DNA ligase D (LigD) performs end remodeling and end sealing reactions during nonhomologous end joining in bacteria. Pseudomonas aeruginosa LigD consists of a central ATP-dependent ligase domain fused to a C-terminal polymerase domain and an N-terminal phosphoesterase (PE) module. The PE domain catalyzes manganese-dependent phosphodiesterase and phosphomonoesterase reactions at the 3 end of the primer strand of a primer-template. The phosphodiesterase cleaves a 3-terminal diribonucleotide to

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... d a primer strand with a ribonucleoside 3-PO 4 terminus. The phosphomonoesterase converts a terminal ribonucleoside 3-PO 4 or deoxyribonucleoside 3-PO 4 of a primer-template to a 3-OH. Here we report that the phosphodiesterase and phosphomonoesterase activities are both dependent on the presence and length of the 5 single-strand tail of the primer-template substrate. Although the phosphodiesterase activity is strictly dependent on the 2-OH of the penultimate ribose, it is indifferent to a 2-OH versus a 2-H on the terminal nucleoside. Incision at the ribonucleotide linkage is suppressed when the 2-OH is moved by 1 nucleotide in the 5 direction, suggesting that LigD is an exoribonuclease that cleaves the 3-terminal phosphodiester. We report the effects of conservative amino acid substitutions at residues: (i) His 42 , His 48 , Asp 50 , Arg 52 , His 84 , and Tyr 88 , which are essential for both the ribonuclease and 3-phosphatase activities; (ii) Arg 14 , Asp 15 , Glu 21 , and Glu 82 , which are critical for 3-phosphatase activity but not 3-ribonucleoside removal; and (iii) at Lys 66 and Arg 76 , which participate selectively in the 3-ribonuclease reaction. The results suggest roles for individual functional groups in metal binding and/or phosphoesterase chemistry.