DNA Polymerase Rebekah Sibbald Guillem Pina Jlia Meli
Structural Biology Year 2015
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Contents 1. Introduction to Polymerases 2. Function 3. Context
4. Families 5. SCOP 6. Architecture 7. Focus on Family A 1.
Sequence and Structural Alignment 2. Secondary Structure 8. General
Mechanism 1. Thumb 2. Finger 3. Hydrophobic Pocket 4. Hydrogen
Bonds 5. Metal Ions
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Introduction to Polymerases What exactly is a polymerase? T7
DNA PolymeraseT7 RNA Polymerase
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Function (Public Domain)
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Lagging Strand Formation Source: Harvard Biotext and Animations
http://sites.fas.harvard.edu/~biotext/animations/replication1.html
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Larger Context
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Even Larger Context
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Multiple Families Although there are multiple types of
polymerase, there are also multiple families Families are based on
sequence homology and crystal structure analysis These families do
not correspond to SCOP classifications Based on comparison of amino
acid sequences, DNA polymerase families all seem to be unrelated
However, based on some common biochemical and structural features,
it is possible that families A, B and C are related Specifically,
possible that exonuclease domains are homologs
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Meet the Families Filee et al., 2001.
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Family Portraits As would be expected, polymerases in different
families look quite different
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SCOP Classification
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Family A in SCOP
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Implications It does not make sense to do sequence or structure
comparison between DNA polymerase families If they are homologous,
the common ancestor is very distant Believe us, we tried...
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Conserved Architecture: The Right Hand Although there is little
conserved sequence or structure between families, there is a
conserved architecture: Finger Domain Template and dNTP
interactions Palm Domain Phosphoryl transfer reaction Thumb Domain
Processivity and translocation Photo: Royal Collection Trust / Her
Majesty Queen Elizabeth II 2013
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Conserved Architecture: The Right Hand Yellow = Thumb Blue =
Finger Pink = Palm
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Family A
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Meet Family A Replicative and repair enzymes Includes
polymerases in the bacteriophage, as well as eukaryotic and
prokaryotic domains We focus on Polymerase I Anyone remember its
function? Answer: Processing Okazaki fragments Bonus: Nucleotide
excision and repair Which means. it has exonuclease and polymerase
functions!
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Arg 573 Arg 659 Lys 663Tyr 671 Phe 667 Asp 610 Ile 614 Glu 615
His 639 Asp 785 Palm Domain Finger Domain Thumb Domain Conserved
residues
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Family A structure: 4 Members of Family A Yellow = Taq
polymerase Blue = T7 DNA Polymerase Pink = E. Coli DNA polymerase I
Green = B. Stearothermophilus DNA polymerase I RMSD = 2.34
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Family A structure: 3 Members of Family A Yellow = Taq
polymerase Pink = E. Coli DNA polymerase I Green = B.
Stearothermophilus DNA polymerase I RMSD = 1.72
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H2H1H HG 6 H2I 78 8 K 9 LM NO O1O2 PQ QR 14 10 11 1213 -strands
-helices Palm Domain Finger Domain Thumb Domain
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General Mechanism To start, primer is already bound to template
Step 1: Primer and template bind to enzyme, which involves a
conformational change Step 2: dNTP binds to primer/template-enzyme
complex Step 3: Conformational change in fingers domain (open to
closed) Step 4: Nucleophilic attack forming a phosphodiester bond
Step 5: Release of inorganic phosphate Finally, enzyme can
dissociate the primer/template (distributive synthesis) or
translocate the template for a new round of synthesis (processive
synthesis)
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General Mechanism The conformational change is the rate
limiting step! Rothwell and Waksman, 2005.
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Conformational Change: Thumb Domain 1. Entire domain rotates by
17 resulting in an opening of the DNA-binding crevice 2. Only
helices H1 and H2 of the domain rotate by 12 to bring the tip of
the thumb domain closer to the DNA There are further conformational
changes (mostly in the H1H2 loop), finally resulting in a cylinder
that almost completely surrounds the DNA.
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Conformational Change: Thumb Domain Blue: unbound DNA state
Pink: bound DNA state
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Conformational Change Conformational change activates the
enzyme complex Involves shift from open structure to closed
structure Open structure: The tip of the fingers domain is rotated
outward by 46, so that a crevice is clearly accessible Closed
structure: Reorientation of the tip of the fingers domain so that
it is oriented inwards towards the template and primer
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Conformational Change: Finger Domain Blue = open Yellow =
closed
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Conformational Change: Finger Domain
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1. Rigid body rotation of helices N, O, O1 and O by 6 causing a
partial closing of the crevice 2. Rotation of N and O helices by 40
The orientation of the O helix is dramatically affected by this
transition, and many residues are directly involved with dNTP
binding and incorporation
Asp 610 Asp 785 Metal Ions interaction (active site)
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Metal Ions
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Conclusions: Requirements for Polymerization Thumb changes
conformation when primer and template bind to enzyme Fingers change
conformation (rate limiting) when nucleotide binds to enzyme
Interactions with incoming nucleotide through a hydrophobic pocket
and hydrogen bonds Metal ion induced nucleophilic attack causing
phosphodiester bond formation
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Arg 573Asp 610 Ile 614 Glu 615 His 639 Arg 659Lys 663 Phe 667
Tyr 671 Asp 785
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Overall Conclusions Common architecture between families In
Family A, conservation is due to functional role There is more
conservation in structure than sequence Sequence is conserved when
residues have a specific function
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References: Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th
edition. New York: W H Freeman; 2002. Li Y, Kerolev S, Waksman G.
Crystal structures of open and closed forms of binary and ternary
complexes of the large fragment of Thermus aquaticus DNA polymerase
I: structural basis for nucleotide incorporation. EMBO J.
1998;17(24):7514-7525. Nakamura T, Zhao Y, Yamagata Y, Hua Y, Yang
W. Watching DNA polymerase g make a phosphodiester bond. Nature.
2012 Jul 12;487(7406):196-201. Filee J, Forterre P, Sen-Lin T,
Laurent J. Evolution of DNA polyemerase Families: Evidence for
Multiple Gene Exchange Between Cellular and Viral Proteins. J Mol
Evol. 2002;54:763-773. Patel PH, Loeb LA. Getting a grip on how DNA
polymerases function. Nat Struct Biol. 2001;8(8):656-9. Steitz TA.
DNA polymerases: structural diversity and common mechanisms. J Biol
Chem. 1999;274(25):17395-8. Astatke M, Grindley ND, Joyce CM. How
E. coli DNA polymerase I (Klenow fragment) distinguishes between
deoxy- and dideoxynucleotides. J Mol Biol. 1998;278(1):147-65.
Sheriff A, Motea E, Lee I, Berdis AJ. Mechanism and dynamics of
translesion DNA synthesis catalyzed by the Escherichia coli Klenow
fragment. Biochemistry. 2008;47(33):8527-37. Ogawa T, Okazaki T.
Discontinuous DNA Replication. Ann Rev Biochem. 1980;49:421-457.
Ramanathan S, Chary KV, Rao BJ. Incoming nucleotide binds to Klenow
ternary complex leading to stable physical sequestration of
preceding dNTP on DNA. Nucleic Acids Res. 2001;29(10):2097-105.
Beese LS, Derbyshire V, Steitz TA. Structure of DNA polymerase I
Klenow fragment bound to duplex DNA. Science. 1993;260(5106):352-5.
Kim Y, Eom SH, Wang J, Lee DS, Suh SW, Steitz TA. Crystal structure
of Thermus aquaticus DNA polymerase. Nature. 1995 Aug
17;376(6541):612-6. Eom SH, Wang J, Steitz TA. Structure of Taq
polymerase with DNA at the polymerase active site. Nature. 1996 Jul
18;382(6588):278-81. Rothwell JP, Waksman G. Structure and
Mechanism of DNA Polymarases. Adv Protein Chem. 2005;71:401-440.
Beese LS, Steitz TA. Structural basis for the 3'-5' exonuclease
activity of Escherichia coli DNA polymerase I: a two metal ion
mechanism. EMBO J. 1991 Jan;10(1):25-33. Patel PH, Suzuki M, Adman
E, Shinkai A, Loeb LA. Prokaryotic DNA polymerase I: evolution,
structure, and "base flipping" mechanism for nucleotide selection.
J Mol Biol. 2001 May 18;308(5):823-37.
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Questions
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1. DNA Polymerases' classic and SCOP classifications are
different, more specifically: a) SCOP classification has more
polymerases in each category. b) SCOP classification is based on
function while classic classification is based on sequence
homology. c) SCOP classification is based on folding of the
polymerase domain while classic classification is based on whole
sequence sequence homology and crystal structure analysis. d) The
classic classification is based in polymerase activity, as well as
SCOPE is, but have different polymerases within them. e) Non of the
above are correct. 2. Regarding DNA polymerases: a) All DNA
polymerases have the same architecture (finger, palm and thumb
domains). b) Only DNA polymerases from the same SCOP family of Taq
polymerase have the same architecture. c) Taq polymerase is the
only polymerase with both polymerase and exonuclease activity. d)
The thumb domain is responsible for the interactions with the
template. e) Non of the above are correct. 3. When performing a
sequence alignment between taq, e. coli, T7 and b.
stearothermophilus (the 4 polymerases obtained using the HMM of
family A), the resulting alignment taking into account the
structural features explained in the presentation: a) shows a lot
of sequence homology throughout the whole sequence alignment but
the finger, thumb and palm domains are not conserved. b) shows a
lot of sequence homology throughout the whole sequence alignment
and the finger, thumb and palm domains are conserved. c) doesn't
show a lot of sequence homology throughout the whole sequence and
neither does the secondary structure, but some key residues are
conserved. d) doesn't show a lot of sequence homology throughout
the whole sequence but secondary structure is highly conserved as
well as the key residues. e) T7 is completely different from the
other family A polymerases and this can be explained because is a
polymerase from a bacteriophage while the others are bacteria
polymerases.
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4. How is the DNA polymerase activated? a) By a conformational
change in the thumb domain after binding of the primer and
template. b) By a conformational change from open to closed in
fingers domain after binding of a dNTP. c) It is always active. d)
The binding of several inductors activates it. e) All of the above
are incorrect. 5. What is the role of the hydrophobic pocket that
is formed in the fingers domain? a) Interacts with the sugar and
the base of the incoming nucleotide. b) Interacts with the
phosphate group of the incoming nucleotide. c) A and B are correct.
d) Interacts with the last base pair of the primer/template. e) All
of the above are incorrect. 6. When does the thumb change its
conformation? a) After binding of the primer and template, forming
a cylinder that surrounds them. b) After binding of a dNTP to the
template/primer-enzyme complex. c) When the template is
translocated for the addition of a new nucleotide. d) Once the
phosphodiester bond between the primer and the dNTP has been
formed. e) All of the above are incorrect. 7. Which residue forms
stacking interactions with the DNA template in the open
conformation of family A polymerases? a) A tyrosine of the O helix
in the fingers domain. b) An aspartate of the active site in the
palm domain. c) A phenylalanine of the H1 helix in the thumb
domain. d) An arginine of the N helix in the fingers domain. e)
Stacking interactions are only formed in the closed
conformation.
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8. How are metal ions involved in DNA polymerase activity? a)
By causing the enzyme to dissociate. b) By stabilizing the
transition state. c) By slowing the reaction progress. d) By
initiation DNA transposition. e) Trick question, they are not
involved. 9. Which of the following is true? a) Different organisms
contain different types of polymerases with the same function. b)
Each organism contains only one type of polymerase. c) All
polymerases are clearly homologous. d) Unlike RNA polymerase, DNA
polymerase involves only one subunit. e) Different organisms
contain different types of polymerases for specialized functions.
10. Which of the following is false concerning DNA polymerase I? a)
Residues with specific functions are more likely to be conserved.
b) DNA polymerase I is specialized in lagging strand formation. c)
Polymerization involves a metal ion mechanism. d) Aside from common
architecture, there are no similarities between polymerases. e) DNA
polymerases share a common architecture.