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!

1 comment:

  1. T7 DNA Ligase catalyzes the formation of a phosphodiester bond between a 5′ phosphate and a 3′ hydroxyl termini in duplex DNA. The enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA.

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