2017 RISE Poster HRS_20Mar2017_2
Graduate Category: Physical and Life Sciences Degree Seeking: PhD in Chemistry Abstract ID# 1791
Role of Distal Residues in DNA Polymerases DinB and Pol Kappa in Bypass of DNA Damage Hannah R. Stern, Caitlyn L. Mills, Mary Jo Ondrechen, Penny J. Beuning Chemistry and Chemical Biology
Opportunity
Approach POOL predicts residues important for enzyme activity
DNA is subject to damage from endogenous and exogenous sources. Replicative DNA polymerases are typically unable to replicate damaged DNA, but specialized DNA polymerases in the Y family possess this ability. Escherichia coli has two Y family polymerases that are specialized to bypass lesions when copying damaged DNA in a process called translesion synthesis (TLS). DinB is one of these polymerases, which is involved in bypassing deoxyguanosine adducts at the N2 position, and is implicated in some antibiotic resistance. There are also four human Y family polymerases, including DNA Pol κ, that have similar function. However, Pol κ is more efficient in copying DNA damage in the extension step of TLS. In order to investigate the importance of particular residues in the extension step of TLS, the computational method POOL was utilized. POOL identified active site residues and residues previously observed to be important for activity. POOL also predicted distant residues that do not have direct contact with substrates that may have catalytic importance, but the residues are in different regions of DinB and Pol κ. In addition, our previous HXMS work suggested a possible allosteric change in both DinB and Pol κ in different distal residues that POOL did not predict. To study the contribution of these distal residues on the extension step of TLS, DinB and Pol κ variants with mutations at the predicted distal positions were constructed and are being assayed for bypass of damage lesions.
DinB and Pol κ are specific for minor groove damage
PDB: 1JDG
PDB: 1D65
PDB: 4IRC
Pol κ
DinB
Nevin, P., et al. (2015). FEBS Journal 282(14): 2646-2660.
Somarowthu, S., H. Yang, D.G.C. Hildebrand, and M.J. Ondrechen, Highperformance prediction of functional residues in proteins with machine learning and computed input features. Biopolymers, 2011. 95(6): 390-400.
PDB: 2OH2
Impact
Results Specific pattern of protection in polymerases due to substrate binding Protection from DNA Fingers
Palm
POOL Results with DinB and Pol κ show half of top 8% residues are conserved
Protection from DNA and dNTP Fingers
Fingers
Palm
E296
DinB Little Finger
Fingers
T248
Thumb Little Finger Palm N-clasp
Thumb
Little Finger
Thumb N-clasp
Little Finger
K289
N-clasp
Palm
Thumb
DinB
R211
Little Finger
Palm
Thumb
Y221
Palm
R330
Pol κ Little Finger
K224
Fingers
N-clasp
Thumb
Pol κ
Nevin, P., et al. (2015). FEBS Journal 282(14): 2646-2660.
• Residues in fingers domain are located near dNTP-binding site • Residues in little finger domain make contact with DNA • Residues in thumb were protected • Regions located distal to the active site • Sites do not make contacts with substrates • Substrate-dependent protection
• Value Proposition The unique feature about my research is: understanding the role of distal residues on enzyme activity and protein dynamics.
• Our goal is to determine which residues contribute to better extension by Pol κ compared with DinB • DinB non-conserved residues include three little finger residues • Non-conserved residues in Pol κ identified as part of the palm and N-clasp • By changing these residues, we can test enzyme activity to determine how these non-conserved residues play a role in the extension step of TLS
• Y-family DNA polymerases contribute to antibiotic resistance through their mutagenic DNA replication, prevent cancer by contributing to DNA damage tolerance, and contribute to chemotherapy resistance. Thus, this research addresses the problems of antibiotic resistance, some cancers, and chemotherapy resistance. Acknowledgments
Support from NSF-MCB-1517290, American Cancer Society RSG-12-161-01-DMC PhRMA Foundation (CLM)
Role of Distal Residues in DNA Polymerases DinB and Pol Kappa in Bypass of DNA Damage Hannah R. Stern, Caitlyn L. Mills, Mary Jo Ondrechen, Penny J. Beuning Chemistry and Chemical Biology
Opportunity
Approach POOL predicts residues important for enzyme activity
DNA is subject to damage from endogenous and exogenous sources. Replicative DNA polymerases are typically unable to replicate damaged DNA, but specialized DNA polymerases in the Y family possess this ability. Escherichia coli has two Y family polymerases that are specialized to bypass lesions when copying damaged DNA in a process called translesion synthesis (TLS). DinB is one of these polymerases, which is involved in bypassing deoxyguanosine adducts at the N2 position, and is implicated in some antibiotic resistance. There are also four human Y family polymerases, including DNA Pol κ, that have similar function. However, Pol κ is more efficient in copying DNA damage in the extension step of TLS. In order to investigate the importance of particular residues in the extension step of TLS, the computational method POOL was utilized. POOL identified active site residues and residues previously observed to be important for activity. POOL also predicted distant residues that do not have direct contact with substrates that may have catalytic importance, but the residues are in different regions of DinB and Pol κ. In addition, our previous HXMS work suggested a possible allosteric change in both DinB and Pol κ in different distal residues that POOL did not predict. To study the contribution of these distal residues on the extension step of TLS, DinB and Pol κ variants with mutations at the predicted distal positions were constructed and are being assayed for bypass of damage lesions.
DinB and Pol κ are specific for minor groove damage
PDB: 1JDG
PDB: 1D65
PDB: 4IRC
Pol κ
DinB
Nevin, P., et al. (2015). FEBS Journal 282(14): 2646-2660.
Somarowthu, S., H. Yang, D.G.C. Hildebrand, and M.J. Ondrechen, Highperformance prediction of functional residues in proteins with machine learning and computed input features. Biopolymers, 2011. 95(6): 390-400.
PDB: 2OH2
Impact
Results Specific pattern of protection in polymerases due to substrate binding Protection from DNA Fingers
Palm
POOL Results with DinB and Pol κ show half of top 8% residues are conserved
Protection from DNA and dNTP Fingers
Fingers
Palm
E296
DinB Little Finger
Fingers
T248
Thumb Little Finger Palm N-clasp
Thumb
Little Finger
Thumb N-clasp
Little Finger
K289
N-clasp
Palm
Thumb
DinB
R211
Little Finger
Palm
Thumb
Y221
Palm
R330
Pol κ Little Finger
K224
Fingers
N-clasp
Thumb
Pol κ
Nevin, P., et al. (2015). FEBS Journal 282(14): 2646-2660.
• Residues in fingers domain are located near dNTP-binding site • Residues in little finger domain make contact with DNA • Residues in thumb were protected • Regions located distal to the active site • Sites do not make contacts with substrates • Substrate-dependent protection
• Value Proposition The unique feature about my research is: understanding the role of distal residues on enzyme activity and protein dynamics.
• Our goal is to determine which residues contribute to better extension by Pol κ compared with DinB • DinB non-conserved residues include three little finger residues • Non-conserved residues in Pol κ identified as part of the palm and N-clasp • By changing these residues, we can test enzyme activity to determine how these non-conserved residues play a role in the extension step of TLS
• Y-family DNA polymerases contribute to antibiotic resistance through their mutagenic DNA replication, prevent cancer by contributing to DNA damage tolerance, and contribute to chemotherapy resistance. Thus, this research addresses the problems of antibiotic resistance, some cancers, and chemotherapy resistance. Acknowledgments
Support from NSF-MCB-1517290, American Cancer Society RSG-12-161-01-DMC PhRMA Foundation (CLM)
