Structure-guided mutational analysis of the OB, HhH, and BRCT domains of Escherichia coli DNA ligase.
NAD(+)-dependent DNA ligases (LigAs) are ubiquitous in bacteria and essential for growth. LigA enzymes have a modular structure in which a central catalytic core composed of nucleotidyltransferase and oligonucleotide-binding (OB) domains is linked via a tetracysteine zinc finger to distal helix-hairpin-helix (HhH) and BRCT (BRCA1-like C-terminal) domains. The OB and HhH domains contribute prominently to the protein clamp formed by LigA around nicked duplex DNA. Here we conducted a structure-function analysis of the OB and HhH domains of Escherichia coli LigA by alanine scanning and conservative substitutions, entailing 43 mutations at 22 amino acids. We thereby identified essential functional groups in the OB domain that engage the DNA phosphodiester backbone flanking the nick (Arg(333)); penetrate the minor grove and distort the nick (Val(383) and Ile(384)); or stabilize the OB fold (Arg(379)). The essential constituents of the HhH domain include: four glycines (Gly(455), Gly(489), Gly(521), Gly(553)), which bind the phosphate backbone across the minor groove at the outer margins of the LigA-DNA interface; Arg(487), which penetrates the minor groove at the outer margin on the 3 (R)-OH side of the nick; and Arg(446), which promotes protein clamp formation via contacts to the nucleotidyltransferase domain. We find that the BRCT domain is required in its entirety for effective nick sealing and AMP-dependent supercoil relaxation.
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Li Kai
LK
Wang
Pravin A
PA
Nair
Stewart
S
Shuman
D000595:Amino Acid Sequence; D001483:Base Sequence; D018360:Crystallography, X-Ray; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000737:chemistry* / Q000235:genetics*; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000201:enzymology*; D017353:Gene Deletion; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009154:Mutation; D020125:Mutation, Missense; D009690:Nucleic Acid Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D011088:DNA Ligases; D000072481:DNA Ligase ATP