Substrate specificity and structure-function analysis of the 3′-phosphoesterase component of the bacterial NHEJ protein, DNA ligase D

Abstract

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 yield a primer strand with a ribonucleoside 3′-PO4 terminus. The phosphomonoesterase converts a terminal ribonucleoside 3′-PO4 or deoxyribonucleoside 3′-PO4 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) His42, His48, Asp50, Arg 52, His84, and Tyr88, which are essential for both the ribonuclease and 3′-phosphatase activities; (ii) Arg 14, Asp15, Glu21, and Glu82, which are critical for 3′-phosphatase activity but not 3′-ribonucleoside removal; and (iii) at Lys66 and Arg76, which participate selectively in the 3′-ribonuclease reaction. The results suggest roles for individual functional groups in metal binding and/or phosphoesterase chemistry. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.