Substrate specificity and structure-function analysis of the 3'-phosphoesterase component of the bacterial NHEJ protein, DNA ligase D.
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.
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Hui
H
Zhu
Stewart
S
Shuman
D001483:Base Sequence; D011088:DNA Ligases / Q000737:chemistry*; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D017931:DNA Primers; D010727:Phosphoric Diester Hydrolases; D011088:DNA Ligases; C493173:DNA ligase D, Mycobacterium tuberculosis