ABSTRACT RESULTS CONCLUSIONS INTRODUCTION
Graduate Category: Physical and Life Sciences Degree Level: Ph.D. Candidate Abstract ID#676
PredicAon and VerificaAon of the Extended AcAve Site in E.coli DNA Polymerase III Timothy A. Coulther, Ramya Parasuram, Mary Jo Ondrechen, Penny J. Beuning Department of Chemistry & Chemical Biology
ABSTRACT
Polymerases catalyze the synthesis of DNA efficiently and with high fidelity in order to preserve the genomic informaAon. Pol III, a replicaAve polymerase in E.coli, is able to insert the correct nucleoAde with an error rate of only 1 in ~105. In order to understand the basis for Pol III catalysis, selecAvity, and fidelity, computaAonal methods were used to predict the funcAonal residues in alpha, the catalyAc subunit of Pol III. Computed electrostaAc and phylogeneAc data about each amino acid residue in Pol III are then uAlized by ParAal Order OpAmum Likelihood (POOL), a machine learning methodology developed at Northeastern, to predict the acAve site residues that parAcipate in the biochemical funcAon of Pol III. POOL predicts an extended acAve site for alpha, where residues outside the immediate vicinity of the substrate contribute to catalysis. Enzyme variants with mutaAons at the POOL-‐predicted residues were assayed for their ability to catalyze DNA synthesis. Many of the variants show diminished or complete loss of acAvity, indicaAng their importance in Pol III funcAon. Work is ongoing to determine kineAc parameters of the variants with diminished catalyAc ability relaAve to the wild-‐type enzyme. The melAng temperatures for alpha and its variants are not significantly different, indicaAng the loss of acAvity is in fact due to local effects rather than loss of structural stability. POOL has allowed us to idenAfy and characterize residues in the extended acAve site of alpha, contribuAng to our understanding of how this polymerase funcAons.
Images
Shell First First Second First Second First Second First First Second Remote First Second First Second
*Known catalyAc residues Distal residues are in red
Variant -‐-‐-‐ -‐-‐-‐ Y340F Y340S -‐-‐-‐ D405N R390A Y686F Y686A R709A K553A K758L D630N D630A R362A R362K E547Q E547A H760L E688Q
AcBvity -‐-‐-‐ -‐-‐-‐ Less acAve Less acAve -‐-‐-‐ InacAve Less acAve AcAve InacAve Less acAve InacAve Less acAve Less acAve AcAve InacAve Less acAve AcAve AcAve InacAve InacAve
• Natural acAve sites can be built in layers so that they are spaAally extended
Verified AcAve Site
• AcAve site residues can be predicted accurately by POOL from a 3-‐D structure • DNA Polymerase III is predicted to have a spaAally extended acAve site • Mutagenesis and acAvity assays confirm the importance of many predicted residues
WT Y340F R390A R709A
Time
Primer extension assays8
Top 15 POOL predicted residues8. Known catalyAc aspartates are colored red. First shell residues are colored purple. Distal residues are colored green. The incoming dNTP is also shown.
CONCLUSIONS
• 3 of the top predicted residues are known to be essenAal for catalysis • 6 of the 12 studied residues showed complete loss of acAvity upon a mutaAon • 11 of the 12 studied residue showed some acAvity loss upon a mutaAon
Time
INTRODUCTION
Alpha is the catalyAc core of E. coli DNA polymerase III, responsible for replicaAve DNA synthesis and a member of the C-‐family polymerases. While the three catalyAc aspartate residues are known, not much else is known about the acAve site since this family shares lille homology with the other polymerase families. To understand the acAve site more fully, the funcAonal residue predictor ParAal Order OpAmum Likelihood1, developed here at Northeastern University, was uAlized. It is a machine learning method that uAlizes inputs from three different programs to generate a complete rank ordered list of funcAonal residues2,3,4. In many cases, including here, POOL predicts an extended acAve site, where residues distal to the substrate sAll play funcAonal roles5,6,7. Since these cannot be readily recognized, computaAonal programs like POOL are necessary for idenAficaAon. Experimental verificaAon through site-‐directed mutagenesis was performed to confirm the computaAonal predicAons.
RESULTS
Extension
Residue Asp403* Asp401* Tyr340 Asp555* Asp405 Arg390 Tyr686 Arg709 Lys553 Lys758 Asp630 Arg362 Glu547 His760 Glu688
Kinetic Parameters for Alpha and Variants Catalytic Efficiency -‐1 Fold Change kcat (min ) KM (µM) -‐1 -‐1 (min µM ) 47 ± 11 116 ± 29 0.40 ± 0.14 -‐ 25 ± 4.6 125 ± 19 0.20 ± 0.05 2 14 ± 8 877 ± 188 0.016 ± 0.011 26 59 ± 22 102 ± 10 0.58 ± 0.22 0.69
13 of the 17 variants assay showed decreased or complete loss of acAvity • Three of the alpha variants with some acAvity have been fully characterized
REFERENCES
1.W. Tong, Y. Wei, L. F. Murga, M. J. Ondrechen and R. J. Williams, PLoS Comput Biol 2009, 5. 2. M. J. Ondrechen, J. G. Cli`on and D. Ringe, Proc Natl Acad Sci U S A 2001, 98. 3. J. A. Capra, R. A. Laskowski, J. M. Thornton, M. Singh and T. A. Funkhouser, PLoS Comput Biol 2009, 5. 4. S. Sankararaman and K. Sjolander, Bioinforma
PredicAon and VerificaAon of the Extended AcAve Site in E.coli DNA Polymerase III Timothy A. Coulther, Ramya Parasuram, Mary Jo Ondrechen, Penny J. Beuning Department of Chemistry & Chemical Biology
ABSTRACT
Polymerases catalyze the synthesis of DNA efficiently and with high fidelity in order to preserve the genomic informaAon. Pol III, a replicaAve polymerase in E.coli, is able to insert the correct nucleoAde with an error rate of only 1 in ~105. In order to understand the basis for Pol III catalysis, selecAvity, and fidelity, computaAonal methods were used to predict the funcAonal residues in alpha, the catalyAc subunit of Pol III. Computed electrostaAc and phylogeneAc data about each amino acid residue in Pol III are then uAlized by ParAal Order OpAmum Likelihood (POOL), a machine learning methodology developed at Northeastern, to predict the acAve site residues that parAcipate in the biochemical funcAon of Pol III. POOL predicts an extended acAve site for alpha, where residues outside the immediate vicinity of the substrate contribute to catalysis. Enzyme variants with mutaAons at the POOL-‐predicted residues were assayed for their ability to catalyze DNA synthesis. Many of the variants show diminished or complete loss of acAvity, indicaAng their importance in Pol III funcAon. Work is ongoing to determine kineAc parameters of the variants with diminished catalyAc ability relaAve to the wild-‐type enzyme. The melAng temperatures for alpha and its variants are not significantly different, indicaAng the loss of acAvity is in fact due to local effects rather than loss of structural stability. POOL has allowed us to idenAfy and characterize residues in the extended acAve site of alpha, contribuAng to our understanding of how this polymerase funcAons.
Images
Shell First First Second First Second First Second First First Second Remote First Second First Second
*Known catalyAc residues Distal residues are in red
Variant -‐-‐-‐ -‐-‐-‐ Y340F Y340S -‐-‐-‐ D405N R390A Y686F Y686A R709A K553A K758L D630N D630A R362A R362K E547Q E547A H760L E688Q
AcBvity -‐-‐-‐ -‐-‐-‐ Less acAve Less acAve -‐-‐-‐ InacAve Less acAve AcAve InacAve Less acAve InacAve Less acAve Less acAve AcAve InacAve Less acAve AcAve AcAve InacAve InacAve
• Natural acAve sites can be built in layers so that they are spaAally extended
Verified AcAve Site
• AcAve site residues can be predicted accurately by POOL from a 3-‐D structure • DNA Polymerase III is predicted to have a spaAally extended acAve site • Mutagenesis and acAvity assays confirm the importance of many predicted residues
WT Y340F R390A R709A
Time
Primer extension assays8
Top 15 POOL predicted residues8. Known catalyAc aspartates are colored red. First shell residues are colored purple. Distal residues are colored green. The incoming dNTP is also shown.
CONCLUSIONS
• 3 of the top predicted residues are known to be essenAal for catalysis • 6 of the 12 studied residues showed complete loss of acAvity upon a mutaAon • 11 of the 12 studied residue showed some acAvity loss upon a mutaAon
Time
INTRODUCTION
Alpha is the catalyAc core of E. coli DNA polymerase III, responsible for replicaAve DNA synthesis and a member of the C-‐family polymerases. While the three catalyAc aspartate residues are known, not much else is known about the acAve site since this family shares lille homology with the other polymerase families. To understand the acAve site more fully, the funcAonal residue predictor ParAal Order OpAmum Likelihood1, developed here at Northeastern University, was uAlized. It is a machine learning method that uAlizes inputs from three different programs to generate a complete rank ordered list of funcAonal residues2,3,4. In many cases, including here, POOL predicts an extended acAve site, where residues distal to the substrate sAll play funcAonal roles5,6,7. Since these cannot be readily recognized, computaAonal programs like POOL are necessary for idenAficaAon. Experimental verificaAon through site-‐directed mutagenesis was performed to confirm the computaAonal predicAons.
RESULTS
Extension
Residue Asp403* Asp401* Tyr340 Asp555* Asp405 Arg390 Tyr686 Arg709 Lys553 Lys758 Asp630 Arg362 Glu547 His760 Glu688
Kinetic Parameters for Alpha and Variants Catalytic Efficiency -‐1 Fold Change kcat (min ) KM (µM) -‐1 -‐1 (min µM ) 47 ± 11 116 ± 29 0.40 ± 0.14 -‐ 25 ± 4.6 125 ± 19 0.20 ± 0.05 2 14 ± 8 877 ± 188 0.016 ± 0.011 26 59 ± 22 102 ± 10 0.58 ± 0.22 0.69
13 of the 17 variants assay showed decreased or complete loss of acAvity • Three of the alpha variants with some acAvity have been fully characterized
REFERENCES
1.W. Tong, Y. Wei, L. F. Murga, M. J. Ondrechen and R. J. Williams, PLoS Comput Biol 2009, 5. 2. M. J. Ondrechen, J. G. Cli`on and D. Ringe, Proc Natl Acad Sci U S A 2001, 98. 3. J. A. Capra, R. A. Laskowski, J. M. Thornton, M. Singh and T. A. Funkhouser, PLoS Comput Biol 2009, 5. 4. S. Sankararaman and K. Sjolander, Bioinforma
