Substrate Specificity and Structure-Function Analysis of the 3′-Phosphoesterase Component of the Bacterial NHEJ Protein, DNA Ligase D

Hui Zhu,Stewart Shuman

doi:10.1074/jbc.m600055200

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 a2'-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, Arg52, His84, and Tyr88, which are essential for both the ribonuclease and 3'-phosphatase activities; (ii) Arg14, 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.

Highlights

The ribonucleotide resection activity is the result of two component steps: (i) the 3Ј-terminal nucleoside is first removed to yield a primer strand with a ribonucleoside 3Ј-PO4 terminus; (ii) the 3Ј-PO4 is hydrolyzed to a 3Ј-OH
By varying the position of the single ribonucleotide, we demonstrate that ligase D (LigD) acts at the terminal phosphodiester
3Ј-Ribonuclease and 3Ј-Phosphatase Activities at a Primer Terminus Depend on the 5Ј Single-strand Tail of a Template DNA Strand— Pseudomonas LigD prefers to remodel the recessed 3Ј ends of the primer strand of a duplex primer-template substrate compared with a single-strand of identical composition (6, 7)

Summary

Introduction

The PE domain catalyzes manganese-dependent hydrolysis of the 3Ј-PO4 of an all-DNA primer-template substrate (7), signifying that the 2Ј-OH of the terminal nucleoside is not required for the phosphomonoesterase reaction. The 3Ј-phosphatase reaction mixtures (80 ␮l) containing 50 mM Tris-HCl (pH 7.5), 5 mM dithiothreitol, 0.5 mM MnCl2, 50 nM 5Ј 32P-labeled D9R1-p primer-template (see Fig. 3C), and 5 ␮M LigD PE domain were incubated at 37 °C.

Results

Conclusion