ABSTRACTEscherichia coliRtcB exemplifies a family of GTP-dependent RNA repair/splicing enzymes that join 3′-PO4ends to 5′-OH ends via stable RtcB-(histidinyl-N)-GMP and transient RNA3′pp5′G intermediates.E. coliRtcB also transfers GMP to a DNA 3′-PO4end to form a stable “capped” product, DNA3′pp5′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 isMyxococcus xanthus, which has six RtcBs. In this study, we purified, characterized, and compared the biochemical activities of threeM. xanthusRtcB paralogs. We found thatM. xanthusRtcB1 resemblesE. coliRtcB in its ability to perform intra- and intermolecular sealing of aHORNAp substrate and capping of a DNA 3′-PO4end.M. xanthusRtcB2 can spliceHORNAp but has 5-fold-lower RNA ligase specific activity than RtcB1. In contrast,M. xanthusRtcB3 is distinctively feeble at ligating theHORNAp substrate, although it readily caps a DNA 3′-PO4end. The novelty ofM. xanthusRtcB3 is its capacity to cap DNA and RNA 5′-PO4ends 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.IMPORTANCERtcB enzymes comprise a widely distributed family of RNA 3′-PO4ligases distinguished by their formation of 3′-GMP-capped RNAppG and/or DNAppG polynucleotides. The mechanism and biochemical repertoire ofE. coliRtcB 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 threeM. xanthusparalogs, 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′-PO4ligase but adept at capping a DNA 3′-PO4end and (ii) able to cap DNA and RNA 5′-PO4ends to form GppDNA and GppRNA, respectively. The GppDNA and GppRNA capping reactions are novel nucleic acid modifications.
A high-throughput screening method for evolving a demethylase enzyme with improved and new functionalities