Rae Joseph Tinao

Beatriz Aira C. Jacob

TEAM JOOB
Chem 145 WFX
DNA Polymerase Beta

The protein DNA polymerase beta (Pol ), with ID number P06746, is an enzyme that plays a key role in
base-excision repair, conducts gap-filling DNA synthesis in a stepwise distributive fashion rather than in a
processive fashion as for other DNA polymerases[1]

. This enzyme can be found in the cytoplasm in normal

conditions, and translocates to the nucleus following DNA damage through either alkylation or oxidation[2]

. Pol
contains alpha helices and beta sheets as secondary structures. Supersecondary structures like
helix-turn-helix and helix-hairpin-helix motifs can be found[3]

. Pol contains four subdomains: 8kD, thumb,

[4]
[5]
palm, and fingers.
It binds with DNA.(

Fig. 1
).
+
The active site is K72 (
Fig. 2
). The (NH
3)
end of Lysine attacks the aldehyde group of the AP site,
[6]
creating a Schiff base intermediate. The intermediate is now prone to attack to the more nucleophilic
nucleotide. Provided that it is the correct base pair, The free nucleotide will attack the Schiff base through the
carbonyl carbon, effectively replacing the NH
end of Lysine with its backbone[7]

3
When this protein is mutated, it will produce different molecular effects and conformational changes.
First mutation given is D190A (
Fig. 3
). Structure-wise, it would not trigger any conformational change. It is only
found in a random coil and does not participate in a secondary structure. However, function-wise, it would have
a dramatic change. The negatively-charged O on the residue is ionically binded to a magnesium ion, which then
forms another ionic bond to the negatively-charged phosphate backbone. This phenomenon helps the enzyme
bind to the DNA. If the D was replaced with A, the ionic bond would be lost and there would be significantly less
force to bind the enzyme to the DNA.
Second is R137Q (
Fig. 4
), where Arginine is located in an alpha helix. This mutation may cause the helix
to unfold. Glutamine will tend to fold in such a way that it will be exposed in a polar environment. Because of this
unfolding, the polymerase will have lower activity. This also affects the interaction with proliferating cell nuclear
antigen (PCNA). It defects the DNA repair capacity of Pol in reconstitution assays, as well as in cellular
extracts. This indicates that polymorphisms in base excision repair genes may contribute to the onset and
development of cancer[8]

.
Third mutation is Y265C (
Fig. 5
). The OH group of the Tyrosine creates an H-bond with the OH group of
T176. Replacing Y with C removes the H-bonding. However, structural implications of this H-bond is unknown
since T176 is already participating in a stable Beta sheet. Also, the mutated cysteine may form a disulfide bridge
with another cysteine separated by tyrosine, a bulky amino acid. This may cause the alpha helix to stabilize.
After isolating the nucleus or cytoplasm from any cell, we can then remove the DNA through
centrifugation. Taking advantage of its positively charged nature, we can use either Free-flow Electrophoresis or
Ion-Exchange Chromatography to isolate the protein of interest. Since the solution would still contain the buffer
along with the protein we can use ultra-filtration to purify Pol . However, since the metal bindings would be
lost, conformational change is expected.
The protein interacts with DNA through scanning the DNA. Arginine can be found on different parts of
surface of the protein, which interacts with the phosphate backbones of the DNA. For the DNAs part, through
hydrogen bonding, it can determine the identity of the amino acid it touched, and therefore it can read the whole
amino acid sequence. If one of its amino acid is different, there will be conformational changes within the
enzyme, and so the DNA may not bind to it. This is how enzyme specificity is achieved.

References:
[1] Bennett R.A., Wilson D.M. III, Wong D., Demple B. Interaction of human apurinic endonuclease and
DNA polymerase beta in the base excision repair pathway.
Proc. Natl. Acad. Sci.

U.S.A.,

1997
, 94:7166-7169
Watson N. Isolations and Use of Mammalian Cell Nuclei.
Sigma-Aldrich Corporation
U.S.A.
[2]
Parsons J.L.
,
Tait P.S.
,
Finch D.
, et.al. Ubiquitin ligase ARF-BP1/Mule modulates base excision repair.
EMBO
J

.,
2009
, 28:3207-3215
[3] Doherty, A.J., Serpell, L.C., Ponting, C.P. The helix-hairpin-helix DNA-binding motif: a structural basis
for non-sequence-specific recognition of DNA.
Nucleic Acids Res.
,
1996
,
24(13),
pp 2488-2497
[4] Murphy, D.L. Jaeger, J. Sweasy, J.B. A Triad Interaction in the Fingers Subdomain of DNA Polymerase
Beta Controls Polymerase Activity.
J. Am. Chem. Soc.,
2011
,
133(16),
pp 6279-6287
[5] P06746 (DPOLB_HUMAN).
UniProtKB.
[6] DeMott M., Beyret E., Wong D., Bales B., Hwang J., Greenberg M., Demple B. Covalent Trapping of
Human DNA Polymerase
by the Oxidative DNA Lesion 2-Deoxyribonolactone.
The Journal of Biological
Chemistry.
2002

.
[7]
Matsumoto Y., Kim K., Katz D., Feng J. Catalytic Center of DNA Polymerase
for Excision of
Deoxyribose Phosphate Groups.
American Chemical Society Publications.
1998

.
[8] Guo, Z. Zheng, L. Dai, H., et.al. Human DNA polymerase polymorphism, Arg137Gln, impairs its
polymerase activity and interaction with PCNA and the cellular base excision repair capacity.
Nucleic Acids Res,
2009
,
37(10),
pp 3431-3441

Appendices:

Fig 1.
DNA polymerase beta

Fig 2.
K72 active site

Fig 3.
D190A

Fig 4.
R137Q

Fig 5.
Y265C