Biophysics 1

Biophysics K. Banu Kö se (50091012) 22.10.
2010
Human DNA topoisomerase I (70 kDa) in complex with the

indenoisoquinoline AI-III-52 and covalent complex with a 22 base
pair DNA duplex
1)
Structure
A) Primary
Chain A (Protein)
1KKPKNKDKDKKVPEPDNKKKKPKKEEEQKWKWWEEERYPEGIKWKFLEHKGP
V F A P P Y E P LYS LYS PRO LYS ASN LYS ASP LYS ASP LYS LYS VAL PRO GLU PRO ASP ASN LYS LYS LYS
LYS PRO LYS LYS GLU GLU GLU GLN LYS TRP LYS TRP TRP GLU GLU GLU ARG TYR PRO GLU GLY ILE LYS
TRP LYS PHE LEU GLU HIS LYS GLY PRO VAL PHE ALA PRO PRO TYR GLU PRO K K P K N K D K D K K V P
E P D N K K K K P K K E E E Q K W K W W E E E R Y P E G I K W K F L E H K G P V F A P P Y E P 174 61 L P
ENVKFYYDGKVMKLSPKAEEVATFFAKMLDHEYTTKEIFRKNFFKDWRKEMTN
E E K N I LEU PRO GLU ASN VAL LYS PHE TYR TYR ASP GLY LYS VAL MET LYS LEU SER PRO LYS ALA GLU
GLU VAL ALA THR PHE PHE ALA LYS MET LEU ASP HIS GLU TYR THR THR LYS GLU ILE PHE ARG LYS ASN
PHE PHE LYS ASP TRP ARG LYS GLU MET THR ASN GLU GLU LYS ASN ILE L P E N V K F Y Y D G K V M K L
S P K A E E V A T F F A K M L D H E Y T T K E I F R K N F F K D W R K E M T N E E K N I 121 I T N L S K C D
F T Q M S Q Y F K A Q T E A R K Q M S K E E K L K I K E E N E K L L K E Y G F C I M D N H K E R I A N F ILE
THR ASN LEU SER LYS CYS ASP PHE THR GLN MET SER GLN TYR PHE LYS ALA GLN THR GLU ALA ARG
LYS GLN MET SER LYS GLU GLU LYS LEU LYS ILE LYS GLU GLU ASN GLU LYS LEU LEU LYS GLU TYR GLY
PHE CYS ILE MET ASP ASN HIS LYS GLU ARG ILE ALA ASN PHE I T N L S K C D F T Q M S Q Y F K A Q T E
A R K Q M S K E E K L K I K E E N E K L L K E Y G F C I M D N H K E R I A N F 181 K I E P P G L F R G R G N
H P K M G M L K R R I M P E D I I I N C S K D A K V P S P P P G H K W K E V R H D N K V T W L LYS ILE
GLU PRO PRO GLY LEU PHE ARG GLY ARG GLY ASN HIS PRO LYS MET GLY MET LEU LYS ARG ARG ILE
MET PRO GLU ASP ILE ILE ILE ASN CYS SER LYS ASP ALA LYS VAL PRO SER PRO PRO PRO GLY HIS LYS
TRP LYS GLU VAL ARG HIS ASP ASN LYS VAL THR TRP LEU K I E P P G L F R G R G N H P K M G M L K R R
I M P E D I I I N C S K D A K V P S P P P G H K W K E V R H D N K V T W L 241 V S W T E N I Q G S I K Y I M
L N P S S R I K G E K D W Q K Y E T A R R L K K C V D K I R N Q Y R E D W K S K E M K V R VAL SER TRP
THR GLU ASN ILE GLN GLY SER ILE LYS TYR ILE MET LEU ASN PRO SER SER ARG ILE LYS GLY GLU LYS
ASP TRP GLN LYS TYR GLU THR ALA ARG ARG LEU LYS LYS CYS VAL ASP LYS ILE ARG ASN GLN TYR ARG
Sayfa 1 / 19
Biophysics K. Banu Kö se (50091012) 22.10.2010
GLU ASP TRP LYS SER LYS GLU MET LYS VAL ARG V S W T E N I Q G S I K Y I M L N P S S R I K G E K D W
Q K Y E T A R R L K K C V D K I R N Q Y R E D W K S K E M K V R 301 Q R A V A L Y F I D K L A L R A G N E
K E E G E T A D T V G C C S L R V E H I N L H P E L D G Q E Y V V E F D F L G K D GLN ARG ALA VAL ALA
LEU TYR PHE ILE ASP LYS LEU ALA LEU ARG ALA GLY ASN GLU LYS GLU GLU GLY GLU THR ALA ASP THR
VAL GLY CYS CYS SER LEU ARG VAL GLU HIS ILE ASN LEU HIS PRO GLU LEU ASP GLY GLN GLU TYR VAL
VAL GLU PHE ASP PHE LEU GLY LYS ASP Q R A V A L Y F I D K L A L R A G N E K E E G E T A D T V G C C S
L R V E H I N L H P E L D G Q E Y V V E F D F L G K D 361 S I R Y Y N K V P V E K R V F K N L Q L F M E N K
Q P E D D L F D R L N T G I L N K H L Q D L M E G L T A K V F R T Y N SER ILE ARG TYR TYR ASN LYS VAL
PRO VAL GLU LYS ARG VAL PHE LYS ASN LEU GLN LEU PHE MET GLU ASN LYS GLN PRO GLU ASP ASP
LEU PHE ASP ARG LEU ASN THR GLY ILE LEU ASN LYS HIS LEU GLN ASP LEU MET GLU GLY LEU THR ALA
LYS VAL PHE ARG THR TYR ASN S I R Y Y N K V P V E K R V F K N L Q L F M E N K Q P E D D L F D R L N T
G I L N K H L Q D L M E G L T A K V F R T Y N 421 A S I T L Q Q Q L K E L T A P D E N I P A K I L S Y N R A N
R A V A I L C N H Q R A P P K T F E K S M M N L Q T K I D A ALA SER ILE THR LEU GLN GLN GLN LEU LYS
GLU LEU THR ALA PRO ASP GLU ASN ILE PRO ALA LYS ILE LEU SER TYR ASN ARG ALA ASN ARG ALA
VAL ALA ILE LEU CYS ASN HIS GLN ARG ALA PRO PRO LYS THR PHE GLU LYS SER MET MET ASN LEU
GLN THR LYS ILE ASP ALA A S I T L Q Q Q L K E L T A P D E N I P A K I L S Y N R A N R A V A I L C N H Q R
A P P K T F E K S M M N L Q T K I D A 481 K K E Q L A D A R R D L K S A K A D A K V M K D A K T K K V V E
S K K K A V Q R L E E Q L M K L E V Q A T D R E E N K Q LYS LYS GLU GLN LEU ALA ASP ALA ARG ARG
ASP LEU LYS SER ALA LYS ALA ASP ALA LYS VAL MET LYS ASP ALA LYS THR LYS LYS VAL VAL GLU SER
LYS LYS LYS ALA VAL GLN ARG LEU GLU GLU GLN LEU MET LYS LEU GLU VAL GLN ALA THR ASP ARG
GLU GLU ASN LYS GLN K K E Q L A D A R R D L K S A K A D A K V M K D A K T K K V V E S K K K A V Q R L
E E Q L M K L E V Q A T D R E E N K Q 541 I A L G T S K L N Y L D P R I T V A W C K K W G V P I E K I Y N K
T Q R E K F A W A I D M A D E D Y E F ILE ALA LEU GLY THR SER LYS LEU ASN PTR LEU ASP PRO ARG ILE
THR VAL ALA TRP CYS LYS LYS TRP GLY VAL PRO ILE GLU LYS ILE TYR ASN LYS THR GLN ARG GLU LYS
PHE ALA TRP ALA ILE ASP MET ALA ASP GLU ASP TYR GLU PHE I A L G T S K L N Y L D P R I T V A W C K
KWGVPIEKIYNKTQREKFAWAIDMADEDYEF
Chain B (DNA)
1AAAAAGACTT
Chain C (DNA)
1CGAAAAATTTTT
Chain D (DNA)
1 AAAAATTTTT CGAAGTCTTT TT
Sayfa 2 / 19
b) Secondary
Chain A (Protein)
Sayfa 3 / 19
TQREKFAWAIDMADEDYEF
Chain B (DNA)
1AAAAAGACTT
Chain C (DNA)
1CGAAAAATTTTT
Chain D (DNA)
1AAAAATTTTTCGAAGTCTTTTT
c) Tertiary
Chain A (Protein)
Sayfa 4 / 19
T Q R E K F A W A I D M A D E D Y E F ILE ALA LEU GLY THR SER LYS LEU ASN PTR LEU ASP PRO ARG ILE
THR VAL ALA TRP CYS LYS LYS TRP GLY VAL PRO ILE GLU LYS ILE TYR ASN LYS THR GLN ARG GLU LYS
PHE ALA TRP ALA ILE ASP MET ALA ASP GLU ASP TYR GLU PHE I A L G T S K L N Y L D P R I T V A W C K
K W G V P I E K I Y N K T Q R E K F A W A I D M A D E D Y E F 765
Chain B (DNA)
1AAAAAGACTT
Chain C (DNA)
1CGAAAAATTTTT
Sayfa 5 / 19
Chain D (DNA)
1AAAAATTTTTCGAAGTCTTTTT
Quaternary
Annotated PDB Quaternary Structure Assembly:1
Hetero Tetrametric
4 subunits of 4 distinct polymers entities (5 molecules in total including ligands)
Sayfa 6 / 19
d) Molecular Function of 1tl8
Synthesis and mechanism of action studies of a series of norindenoisoquinoline topoisomerase I

poisons reveal an inhibitor with a flipped orientation in the ternary DNA-enzyme-inhibitor complex as
determined by X-ray crystallographic analysis.
91.8% (516/562) of all residues were in favored (98%) regions.

Sayfa 7 / 19
98.4% (553/562) of all residues were in allowed (>99.8%) regions.

There were 9 outliers (phi, psi):
202 LYS (83.2, 62.2)
212 PRO (-56.6, 88.3)
213 GLU (4.7, 121.8)
236 GLU (-8.1, -51.9)
407 ASP (-51.7, 93.5)
467 SER (-1.5, 133.6)
638 LYS (-37.2, 91.0)
675 MET (86.1, 101.8)
724 LEU (65.5, 102.3)
DNA topoisomerase 1 is an enzyme that in humans is encoded by the TOP1 gene.
This gene encodes a DNA topoisomerase, an enzyme that controls and alters the topologic states of
DNA during transcription. This enzyme catalyzes the transient breaking and rejoining of a single
strand of DNA which allows the strands to pass through one another, thus altering the topology of
DNA. This gene is localized to chromosome 20 and has pseudogenes which reside on chromosomes 1
and 22.
We demonstrate that five topoisomerase I (Top 1) inhibitors (two indenoisoquinolines, two

camptothecins, and one indolocarbazole) each intercalate between the base pairs flanking the
cleavage site generated during the Top1 catalytic cycle and are further stabilized by a network of
hydrogen bonds with Top1. The interfacial inhibition paradigm described for Top1 inhibitors can be
generalized to a variety of natural products that trap macromolecular complexes as they undergo
catalytic conformational changes that create hotspots for drug binding. Stabilization of such
conformational states results in uncompetitive inhibition and exemplifies the relevance of screening
for ligands and drugs that stabilize (“trap”) these macromolecular complexes
(DNA Topoisomerase I Inhibitors: Chemistry, Biology, and Interfacial Inhibition/ Yves Pommiera/Laboratory of Molecular
Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
Chem. Rev., 2009)
e)
Crystallization Experiments
Method VAPOR DIFFUSION, SITTING DROP pH 6.4 Temperature 289.0 Details PEG 8000, MES,
Ammonium Sulfate, pH 6.40, VAPOR DIFFUSION, SITTING DROP, temperature 289K
Sayfa 8 / 19
Crystal Data / Unit Cell
Length (Å) Angle (°)
a= 56.95 α = 90
b= 114.14 β = 94.18
c= 73.5 γ= 90
2) DNA TOPOLOGY
The double-stranded structure of DNA has many implications for biological

function. Replication, at first appeared facilitated because genetic information is
encoded twice in the DNA structure, once on each strand, however, the two parental
strands must be separated and unwound to be copied.
Transcriptional enzymes must decide which of two complementary strands
contains the correct information to copy. Transcription also involves transient
unwinding of the DNA helix in a local region in order to be able to copy one strand.
Metabolic events involving unwinding impose great stress on the DNA because
of the constraints inherent in the double helix. Today we will discuss the topology of
DNA and tomorrow we will cover enzymes that can alter the topological state of DNA
without changing its primary structure. These enzymes are called DNA
topoisomerases.
Sayfa 9 / 19
In addition to the requirement to unwind DNA for replication and for

transcription, there is an absolute requirement for the correct topological tension in the
DNA (super-helical density) in order for genes to be regulated and expressed normally.
This supercoiling or writhing of circular DNAs was a result of the DNAs being
underwound with respect to the relaxed form of DNA. There are actually fewer turns in the
DNA helix than one would expect given the natural pitch of DNA in solution
(10.4 base pairs per turn).
When a linear DNA is free in solution it assumes a pitch which contains 10.4
base pairs per turn. This is less tightly wound than the 10.0 base pairs per turn in the
Watson and Crick B-form DNA.
In order to understand the origin of supercoiling; imagine a linear DNA 5200
base pairs in length. If the DNA were in the B-form one would expect the two strands of
the helix to be wrapped around each other 500 times (5200 bp/10.4 bp/turn). Imagine a
linear DNA in which the two ends become connected to form an open circle. This is
referred to as a relaxed circular DNA. On the other hand, if the linear DNA were
unwound 10%, say 50 turns, before its ends were joined, then the DNA molecule
would be under stress. When the molecule is free in solution it will coil about itself in
space as the two strands simultaneously twist about each other in order to return to
equilibrium value of 10.4 base pairs per turn.
DNA that is underwound is referred to as negatively supercoiled. The helices
wind about each other in a right handed path in space.
DNA that is overwound also will relax and assume a supercoiled conformation
but this is referred to as a positively supercoiled DNA helix. Positively coiled DNA has
its DNA helices wound around each other in a left-handed path in space.
The total number of times one strand of the DNA helix is linked with the other
in a covalently closed circular molecule is known as the linking number Lk.
Sayfa 10 / 19
1. The linking number is only defined for covalently closed DNA and its value is
fixed as long as the molecule remains covalently closed.
2. The linking number does not change whether the covalently closed circle is
forced to lie in a plane in a stressed conformation or whether it is allowed to
supercoil about itself freely in space.
3. The linking number Lk of a circular DNA can only be changed by breaking a

phosphodiester bond in one of the two strands, allowing the intact strand to pass
through the broken strand and then rejoining the broken strand.
4. Lk is always an integer since two strands must always be wound about each
other an integral number of times upon closure.
The linking number of a covalently closed circular DNA can be resolved into
two components called the twists Tw and the writhes Wr.
Lk = Tw + Wr
The twists Tw are the number of times that the two strands are twisted about
each other while the writhes Wr is the number of times that the DNA helix is coiled
about itself in three-dimensional space.
Unlike the Twist and the Linking number, the writhe of DNA only depends on
the path the helix axis takes in space, not on the fact that the DNA his two strands. If
the path of the DNA is in a plane, the Wr is always zero. Also if the path of the DNA
helix were on the surface of a sphere (like the seams of a tennis ball or base ball) then
the total Writhe can also be shown to be zero.
Writhes
Sayfa 11 / 19
DNA GEOMETRY
A-DNA / B-DNA
When DNA fibres are formed at low humidity DNA assumes a less hydrated form. (A-form)
This structure was determined at the same time as the B-form. This too is a double helix
structure, but is formed at a lower hydration than B-DNA (i.e.<75% humidity). Once again
this structure was the result of X-ray difractioin from DNA fibres so the position of the base
pairs is an average over the whole fibre.
The helix is wider, flatter and more compressed than the B-DNA helix. If synthetic RNA
molecules which are complementary in sequence form a double helix they form an A-type
double helix. This is due to the fact that the -OH group in the 2' position of the ribose would
be sterically hindered in the B-form helix. However remember that naturally occuring RNA
molecules are not normally complementary. (Why?)
A-DNA B-DNA
pitch=2.8nm pitch=3.6nm
11 base pairs/turn 10 base pairs/turn
sugar=C3'-endo sugar=C2'-endo
The planes of the bases are tilted 20 degrees to the helix axis, and there is a very deep major
groove, and a very shallow minor groove.
The physiological relevance of the A-form of DNA is not established. Most of the DNA in
chromatin is thought to be in the B-form.
Sayfa 12 / 19
Sayfa 13 / 19
Sayfa 14 / 19
Oligonucleotide Structures
More recently small, self-complimentary pieces of DNA have been co-crystallised and their
stuctures determined by X-ray crystallography. These are true crystalls and the positions of
each individual atom and base-pair can be determined. This contrasts with the fibre difraction
patterns which only give an average picture for all base pairs. Analysis of these crystalls has
shown that DNA can have quite large local deviations from the canonical Watson and Crick
structure and that these deviations appear to be dependent on the sequence of base pairs. The
crystal structure of the B-DNA decamer:
shows there are:
 About 10.1 base pairs/turn

 An average helical twist of 35.6o
 An average rise/residue of 0.34nm
Sayfa 15 / 19
These average values are quite close to the values for those for B-DNA determined from fibre
difraction. However there are large variations from these average values for individual base
pairs in the sequence, such that:
 The rise/residue varies from 0.30nm to 0.42nm

 The helical twistvaries from 32o to 40o
 The sugar conformation varies, some are C3'-endo, some are C2'-
endo, and some assume an intermediate conformation
 The base roll (angle to helix axis) varies
 Individual bases in a base pair have propeller twist
 Bases in a base pair are at a dihedral angle to each other
The variations in geometry appear to be sequence specific, purines favouring the C2'-endo
sugars, and pyrimidines the C3'-endo sugars. The conformation of a residue is also affected by
what it's neighbours conformation is (i.e. the AT pairs in the middle have a slightly different
conformation to those next to the GC's).
Use the mouse to rotate and zoom the image and convince yourself of the changes in propeller
twist and dihedral angle. The changes in sugar conformation are not quite so easy to spot.
The helix is curved by approximately 19o and not straight. (This is not easy to spot either if
you have not had a lot of experience looking at structures of this type.) DNA helices are
reasonably flexible to bending about small angles, so it is not known whether the curve seen is
due to natural conditions or an artifact of the crystallisation process.
There is a row of tightly bonded water molecules in the major groove in the AT-rich region. It
is not possible to say if these water molecules are present in native DNA as fibre difraction
does not enable water molecules to be identified. It is thought these water molecules may
provide a "spine of hydration" which stabilises the structure in the B-form and may be lost in
the transition to A-form. This is speculation however.
DNA is easily deformed, and does not have a precisely regular structure. Geometric variations
apear to be sequence specific...
If the geometry of DNA changes according to sequence, there may be a

geometrical mechanism for recognition of sequence.
A protein could initially recognise a particular sequence from the shape
of the DNA it binds to.
Sayfa 16 / 19
Sayfa 17 / 19
Z-DNA
The third and final geometry of DNA is that of the so-called Z-DNA.
This is sometimes seen when the sequence is of alternating purines and pyrimidines and was
first seen in the structure of the alternating deoxynucleotide dCpGpCpGpCpG.
If this structure displays the hydrogens-white dots- use the pull down menu to turn them off.
(OPTIONS submenu)
The Z structure was first shown in oligonucleotides, and an anti-parallel double helix forms ...
 Left handed
 12 base pairs/turn
BUT the helix is:  Pitch is 4.5nm per turn
 Deep minor groove
 Little or no major groove
This structure is formed by rotating the N-glycosyl bonds of G residues 180o with respect to
their conformation in B-DNA. In Z DNA all of the purines are in the "syn" conformation and
all the pyrimidines are in the "anti" conformation. Because the purines are "flipped" because
of the 180o rotation the pyrimidines must also "flip" to keep the base pairing intact. However
pyrimidine nucleotides do not readily assume the "syn" configuration because of steric
hinderance between the oxy group at position 2 of the pyrimidine and the atoms of the sugar.
Sayfa 18 / 19
In order to keep the base pairs intact the whole nucleoside (base plus sugar) has to flip 180o.
Despite these quite drastic conformational changes it is topologically possible for the G bases
to go syn and the C nucleosides to rotate 180o without breaking and reforming the hydrogen
bonds. This means that the B to Z structural transition can occur without any separation of the
helix.
It is not known whether Z-DNA is biologically significant, although antibodies raised to

oligonucleotides of this structure, have bound to some DNA regions. At least one DNA
binding protein has been found which is specific to Z-DNA. This form of DNA is only found
in high salt concentrations, because a high concentration of cations are required to maintain
the stability between the phosphate backbones (which are in close proximity to each other in
Z DNA).
Source:
http://www.pdb.org/pdb/explore/explore.do?structureId=1TL8
http://pubs.acs.org/doi/abs/10.1021/cr900097c
http://www.ebi.ac.uk/pdbsum/1tl8
http://books.google.com.tr/books?
id=WGBAGyzvQOUC&pg=PA25&dq=DNA+TOPOLOGY+AND+GEOMETRY&source=gbs_toc_r&cad=4#v
=onepage&q=DNA%20TOPOLOGY%20AND%20GEOMETRY&f=false
http://www.ima.umn.edu/2007-2008/T9.15.07/activities/Olson-Wilma/IMA_tutorial_1.pdf
Sayfa 19 / 19

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Biophysics 1

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Biophysics K. Banu Kö se (50091012) 22.10.

Human DNA topoisomerase I (70 kDa) in complex with the

Annotated PDB Quaternary Structure Assembly:1

4 subunits of 4 distinct polymers entities (5 molecules in total including ligands)

d) Molecular Function of 1tl8

Synthesis and mechanism of action studies of a series of norindenoisoquinoline topoisomerase I

91.8% (516/562) of all residues were in favored (98%) regions.

98.4% (553/562) of all residues were in allowed (>99.8%) regions.

DNA topoisomerase 1 is an enzyme that in humans is encoded by the TOP1 gene.

We demonstrate that five topoisomerase I (Top 1) inhibitors (two indenoisoquinolines, two

Crystal Data / Unit Cell

Length (Å) Angle (°)

The double-stranded structure of DNA has many implications for biological

In addition to the requirement to unwind DNA for replication and for

3. The linking number Lk of a circular DNA can only be changed by breaking a

shows there are:

 About 10.1 base pairs/turn

 The rise/residue varies from 0.30nm to 0.42nm

If the geometry of DNA changes according to sequence, there may be a

It is not known whether Z-DNA is biologically significant, although antibodies raised to

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