Human Topoisomerase I and DNA
Complex
Nat Fox '16 and Yohanna Ewing '16
Contents:
I. Introduction
Model View:
Color Scheme:
Human
Topoisomerase I (topo I) is a nuclear protein that relieves the
tension in overwound DNA helices, a result of the transcription and
translation processes.1 This unwinding process is critical for
proper DNA replication, transcription, and maintenance, making
Topoisomerase I a vitally important protein. Topo I achieves DNA
relaxation by making a cut to one strand of the double helix. The
catalytic nucleophilic tyrosine (Tyr 723) attacks the electrophilic
phosphodiester DNA backbone introducing a break in the strand and
forming a covalent bond between broken strand and the protein. This
allows the free end of the strand to rotate in a "Controlled
Rotation" manner, relieving helix tension1. The
goal of this tutorial is to illustrate how topo I binds to DNA and
carries out the chemical reactions required for DNA relaxation.
II. General Structure
Model View:
Color Scheme:
Topo I is a
heterodimer composed of 4 domains: the N-terminus, core (subdomain I, subdomain
II, and subdomain III),
C-terminal domain,
and the linker domain.1.
The N-terminus domain, not pictured, is highly charged and
thought to be unstructured. Core subdomains
I and II contain
the CAP (residues 202-434)
and subdomain III contains
the CAT (residues 435-635)
which
the double stranded DNA and position the active site
around the target phosphate group.2 Core
subdomain III contains three of the amino acids
(His632, Arg590, and Arg488) involved in the active site.
The C-terminal domain contains
the catalytic tyrosine and alpha-21 helix necessary for
the formation of the active complex. The topo I dimer is
the result of the three salt bridges and nine hydrophobic
pairwise interactions formed between alpha helices,
alpha-18 and alpha-19, in the
linker domain.2
III. DNA Binding
Model View:
Color Scheme:
DNA binding of topo I to the dsDNA is the result of
a coulombic protein/DNA charge interaction (positive=blue,
neutral=white, negative=red)
between the positively charged CAP, CAT and the linker domain with the negatively
charged sugar-phosphate backbone.1 Topo 1 initiates its
transition into the active state when the CAP
and CAT regions encircle and
bind to the DNA. This DNA/protein interaction shift the alpha helix
residues Thr597 and Leu602 of
alpha-16 and Val626, Leu629, and Cys630 of alpha-17 of
the CAT and catalytic Tyr723
of alpha-21 of the C-terminal
domain to create the binding
where catalytic Tyr723
is rotated from buried to active position.2 Lys650
and Arg708 make hydrogen
bonding
with phosphates on
the target cut strand. These residues bond 9-10 nucleotides from
the cleavage site on the scissile strand and 7 to 10 nucleotides
on the intact strand which further stabilizes the active site. The linker domain contains an
additional seven positively charged and seven polar residues that
do not directly interact with the sugar-phosphate backbone. These
residues likely play a role in the "controlled rotation" mechanism
of human topoisomerase I.1
IV. DNA Cleavage
Model View:
Color Scheme:
Once the catalytic tyrosine is in position, DNA cleavage is
carried out by a nucleophilic
attack. The hydroxyl group
on Tyr723 attacks the electrophilic
phosphate on the sugar-phosphate backbone. This results
in a covalent bond between Tyr723 and the 3' end of the cleaved
strand.1 The hydroxyl
group binds to the phosphate to form a five substituent
transition state which is stabilized by hydrogen bonds between
water and the hydroxyl group of Tyr723 . Click
for Mechanism. This results in a covalent bond between
Tyr723 and the 3' end of the cleaved strand. The hydroxyl group
binds to the phosphate to form a five membered transition state
which is stabilized by hydrogen bonds between water and the
hydroxyl group of Tyr723 . Additionally, this hydrogen bonding
induces a more basic nucleophile, increasing the rate of the
reaction. The phosphate is held in place by hydrogen bonding
between Oxygen 1 (O1) and Arg488, O1 and Arg590, and His623 and
O2.
Additionally, His632 acts as an
acid by donating a proton to
O5, the leaving group. DNA
relaxation results from the the rotation of the free end (O5)
of broken sugar-phosphate backbone around the intact strand of
DNA.6 Mutations in these amino acids result in a nonfunctional
active site.
V. Implications
Model View:
Color Scheme:
Human topoisomerase I is a popular target of anticancer therapies and
research because of its important role in transcription and
translation.3 Camptothecins, indolocarbazoles and
indenoisoquinolines are the three major classifications of topo I
specific therapies.4 These molecules interfere with the
catalytic Tyr723 in the binding pocket to form a DNA lesion.3
The mechanism for topoisomerase inhibition is best understood in
camptothecins, the first group of topo I specific compounds
discovered. Camptothecins are planer pentacyclical molecules with a
alpha-hydroxyacetone.3 When there is a guanine base on the
5' end of the broken DNA strand, the drug is able to bind to the
DNA/protein which results in a ternary complex. This complex is
stabilized by base stacking between the cyclical campothecin and
guanine. The stabilized complex forms a DNA lesion which holds the
DNA/topo I the intermediate cleaving position inhibiting cancerous
cell proliferation.3 Different topo I inhibitors are
effective in particular cancerous cell types. As of 2006, two
camptothecin derived anti-cancer drugs have been approved by the
Federal Drug Administration: Topotecan (Hycamin) targets ovarian and
lung cancers and irinotecan (CPTII, Campto) targets colon cancer.3
These targeted therapies are more successful than traditional
chemotherapy and have higher cure rate.5 Therefore,
understanding human topoisomerase DNA/protein binding interaction can
lead to more targeted specific therapies and higher remission and cure
rates.
VI. References
1. Stewart, Lance, Matthew R. Redinbo, Xiayang
Qui, Wim G.J. Hol, and James J. Champoux. 1998. A Model for the
Mechanism of Human Topoisomerase I. Science 279, 1534-1541.
2. Lesher, Diem-Thu T., Yves Pommier, Lance Stewart, and Matthew
R. Redinbo. 2002. 8-Oxoguanine Rearranges the Active Site of Human
Topoisomerase I. PNAS 99 (19):12102-12107.
3. Moisan, F., Longy, M., Robert J, and. Morvan V. Le. 2006.
Identification of Gene Polymorphisms of Human DNA Topoisomerase I
in the National Cancer Institute Panel of Human Tumour Cell Lines.
British Journal of Cancer 99(7)
4. Moukharskaya J. 2012. Topoisomerase 1 Inhibitors and Cancer
Therapy. Hematology/oncology clinics of North America. 26:507-507.
5. Arakawa, Yasuhiro, Koji Ozaki, Yutaka Okawa, and Hisashi
Yamada. 2013. Three Missense Mutations of DNA Topoisomerase I in
Highly Camptothecin-Resistant Colon Cancer Cell Sublines. Oncology
Reports 30. 1053-1058
Back to Top