′-3′-cyclophosphate end. First, RtcB reacts with GTP and becomes charged by guanylylation at a conserved histidine residue located at the...

The neurodegenerative diseases ALS and SMA are linked at the molecular level via the ASC-1 complex

“…Finally, this machinery contains multiple complexes that function in DDR. These include the tRNA ligase complex (83,84), NELF (85), P-TEFb (86) and TFIIF (87), and importantly, these complexes dissociate from the RNAP II/U1 snRNP machinery in the different ALS protein KOs. Thus, a possible cause of ALS/SMA pathogenesis is disruption of DDR due to loss of interactions between these DDR complexes/proteins from the RNAP II/U1 snRNP machinery.…”

Recognition and repair of chemically heterogeneous structures at DNA ends

“…This raises important questions: (1) How does ribose chemistry influence DNA transactions carried out by known repair factors, and (2) what additional factors operate in the RNA‐DNA realm to preserve integrity of the genome? Further, the identification of an RNA ligase that can function to join 3′‐phosphorylated or 2′‐3′ cyclic phosphate ends to 5′‐OH termini in the context of DNA [Das et al, ] suggests that our de facto views of DNA end joining may require revision in the context of RNA‐DNA [Huang and Pommier, ]. Given the abundance of RNA incorporation in the genome it seems likely that other known and yet to be discovered factors may play roles in detection and resolution of RNA‐derived lesions and act in RNA‐DNA damage responses (RDDRs) similar to that recently documented for APTX [Tumbale et al, ].…”