214 MaundersIn vitro a variety of activities have been detected. These include the
joining of hydrogen-bonded cohesive DNA termini (with either 5' or
3' projecting single-stranded regions), the joining of blunt-ended
double-stranded DNA molecules, the resealing of single-stranded
"nicks" in DNA duplexes, and the interconversion of ATP and AMP
by the exchange of pyrophosphate groups.
The two most intensively studied and widely used DNA ligases
are that from the E. coli bacterium (EC 6.5.1.2) (11,12) and that
occurring in E. coli that has been infected with bacteriophage T4 (EC
6.5.1.1) (13). This section of the chapter will concentrate largely on
these two enzymes.
E. coli DNA ligase has been purified from several strains that over-
produce this enzyme (11,12,14). The T4 enzyme is prepared from E.
coli that has been infected with either wild-type or replication-defi-
cient T4 phage (15). Both the E. coli and T4 DNA ligase genes have
been cloned into recombinant ~, vectors.
2.1. The Enzyme
E. coli DNA ligase is a monomeric enzyme of asymmetric shape
(S20,w = 3.9S) with a mol mass of 77,000 Da as determined by sedimen-
tation equilibrium (14), or 74,000 + 3000 Da by polyacrylamide gel
electrophoresis (PAGE) (16). T4 DNA ligase is also an elongated
monomer (S20,w = 3.5) of mol mass 68,000 + 6800 Da determined by
gel filtration (18) or 63,000 + 3200 Da by PAGE (17). T4 DNA ligase
exhibits five species on isoelectric focusing. The adenylated form of
the enzyme displays a major band with a pI of 6.0, and the non-
adenylated form has a value of 6.2 for this band (15).
2.2. Enzyme Reaction
2.2.1. Nucleic Acid Substrate
DNA ligase requires as a substrate two DNA termini, the 5' termi-
nus, carrying a phosphate group, and the 3' terminus, a hydroxyl group.
These termini must reside on a double-stranded molecule (DNA:DNA
or DNA:RNA). Both strands of the duplex may terminate, in the form
of a staggered end or a blunt end, and the ligase then requires a second
similar double-stranded terminus and proceeds to join the two in an inter-
molecular reaction. Alternatively, the two termini may be provided by a
nick in just one strand of a duplex, which the enzyme will then seal.