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

Abstract

ABSTRACT Escherichia coli RtcB exemplifies a family of GTP-dependent RNA repair/splicing enzymes that join 3′-PO 4 ends to 5′-OH ends via stable RtcB-(histidinyl- N )-GMP and transient RNA 3′ pp 5′ G intermediates. E. coli RtcB also transfers GMP to a DNA 3′-PO 4 end to form a stable “capped” product, DNA 3′ pp 5′ G. RtcB homologs are found in a multitude of bacterial proteomes, and many bacteria have genes encoding two or more RtcB paralogs; an extreme example is Myxococcus xanthus , which has six RtcBs. In this study, we purified, characterized, and compared the biochemical activities of three M. xanthus RtcB paralogs. We found that M. xanthus RtcB1 resembles E. coli RtcB in its ability to perform intra- and intermolecular sealing of a HO RNAp substrate and capping of a DNA 3′-PO 4 end. M. xanthus RtcB2 can splice HO RNAp but has 5-fold-lower RNA ligase specific activity than RtcB1. In contrast, M. xanthus RtcB3 is distinctively feeble at ligating the HO RNAp substrate, although it readily caps a DNA 3′-PO 4 end. The novelty of M. xanthus RtcB3 is its capacity to cap DNA and RNA 5′-PO 4 ends to form GppDNA and GppRNA products, respectively. As such, RtcB3 joins a growing list of enzymes (including RNA 3′-phosphate cyclase RtcA and thermophilic ATP-dependent RNA ligases) that can cap either end of a polynucleotide substrate. GppDNA formed by RtcB3 can be decapped to pDNA by the DNA repair enzyme aprataxin. IMPORTANCE RtcB enzymes comprise a widely distributed family of RNA 3′-PO 4 ligases distinguished by their formation of 3′-GMP-capped RNAppG and/or DNAppG polynucleotides. The mechanism and biochemical repertoire of E. coli RtcB are well studied, but it is unclear whether its properties apply to the many bacteria that have genes encoding multiple RtcB paralogs. A comparison of the biochemical activities of three M. xanthus paralogs, RtcB1, RtcB2, and RtcB3, shows that not all RtcBs are created equal. The standout findings concern RtcB3, which is (i) inactive as an RNA 3′-PO 4 ligase but adept at capping a DNA 3′-PO 4 end and (ii) able to cap DNA and RNA 5′-PO 4 ends to form GppDNA and GppRNA, respectively. The GppDNA and GppRNA capping reactions are novel nucleic acid modifications.