DNA Polymerase Rebekah Sibbald Guillem Pina Júlia Melià Structural Biology Year 2015

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

Introduction to Polymerases What exactly is a polymerase? T7 DNA PolymeraseT7 RNA Polymerase

Function (Public Domain)

Lagging Strand Formation Source: Harvard Biotext and Animations

Larger Context

Even Larger Context

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

Meet the Families Filee et al., 2001.

Family Portraits As would be expected, polymerases in different families look quite different

SCOP Classification

Family A in SCOP

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...

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

Conserved Architecture: The Right Hand Yellow = Thumb Blue = Finger Pink = Palm

Family A

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 it’s function? Answer: Processing Okazaki fragments Bonus: Nucleotide excision and repair Which means…. it has exonuclease and polymerase functions!

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

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

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

H2H1H HG 6 H2I 78 8 K 9 LM NO O1O2 PQ QR β -strands α -helices Palm Domain Finger Domain Thumb Domain

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)

General Mechanism The conformational change is the rate limiting step! Rothwell and Waksman, 2005.

Conformational Change: Thumb Domain 1. Entire domain rotates by 17º resulting in an opening of the DNA-binding crevice 2. Only helices H1 and H2 of the domain rotate by 12º 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.

Conformational Change: Thumb Domain Blue: unbound DNA state Pink: bound DNA state

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 46º, 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

Conformational Change: Finger Domain Blue = open Yellow = closed

Conformational Change: Finger Domain

1. Rigid body rotation of helices N, O, O1 and O by 6º causing a partial closing of the crevice 2. Rotation of N and O helices by 40º The orientation of the O helix is dramatically affected by this transition, and many residues are directly involved with dNTP binding and incorporation

Conformational Change: Finger Domain

Tyr 671 Phe 667 Ile 614 Hydrophobic Pocket

Hydrophobic pocket

Arg 573 Arg 659 Lys 663 Glu 615 His 639 Nucleotide stabilization (H bonds)

Hydrogen Bonds

Asp 610 Asp 785 Metal Ions interaction (active site)

Metal Ions

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

Arg 573Asp 610 Ile 614 Glu 615 His 639 Arg 659Lys 663 Phe 667 Tyr 671 Asp 785

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

References: Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 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): Nakamura T, Zhao Y, Yamagata Y, Hua Y, Yang W. Watching DNA polymerase g make a phosphodiester bond. Nature Jul 12;487(7406): 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: Patel PH, Loeb LA. Getting a grip on how DNA polymerases function. Nat Struct Biol. 2001;8(8): Steitz TA. DNA polymerases: structural diversity and common mechanisms. J Biol Chem. 1999;274(25): 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): 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): Ogawa T, Okazaki T. Discontinuous DNA Replication. Ann Rev Biochem. 1980;49: 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): Beese LS, Derbyshire V, Steitz TA. Structure of DNA polymerase I Klenow fragment bound to duplex DNA. Science. 1993;260(5106): Kim Y, Eom SH, Wang J, Lee DS, Suh SW, Steitz TA. Crystal structure of Thermus aquaticus DNA polymerase. Nature Aug 17;376(6541): Eom SH, Wang J, Steitz TA. Structure of Taq polymerase with DNA at the polymerase active site. Nature Jul 18;382(6588): Rothwell JP, Waksman G. Structure and Mechanism of DNA Polymarases. Adv Protein Chem. 2005;71: 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 Jan;10(1): 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 May 18;308(5):

Questions

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.

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.

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.