section 24.5
DNA Repair
557
A GTG ACTTA G
TC A C TG A A TC
Ligase
A GTG ACTTA G
TC A C TG A A TC
Deamination
Polymerase I
A GTG ACTTA G
A GTG ACTTA G
TC A U TG A A TC
TCA TGAATC
Uracil W-glycosyiase
U
A GTG ACTTA G
TCA TG AATC
' AP endonuclease
FIGURE 24-13
Scheme for repair of cytosine deamination. The same mechanism could
remove a uracil that is accidentally incorporated.
Alterations of DNA Molecules
Several agents can break phosphodiester bonds. Among
the more common are peroxides and various metal ions
(e.g., Fe2+, Cu2+). Ionizing radiation also efficiently pro-
duces strand breaks. DNases present in cells probably also
occasionally break phosphodiester bonds. Double-strand
breakage, i.e., two single-strand breaks opposite one an-
other, results from exposure to all forms of ionizing radi-
ation. Single-strand breaks can be repaired by DNA lig-
ases, although sometimes additional enzymes are required.
Double-strand breaks rarely are repaired.
Bases can be changed into different compounds by a
variety of chemical and physical agents. For instance, ion-
izing radiation can break purine and pyrimidine rings and
can cause several types of chemical substitutions, the most
common being in guanine and thymine. The best studied
altered base is the dimer formed by covalent linkage of
two adjacent pyrimidine rings; these are produced by ultra-
violet (UV) irradiation. The most prevalent of these dimers
is the
thymine dimer.
The significant effects of the pres-
ence of thymine dimers are the following:
1. The DNA helix becomes distorted as the thymines,
which are in the same strand, are pulled toward one
another (Figure 24-14).
2. As a result of the distortion, hydrogen bonding to
adenines in the opposite strand is significantly
CGATAACTAG
1
i
I
I
I
I
I
I
I
!
GCTATTGATC
FIGURE 24-14
Ultraviolet radiation damage of two adjacent thymines of DNA. Distortion
of the DNA helix caused by two thymines moving closer together when
joined in a dimer. The dimer is shown as two joined lines.
U
V
weakened, causing inhibition of advance of the
replication fork.
General Mechanisms for Repair of DNA
Repair of damaged bases was first observed and is best
understood in bacteria. It is a widespread and probably
universal phenomenon in both prokaryotes and eukary-
otes. Some systems that repair dimers repair other types
of DNA damage also.
Four major pathways for DNA repair exist that can be
subdivided into two classes: light-induced repair (photore-
activation) and light-independent repair (dark repair). The
latter can be accomplished by three distinct mechanisms:
1. Excision of the damaged nucleotides (excision repair),
2. Reconstruction of a functional DNA molecule from
undamaged fragments (recombinational repair), and
3. Disregard of the damage (SOS repair).
Photoreactivation
Photoreactivation
is a light-induced (300-600 nm) en-
zymatic cleavage of a thymine dimer to yield two thymine
monomers. It is accomplished by
photolyase,
an enzyme
that acts on dimers contained in single- and double-
stranded DNA.
The enzyme-DNA complex absorbs light and uses the
photon energy to cleave specific C-C bonds of the cy-
clobutylthymidine dimer. Photolyase is also active against
cytosine dimers and cytosinethymine dimers, which are
also formed by UV irradiation but much less frequently.
Excision Repair
Excision repair is a multistep enzymatic process. Sev-
eral mechanisms are known, but only two will be described
(Figure 24-15). All require an early incision step, in which
a nuclease recognizes the distortion produced by a thymine
dimer and makes a cut in the sugar-phosphate backbone.
Following this, a DNA polymerase mediates a strand dis-
placement step. In
E. coli,
two cleavages occur; the first
is 12 nucleotides from the 5' side of the dimer and the
second is 4-5 nucleotides from the 3' side. Each cut pro-
duces a 3'-OH and a 5'-P group. The 3'-OH group of the
first cut is recognized by pol I, which then synthesizes a
new strand, displacing the dimer-containing DNA strand.
When the second cut is reached, the displaced fragment
falls away and DNA ligase joins the 3'-OH and 5'-P groups.
In
Micrococcus luteus,
the first step is breakage of the
N-glycosidic bond of the thymine at the 5' end of the dimer
(by a dimer-specific glycosylase), leaving a free deoxyri-
bose which is removed, leaving a free 3'-OH group. As
