Abstract
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.
Highlights
The essential constituents of the HhH domain include: four glycines (Gly455, Gly489, Gly521, Gly553), which bind the phosphate backbone across the minor groove at the outer margins of the LigA-DNA interface; Arg487, which penetrates the minor groove at the outer margin on the 3-OH side of the nick; and Arg446, which promotes protein clamp formation via contacts to the nucleotidyltransferase domain
We find that the BRCA1-like C-terminal (BRCT) domain is required in its entirety for effective nick sealing and AMP-dependent supercoil relaxation
All LigA enzymes have a modular structure in which a central ligase catalytic core, composed of a nucleotidyltransferase (NTase) domain and an oligonucleotide-binding (OB) domain, is flanked by an N-terminal “Ia” domain and three C-terminal modules: a tetracysteine zinc finger domain, a helix-hairpin-helix (HhH) domain, and a BRCA1-like C-terminal (BRCT) domain (4 – 8)
Summary
All LigA enzymes have a modular structure in which a central ligase catalytic core, composed of a nucleotidyltransferase (NTase) domain (amino acids 70 –316 in E. coli LigA) and an oligonucleotide-binding (OB) domain (amino acids 317– 404), is flanked by an N-terminal “Ia” domain (amino acids 1– 69) and three C-terminal modules: a tetracysteine zinc finger domain (amino acids 405– 432), a helix-hairpin-helix (HhH) domain (amino acids 433–586), and a BRCA1-like C-terminal (BRCT) domain (amino acids 587– 671) (4 – 8). In E. coli of the 5Ј-PO4 nick strand, with van der Waals contacts from LigA, these polar side chains (Asp452, Arg487, Glu519, or Asp551) Ile384 C␥1 to the deoxyribose O4 (3.06 Å) and C5 (3.94 Å) and straddle or penetrate into the DNA minor groove.
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