DNA Ligases

Ligation → a process that involves the formation of 4 phosphodiester (PD) bonds -- 2 at each end of the molecule between neighbouring 3'-OH and 5'-PO4

DNA Ligases

• Exquisite specificity for nicked DNA
• In E. coli:
• homologous and monomeric
• polypeptide chain with molecular weight of 75 KDa
• require NAD+ as a co-factor
• In T4 phages, mammalian and plant cells:
• polypeptide chain with molecular weight of 68 KDa
• requires ATP as a co-factor and energy source

Reaction

1. Formation of an adenylate-enzyme complex involving NAD+/ATP
2. Release of nicotinaminde mononucleotide (NMN, in case of NAD+) or Pyrophosphate (Pi, in case of ATP) while an AMP residue binds covalently to the Ɛ-amino group of lysine in the enzyme via a phosphoric acid amide bond or "phosphoamidite bond". This occurs simultaneously with (1)
3. 5'-PO4 of DNA subsequently activated by transfer of adenylate residue
4. Nucleophilic substitution of 3'-OH at the activated 5'-PO4 residue
5. Release of AMP

Substrates

• Physiological → breakage point at a PD bond between neighboring 3'-OH and 5'-PO4 ends held together by an intact complementary strand (E. coli, T4)
• Open and staggered PD bonds formed through reassociation of the protruding termini of different DNA molecules held together by basepairing between 2, 3, 4 protruding nucleotides (again E. coli, T4)
• T4 DNA ligase → blunt end ligation of double stranded DNA (dsDNA) molecules
• T4 DNA ligase → nicks in the RNA chains of dsRNA-DNA hybrids (i.e annealing of RNA termini with DNA strands)

** single stranded (ss) polynucleotides are ligated by RNA ligase (eg. in T4)

Architecture

• 2 or more domains uniquely arranged
• 5 classes of motifs recently detected:
1. NBD → nucleotide binding domain / AD → adenylation domain
2. OB fold → oligomer binding fold
3. ZFM → zinc finger motif
4. HhH → helix-hairpin-helix motif
5. BRCT → BRCA1 C-terminus domain

Adenylation domain

• Main enzyme has 2 domains:
• Larger N-terminal → domain 1
• In T7 (ATP dependent ligase):
• 3 main anti-parallel β-sheets flanked by 6 α-helices
• contains ATP-binding site situated in a pocket beneath the β-sheet
• has intrinsic adenylation activity
• In other NAD+ dependent ligases:
• contains a sub-domain that is mainly α-helical
• Smaller C-terminal → domain 2

OB fold domain

• Derivative of the Greek key motif
• Conserved, connects to Domain 1
• In T7:
• binds dsDNA
• dramatically enhances adenylation activity of Domain 1 by undergoing conformational change → 13A movement of Domain 2 towards Domain 1

Zinc Finger Motif

• 4 cystine conserved in C-terminal region of NAD+ dependent ligases which tetrahedrally ligand a zinc ion
• acts as a DNA recognition module that recognizes specific DNA sequences
• also plays a structural role in proteins by offering support for subdomain 3b and domain 4

HhH motif

• 4 copies of conserved HhH observed in NAD+ dependent ligases
• consists of 2 helices + 2 type II β-turns
• implicated in non-sequence specific binding
• in Tfi, 4 HhH form a compact structure (subdomain 2b)
• hairpins located in a linear chain at the bottom of a subdomain
• rich in positively charged residues
• forms 1 of 2 DNA binding sequences (eg. In Tif)

BCRT domain

• found in both NAD+ as well as ATP dependent ligases
• in Tfi, it consists of a 4-stranded parallel β-sheets flanked by 3 α-helices
• may act as a signal tranducer that transmits signals from DNA damage detectors to other components of the DNA repair machinery via specific protein-protein interactions
• mobile when in open conformation and restricted mobility in closed conformation
• acts as a gate which regulates DNA binding and release

Nick Recognition

• In T7 ligase:
• dsDNA binds predominantly in the positively charged interdomain cleft lined by conserved motifs and residues with strong positive potential
• domain 2 acts as a movable thumb that can open/close in response to ligand association/dissociation
• domain 1 has a higher affinity for DNA than domain 2
• however, both domains 1 + 2 are the minimal unit required for all the ATP dependent ligases and for NAD+ dependent bacterial DNA ligases
• Motif I → contains active site Lysine (K34, K116) which forms the covalent AMP adduct
• Motif III → contains a glutamate residue (E93, E114) which forms H-bonds with ribose of ATP
• Motif IIIa → contains Tyrosine (Y149, Y221) which is stacked against the adenine ring and the essential Lysine in motif V
• Motif V → contains the essential Lysine
• Larger ligases have additional domains* that enhance certain properties of the enzyme:
• DNA binding
• nick recognition
• targeting of enzyme to sites of DNA damage
• replication and recombination

*These additional domains are not directly involved in catalysis!