Ultraviolet
Damage
Endonuclease
Q Tashiro '15 and Rachel Rhee '15
Contents:
I.
Introduction
Cells
have
developed a range of repairing mechanisms in order to guard
genetic integrity against normal metabolic activities and
environmental
factors such as UV light and radiation. Such damage can form
molecular
lesions that cause structural damage to the DNA molecule and can
alter
or eliminate the cell's ability to transcribe the gene the DNA
encodes3.
Thus, the cell's DNA repair system is constantly active as it
responds
to damage in the DNA structure. DNA is most commonly cleansed of
damaged bases by
repair systems that remove and replace the altered bases including
direct reversal, nucleotide excision repair (NER), base excision
repair
(BER), and ultraviolet damage endonuclease repair (UVDE). The ultraviolet
damage
endonuclease repair system
distinguishes
itself by recognizing and subsequently nicking DNA
containing different types of damage with the single,
multifunctional
UVDE enzyme2.
The UVDE repair system
involves an endonuclease activity to catalyze the hydrolysis
of ester
linkages within nucleic acids by creating internal breaks and
a metal
ion binding site that interacts selectively and non-covalently
with
metal ions within the active site2
UV
light
induces damage in the cell resulting in two major types of
photodamage including cyclobutane pyrimidine dimers (CPDs) and
photoproducts (PPs) that will eventually lead to cytotoxic and
mutagenic damage2.
Cellular DNA have developed repair
mechanisms that are able to restore photoproducts bases back to
their
original undamaged states.
Specifically, the UVDE of Thermus
thermophilus
is thought to initiate the first step in an alternative excision
repair
pathway for the removal of UV light-induced DNA damage. The UVDE
functions as a broad specificity, ATP-independent 5’
endonuclease for initiating the repair of DNA lesions, CTDs, and UV
photoproducts3.
The enzyme performs the initial step in an
alternative excision repair pathway by cleaving the phosphodiester
backbone of substrates containing both CPDs and PPs immediately
adjacent to the 5’ phosphate of the damaged nucleotides2.
Most
proteins involved in DNA repair have very narrow specificity for
addressing only one damage. However, the UVDE is only a single
protein
that has a relatively broad substrate specificity, which makes it
an
interesting model to study DNA repair. The
closest
homolog of the UVDE is Endo IV and while they share major
structural features, the UVDE is much more versatile in its ability
to
repair DNA damage. Preliminary work has shown that the structural
flexibility from the more substantial rearrangements of UVDE upon
DNA
binding might explain its broader substrate specificity compared to
that of Endo IV2.
II.
General
Structures
The
general
structure of the UVDE protein includes a single-domain TIM
barrel (lacking the α8 helix) of the prototypical TIM-barrel
fold (Paspaleva). The TIM barrel is a conserved protein fold
consisting
of 8
α-helices and 8
parallel β- strands
and
is considered one of
the most common protein folds4.
The UVDE is classified as a member of the TIM-barrel family of
divalent metal-dependent enzymes due to the three anomalously
scattering metal
ions
,
located closely to the C terminus and due to the close proximity
of
the protein’s N and C termini2.
The
UVDE structure reveals a novel use of the TIM barrel fold for
binding DNA for damage recognition and catalysis. The enzyme must
bind
and scan normal DNA via electrostatic complementarity and hydrogen
bonding to the DNA phosphate backbone from β
barrel loops and α-helical
dipoles
identically positioned by the α8β8
framework1.
DNA damage detection proceeds by insertion of side chains from
minor
groove recognition loops to provide DNA backbone compression and
flipping of the target AP site and its opposing nucleotide out of
the
helix1.
III.
Base
Flipping
Two
residues
that stick out (Gln
104
and Tyr 105)
are
called
the probing finger and are responsible for probing the DNA for
damage. These residues function in close proximity of the metal
coordination site and
present
the scissile phosphodiester bond at the 5` side
of the lesion
to the active site2.
A small rearrangement
of two residues
at its C terminus, Lys-273
and Glu-274,
create a pocket to fit both the
damaged base and the flipped-out base opposing this damage
.
IV.
DNA
Binding
DNA Binding site
A
distinct crescent-shaped groove
formed by the C-terminal end of the TIM
barrel forms the enzyme active site. The wide groove
(29 Å)
which contains
extensive positive
charges on both
sides and three active site ions
impart
an overall net positive electrostatic potential throughout the
groove
that
complements the
negatively charged
DNA phosphate backbone. This pocket allows the
UVDE to comfortably harbor the kinked duplex.
At
the
bottom of the DNA binding groove there is a loop, formed from the
previously mentioned probing fingers, Gln
104
and Tyr 105,
that also serve to
stabilize the kink in the DNA duplex at the position
of damage. This overall structure demonstrates how the TIM barrel
fold
can be optimized for DNA binding, therefore is a fold that recurs in
other proteins that bind to, modify, or repair nucleic acid
substrates.
A homolog of UVDE, Endo IV, is shown bound to a damaged DNA. The
binding to dsDNA
is
mediated via five DNA recognition loops (R
loops)
that
emanate from the
positively charged C-terminal end of the central
β
barrel. The position of the DNA against the protein shows how it
fits
along
the pocket of the DNA.
Significant side chain rearrangements of β2–α2
residues
Asn-35,
Gln-36,
Arg-37,
Gln-38,
and
Trp-39 pack
against
the hydrophobic carbon atoms of the minor groove atoms to
stabilize the extrahelical
nucleotides and the 90°
bend in the DNA
2. The
enzyme facilitates
contacts
via
electrostatic
complementarity
and hydrogen bonding to contacts anchor the flipped-out
abasic nucleotide to the enzyme active site and position its 5`
phosphate such that it is intimately ligated to all three ions1.
The rigid TIM barrel framework
positions these five R loops to provide extensive charge, chemical,
and
shape complementarity for the
entire interface
2.
V.
Role
of Metal Ions in the Active Site
Metal
ions
that are present within close proximity of the protein’s
N and C termini, classifies UVDE as a member of the TIM-barrel
family
of divalent metal-dependent enzymes2.
The
first
metal ion
is
octahedrally
coordinated by the side chain
interactions between Glu-175,
Glu-269, His-231,
and Asp-200
residues
and two oxygen atoms from a phosphate ion.
The second metal ion
forms
a distorted
bipyramidal coordination by His-101,
His-143,
and Glu-175,
along with two oxygen atoms from the
phosphate ion.
The
third metal ion
has
an irregular
four-fold coordination by His-244,
His-203,
one water molecule and one oxygen atom from the phosphate
ion.
Studies
show that the UVDE active site metal ions are likely to be directly
involved in phosphodiester cleavage, also observed in other DNA
repair
enzymes such as Endo IV. These positively charged metal ions can act
as
a Lewis acid to stabilize a water-derived hydroxide attacking the
phosphodiester backbone of DNA which will counteract the developing
negative charge on the DNA during cleavage reaction2. T.
thermophilus
UVDE
(Tth-UVDE)
shows high incision on substrates containing CPD or 6-4PP
however incision of the abasic sites is much less efficient2.
For
the
UVDE to incise DNA containing CPD and 6-4PP UV lesions, Mn2+
is required for the UVDE catalytic activity. Ongoing studies have
shown
that the specific divalent metals present in the active site vary
depending on other UVDE homologs. For instance, the S.
pompe
protein
can
only utilize Mg2+
as a catalytic cofactor2
however, T.
thermophilus
UVDE
is
not able to stimulate enzymatic activity with Mg2+.
3...2...1!
VI.
References
1.
Hosfield DJ, Guan Y, Haas BJ, Cunningham RP, Tainer JA. Structure of
the DNA repair enzyme endonuclease IV and its DNA complex:
Double-nucleotide flipping at abasic sites and three-metal-ion
catalysis. Cell.
1999;98(3):397-408.
2. Paspaleva K, Thomassen E, Pannu NS, Iwai S, Moolenaar GF, Goosen N,
Abrahams JP. Crystal structure of the DNA repair enzyme ultraviolet
damage endonuclease. 2007;15:1316-1324.
3. Siede W, Doetsch PW, Wah Kow Y, Doetsc PW. DNA
damage
recognition.
1st
ed.
New York: Taylor & Francis Group; 2005.http://books.google.com/books?hl=en&lr=&id=JB6OKFsBjNQC&oi=fnd&pg=PR3&dq=UVDE+protein&ots=li1I1Xqj0B&sig=8jdsETEJhNhoQTvle
7aQrM7AewI#v=onepage&q=UVDE%20protein&f=false.
4.
Wierenga
RK. The TIM-barrel fold: A versatile framework for efficient
enzymes. FEBS
Lett.
2001;492(3):193-198.
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