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

Escherichia coli RtcB exemplifies a family of GTP-dependent RNA repair/splicing enzymes that join 3'-PO4 ends to 5'-OH ends via stable RtcB-(histidinyl-N)-GMP and transient RNA3'pp5'G intermediates. E. coli RtcB also transfers GMP to a DNA 3'-PO4 end 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 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 HORNAp substrate and capping of a DNA 3'-PO4 end. M. xanthus RtcB2 can splice HORNAp but has 5-fold-lower RNA ligase specific activity than RtcB1. In contrast, M. xanthus RtcB3 is distinctively feeble at ligating the HORNAp substrate, although it readily caps a DNA 3'- PO4 end. The novelty of M. xanthus RtcB3 is its capacity to cap DNA and RNA 5'-PO4 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.