Dual mechanisms whereby a broken RNA end assists the catalysis of its repair by T4 RNA ligase 2

T4 RNA ligase 2 ( Rnl2) efficiently seals 3'-OH/5'-PO4 RNA nicks via three nucleotidyl transfer steps. Here we show that the terminal 3'-OH at the nick accelerates the second step of the ligase pathway (adenylylation of the 5'-PO4 strand) by a factor of 1000, even though the 3'-OH is not chemically transformed during the reaction. Also, the terminal 2'-OH at the nick accelerates the third step ( attack of the 3'-OH on the 5'-adenylated strand to form a phosphodiester) by a factor of 25-35, even though the 2'-OH is not chemically reactive. His-37 of Rnl2 is uniquely required for step 3, providing a similar to 10(2) rate acceleration. Biochemical epistasis experiments show that His-37 and the RNA 2'-OH act independently. We conclude that the broken RNA end promotes catalysis of its own repair by Rnl2 via two mechanisms, one of which ( enhancement of step 3 by the 2'-OH) is specific to RNA ligation. Substrate-assisted catalysis provides a potential biochemical checkpoint during nucleic acid repair.