Rei Mitsuyama '15 and Holden Richards '15
Removal and replacement of the altered bases is the most common
method cells employ to rid their DNA of damaged bases. Base excision
reapir (BER) and nucleotide excision repair (NER) are the two
principle repair pathways. The first enzyme in the BER process is
glycosylase. Uracil DNA glycosylase (UDG) is one of 11 different human
DNA glycosylases that have been identified5.
Though functionally only present in RNA, uracil is
occasionally misincorporated into DNA. This most commonly is a
result of cytosine deamination. UDG scans the minor groove for
damage and once it finds the error, it hydrolyzes the glycosidic
bond to release the base from the sugar-phosphate backbone.
Remarkabley, it is able to all of this without the aid of other
cofactors. It then leaves the AP site (apyrimidinic site) open for
an AP endonuclease to cut the backbone at the 5' end of the AP site.
This marks the initiation of this BER5.
II. General Structure
UDG is a
composed of four parallel beta sheets
surrounded by eight alpha helices in
the following order: α1, α2, α3, β1, α4, α5, β2, α6, β3, α7, β4, α81.
The active site is comprised of
. The Leu272 loop is involved in base
stacking interactions with the DNA. Along with the 4-Pro
loop and the Gly-Ser
loop , it compresses backbone phosphates, bending the DNA.
comprised of residues Tyr275
and Arg276 are also part of the
Leu272 Loop. The uracil specificity region
is responsible for recognition of the correct flipped-out base while
the water-activating loop is vital in
III. DNA Binding
Uracil-DNA glycosylase is lesion-specific (as the name
in DNA. For DNA to be bound by UDG, it must be in the B form.
This makes sense because UDG acts on newly synthesized DNA
containing a misincorporated base (uracil). The exact mechanism by
which UDG scans the DNA remains unknown. However, it seems to
involve the serine residues of
- the Leu272 loop, 4-Pro
loop, and Gly-Ser
loop. The damaged base is flipped out, sitting in the
specificity pocket of the glycosylase, which makes it project away
from the double helix. These Ser residues form
with the phosphates both 5' and 3' to the uracil. This
interaction compresses the DNA backbone, bringing the UDG into
closer proximity of the uracil, aiding its recognition. Initial
detection of uracil misincorporation by backbone compression is
coupled to the minor groove reading head residues Tyr275
and Arg276, which make
hydrogen bonds to the uracil
N3. The closest interaction between the enzyme and the
uracil involves two
with the Asn204 of the uracil specificity region3. This hydrogen
bonding is thought to be the strongest and most important in
recognizing the uracil lesion. It is believed that these hydrogen bonds
allow UDG to distinguish between cytosine and uracil4. The total enzyme-DNA interface is
small and most of the interactions involve this flipped-out base.
IV. Enzymatic Activity
(~27 angstroms long between Ser247 and Val164
before binding) has a conical shape that can accommodate a DNA
double helix at its wide end, which is about 21 angstroms (before
binding) between His212 and Leu272,
but not at its narrow end, which is only 10 angstroms between Pro150 and Pro165. This suggests that
the groove floor cannot directly bind dsDNA without significant
conformational change. However, no conformational change has been
observed, suggesting that DNA only binds at the wide end of the
enzyme's active site4.
In order for the misincoprporated
uracil to be situated in the active site, it must be
into an extrahelical conformation. It was hypothesized that this was
caused by a "push" from residue
emanating from the Leu272 loop,
between beta sheet 4 and alpha helix 8, as it penetrates the DNA through the minor groove and
replaces the flipped-out uracil2. However, the crystal structures
have confirmed that this "push" is not essential for
base-flipping, as an extrahelical uracil has been observed in
crystal structures without the presence of Leu272 within the DNA
double helix. Nevertheless, the "push" has been linked to
enhanced efficiency of excision3. This can be seen as a
consequence of two major actions of Leu272.
The insertion of Leu 272 into the double helix stabilizes the
extrahelical-uracil conformation by forming new
interactions, allowing this conformation to be favorable. It also
aids the formation of the recognition pocket as the insertion
brings conserved residue His268
close enough to
with the uracil O23.
The ultimate reaction catalyzed by
UDG cleavage of the glycosidic bond between the uracil base
and its corresponding deoxyribose sugar, releasing uracil
from the DNA backbone.
seems to be chiefly responsible, through its interaction with water.
Once UDG binds DNA, the His148
rotates into a conformation that allows it to deprotonate
molecule. This activates the water molecule by giving it a negative
charge and thereby making it a strong nucleophile. Then, by
nucleophilic attack, the water molecule breaks the
glycosydic bond, freeing the uracil base. To aid in this process,
Asp145 hydrogen bonds
with N3 of the base,
polarizing the glycosidic bond, which makes it
more susceptible to this attack1.
(1) Mol, Clifford D., Andrew S. Arvai,
Geir Slupphaug, Bodil Kavli, Ingrun Alseth, Hans Krokan,
and John A. Tainer. 1989. Crystal structure and mutational
analysis of human uracil-DNA glycosylase: Structural basis
for specificity and catalysis. Cell Press
(2) Mol, Clifford D., Andrew S. Arvai,
Russell J. Sanderson, Geir Slupphaug, Bodil Kavil, Dale W.
Mosbaugh, and John A. Tainer. 1995. Crystal Structure of
Human Uracil-DNA Glycosylase in Complex with a Protein
Inhibitor: Protein Mimicry of DNA. Cell Press
(3) Parikh, Sudip S., Clifford D. Mol, Geir
Slupphaug, Sangeeta Bharati, Hans E. Krokan, and John A.
Tainer. 1998. Base excision repair initiation revealed by
crystal structures and binding kinetics of human uracil-DNA
glycosylase of DNA. The EMBO Journal 17:5214-5226.
(4) Pearl, Laurence H. 1987. Structure
and function in the uracil-DNA glycosylase superfamily.
(5) Watson, James D. Alexander Gann,
Tania A. Baker, Michael Levine, Stephen P. Bell, and
Richard Losick. 2014. Molecular Biology of the Gene. Cold
Spring Harbor Laboratory Press 7:326-329.
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