Structural Insights Into DNA Repair by RNase T- An Exonuclease Processing 39 End of Structured DNA in Repair Pathways

Abstract

DNA repair mechanisms are essential for preservation of genome integrity. However, it is not clear how DNA are selected and processed at broken ends by exonucleases during repair pathways. Here we show that the DnaQ-like exonuclease RNase T is critical for Escherichia coli resistance to various DNA-damaging agents and UV radiation. RNase T specifically trims the 3 end of structured DNA, including bulge, bubble, and Y-structured DNA, and it can work with Endonuclease V to restore the deaminated base in an inosine-containing heteroduplex DNA. Crystal structure analyses further reveal how RNase T recognizes the bulge DNA by inserting a phenylalanine into the bulge, and as a result the 3 end of blunt-end bulge DNA can be digested by RNase T. In contrast, the homodimeric RNase T interacts with the Y-structured DNA by a different binding mode via a single protomer so that the 3 overhang of the Y-structured DNA can be trimmed closely to the duplex region. Our data suggest that RNase T likely processes bulge and bubble DNA in the Endonuclease V-dependent DNA repair, whereas it processes Y-structured DNA in UV-induced and various other DNA repair pathways. This study thus provides mechanistic insights for RNase T and thousands of DnaQ-like exonucleases in DNA 3-end processing. Author Summary DNA repair relies on various enzymes, including exonucleases that bind and trim DNA at broken ends. However, we know little about how an exonuclease precisely selects and trims a DNA broken end in specific repair pathways. In this study, the enzyme RNase T, previously known for its involvement in processing RNA substrates, is shown to also possess DNase activity. RNase T is a DnaQ-like exonuclease and is characterized in this work as the exoDNase responsible for trimming the 3 ends of structured DNA in various DNA repair pathways. Based on the high-resolution crystal structures of RNase T-DNA complexes, an insightful working model is provided showing how RNase T processes bulge, bubble, and Y- structured DNA in various DNA repair pathways. RNase T thus represents a unique structure-specific exonuclease with multiple functions not only in processing 3 overhangs of duplex RNA during RNA maturation, but also in processing 3 ends of bubble, bulge, and Y-structured DNA during DNA repair. These findings advance our understanding of the precise function of an exonuclease in DNA repair and suggest possible roles for thousands of members of DnaQ superfamily exonucleases in DNA repair and replication.