E. coli
Undergraduate Category: Physical and Life Sciences Degree Level: B.S., Biochemistry Abstract ID #733
Characteriza0on of DNA Alkyla0on in E. coli Samir Baig, Sirine Bellou, Meghan Travers, Charles Conway, Caitlin Kramer, Mark Muenter, and Penny Beuning Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115
ABSTRACT
DNA alkylation is the process by which alkyl groups are covalently attached to DNA bases forming lesions that can cause mutations and lead to cancer. DNA alkylation is ubiquitous and can occur endogenously via cellular metabolism and exogenously from exposure to chemical and environmental agents. Alkylating agents are used in cancer chemotherapy as cytotoxic agents, but also can be present in industrial processes and as impurities in drug synthetic pathways. Hence, a comprehensive understanding of the DNA alkylation process is extremely important. Repair of DNA alkylation involves various processes including Base Excision Repair (BER), Nucleotide Excision Repair (NER), Mismatch Repair (MMR), Direct Repair (DR), and bypass by specialized DNA polymerases. Our research aims to determine the survival of E. coli cells upon treatment with styrene oxide, benzyl bromide, and chloroacetaldehyde, three alkylating agents found in various industrial processes (plastics, chemical, pharmaceutical) and identify how cellular responses to these agents correlate with the identity and frequency of adducts. The survival of wild-type E. coli and knockouts of twenty-seven specific genes associated with the various DNA repair processes are being examined following exposure to each alkylating agent to determine which genes are required for resistance to alkylation damage and to what degree. We have identified a number of DNA repair genes that are critical for survival in response to a given agent, including genes involved in nucleotide excision repair and direct repair. We plan to profile the DNA adducts formed from benzyl bromide and the adducts that persist in certain knockout strains via HPLC-(ESI)-MS. This work will allow us to identify the range of adducts formed and the contribution of specific DNA repair pathways to repair defined adducts.
AIM To determine the range of adducts formed in E. coli by different alkyla@ng agents and their suscep@bility to repair.
BACKGROUND
Response in E. coli SOS
Alkyla0ng Agents
Adap0ve Reponse in E. coli
7 mL LB 140µL o/n
35-40 min @ 37oC OD600 0.1-0.2 750 µL/OD = VSC
CH3
Alkylated DNA
CH3
Alkyla@ng agents alkylate DNA via SN1 or SN2 mechanisms, disrup@ng normal Watson-‐Crick DNA base pairing and leading to DNA damage in the form of various adducts. Accumula@on of these adducts can lead to impairment and/or loss of cellular func@on. Above, atoms colored red indicate sites most open methylated by SN1 alkyla@ng agents, while atoms in green indicate sites commonly modified by SN2 alkyla@ng agents. Atoms colored orange represent sites open methylated in single stranded DNA, while those colored blue designate exocyclic amino groups important in forma@on of cyclized DNA adducts.
Alkylated Ada protein Repaired DNA
ada
Ada DNA Alkyltransferase
Part A. Cloning The Gene Plasmid DNA
WT E.coli
Cut plasmid DNA using same restric@on enzymes as gene Dephosphorylate to prevent plasmid from liga@ng to itself Amplify gene from WT using designed plasmids containing restric@on sites
Cut Plasmid DNA
E. Coli with plasmid DNA Ligate and transform Into cells
Isolate plasmid DNA from cells
Plasmid DNA with gene
Gene from amplifica@on in large amounts with s@cky ends for liga@on
Percent Survival of E. coli strain dele@ons exposed to alkyla@ng agents:
1) Zone of inhibi@on assays
(1) Chloroacetaldehyde (2) Styrene Oxide (3) Benzyl Bromide
Percent Survival (Log10 Scale/Error Analysis) Versus Time of E. coli AB1157 Derivatives Versus WT Exposed to Benzyl Bromide (0.14 mg/mL) 100.000 AB1157 WT Delta ada
% Survival (Log10)
Delta recJ
Delta recN Delta rnt Delta ruvA
10.000
Delta sbmC Delta sulA Delta symE Delta umuC Delta uvrA Delta uvrD Delta uvrY
% Survival (log Scale)
Delta dinB
10
Delta umuD
WT
Delta umuDC
1.000
Delta dinB Delta umuDC
0
Exposure Time (Minutes)
(dG)
Frick, L.E.; Delaney, J.C.; Wong, C.; Drennan, C.L.; Essigmann, J.M. Allevia@on of 1,N6-‐ethanoadenine genotoxicity by the Escherichia coli adap@ve response protein AlkB Proc. Nat. Acad. Sci. USA 104(3) (2007) 755-‐76 New Research Direc@ons in DNA Repair, Chapter 5: “Direct Repair in Mammalian Cells” by S. L. Nay and T. R. O’Connor (edited by Clark Chen, ISBN 978-‐953-‐51-‐1114-‐6, Published: May 22, 2013) 123-‐162 Lehninger, et al. Principles of Biochemistry, 2nd Edi@on Quinlivan, E. P.; Gregory, J. F., 3rd, DNA diges@on to deoxyribonucleoside: a simplified one-‐step procedure. Analy@cal biochemistry 2008, 373 (2), 383-‐5.
ACKNOWLEDGEMENTS
Delta uvrC
Delta yebG
100
+
REFERENCES
100
50
2) Protein purifica@on
Delta ybfE
0
in vitro
f ybfE Characteriza0on o
% Survival Versus Time
0.1
Glycosylase
RESULTS
Delta recE
1
Dioxygenase
in vivo:
Delta recA
0 30 60 90 min.
AlkA
The normal survival assay will be conducted with the transformants from above and % survival will be examined to see if complementa@on was successful. The expected result is that WT will do the best, dele@on will do the worst with empty vector about the same and the dele@on strain with the plasmid+gene will do beher than the dele@on. This will confirm that the gene is cri@cal for repair of alkylated DNA
Delta mug
1. Centrifuge T0 3 min/6K rcf 2. Remove SN and wash/resuspend in 500 µL of 0.85% saline 3. Store on Ice 4. Repeat all steps for all timepoints
AidB AlkB
Several plasmids need to be transformed into strains of E. coli for complementa@on The transformants needed for complementa@on are as follows: a) WT b) Dele@on strain, e.g. ΔumuDC c) Dele@on strain transformed with gene-‐containing plasmid d) Dele@on strain transformed with emplty plasmid (no gene inserted)
Delta nfo
180 µL 0.85% saline into Rows C à G 100 µL washed RSP into Row B Serial Dilutions: 20 µL B à C; C à D…..F à G Plate 10 µL from G à B (bottom of plate à top)
aidB
• In vitro studies by LC-‐(ESI)-‐MS: React free nucleosides (dG; dA; dC; dT) with benzyl bromide and iden@fy/profile DNA adducts formed • In vivo studies by LC-‐(ESI)-‐MS: Analyze damaged DNA (DNA extrac@on; diges@on to nucleosides) from specific E. coli strain dele@ons showing high sensi@vity to benzyl bromide Examples: AB1157(ΔybfE and ΔrecA)
Part B. Survival Assays
To confirm that these genes are cri@cal for the repair of alkylated DNA and there isn’t some other factor involved, we have designed a protocol that allows the gene to be expressed in cells that have the gene deleted using plasmids
Delta mutS
1. 2. 3. 4.
alkA
Ada regulon
Delta dinG
S
alkB
COMPLEMENTATION STUDIES EXPERIMENTAL DESIGN
1. Centrifuge 10 min @ 3.8k rcf 2. Remove supernatant (SPN) 3. Resuspend pellet in 1 mL LB (New 15 mL snap caps) 4. 900 µL resuspension (RSP) à “S” eppie 5. 90 µL “S” RSP à T0 eppie 6. 90 µL AA into “S” eppies; mix
0
• Dele@on of recA and ybfE confers high sensi@vity styrene oxide, benzyl bromide, and chloroacetaldehyde and benzyl bromide • Complementa@on studies to confirm percent survival (are high sensi@vi@es (high cellular death) due to the specific gene dele@on?
Ada protein
Delta mutM
96-Well Plate Preparation: Using Multi-channel pipettes…...
• Strains are generally more resistant to styrene oxide and chloroacetaldehyde compared to benzyl bromide
Alkylating agent
SURVIVAL ASSAY EXPERIMENTAL DESIGN
CONCLUSIONS/FUTURE WORK
30
60
Exposure Time (minutes)
90
Delta alkB
Dr. Roger Giese, Barneh Ins@tute – Northeastern University Dr. April Gu, Civil and Environmental Engineering – Northeastern University Man Hu, Civil and Environmental Engineering – Northeastern University Dr. Keith McCarthy, Process R&D Rhodes Technologies Dr. Ewa Sokol, Process R&D Rhodes Technologies
Characteriza0on of DNA Alkyla0on in E. coli Samir Baig, Sirine Bellou, Meghan Travers, Charles Conway, Caitlin Kramer, Mark Muenter, and Penny Beuning Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115
ABSTRACT
DNA alkylation is the process by which alkyl groups are covalently attached to DNA bases forming lesions that can cause mutations and lead to cancer. DNA alkylation is ubiquitous and can occur endogenously via cellular metabolism and exogenously from exposure to chemical and environmental agents. Alkylating agents are used in cancer chemotherapy as cytotoxic agents, but also can be present in industrial processes and as impurities in drug synthetic pathways. Hence, a comprehensive understanding of the DNA alkylation process is extremely important. Repair of DNA alkylation involves various processes including Base Excision Repair (BER), Nucleotide Excision Repair (NER), Mismatch Repair (MMR), Direct Repair (DR), and bypass by specialized DNA polymerases. Our research aims to determine the survival of E. coli cells upon treatment with styrene oxide, benzyl bromide, and chloroacetaldehyde, three alkylating agents found in various industrial processes (plastics, chemical, pharmaceutical) and identify how cellular responses to these agents correlate with the identity and frequency of adducts. The survival of wild-type E. coli and knockouts of twenty-seven specific genes associated with the various DNA repair processes are being examined following exposure to each alkylating agent to determine which genes are required for resistance to alkylation damage and to what degree. We have identified a number of DNA repair genes that are critical for survival in response to a given agent, including genes involved in nucleotide excision repair and direct repair. We plan to profile the DNA adducts formed from benzyl bromide and the adducts that persist in certain knockout strains via HPLC-(ESI)-MS. This work will allow us to identify the range of adducts formed and the contribution of specific DNA repair pathways to repair defined adducts.
AIM To determine the range of adducts formed in E. coli by different alkyla@ng agents and their suscep@bility to repair.
BACKGROUND
Response in E. coli SOS
Alkyla0ng Agents
Adap0ve Reponse in E. coli
7 mL LB 140µL o/n
35-40 min @ 37oC OD600 0.1-0.2 750 µL/OD = VSC
CH3
Alkylated DNA
CH3
Alkyla@ng agents alkylate DNA via SN1 or SN2 mechanisms, disrup@ng normal Watson-‐Crick DNA base pairing and leading to DNA damage in the form of various adducts. Accumula@on of these adducts can lead to impairment and/or loss of cellular func@on. Above, atoms colored red indicate sites most open methylated by SN1 alkyla@ng agents, while atoms in green indicate sites commonly modified by SN2 alkyla@ng agents. Atoms colored orange represent sites open methylated in single stranded DNA, while those colored blue designate exocyclic amino groups important in forma@on of cyclized DNA adducts.
Alkylated Ada protein Repaired DNA
ada
Ada DNA Alkyltransferase
Part A. Cloning The Gene Plasmid DNA
WT E.coli
Cut plasmid DNA using same restric@on enzymes as gene Dephosphorylate to prevent plasmid from liga@ng to itself Amplify gene from WT using designed plasmids containing restric@on sites
Cut Plasmid DNA
E. Coli with plasmid DNA Ligate and transform Into cells
Isolate plasmid DNA from cells
Plasmid DNA with gene
Gene from amplifica@on in large amounts with s@cky ends for liga@on
Percent Survival of E. coli strain dele@ons exposed to alkyla@ng agents:
1) Zone of inhibi@on assays
(1) Chloroacetaldehyde (2) Styrene Oxide (3) Benzyl Bromide
Percent Survival (Log10 Scale/Error Analysis) Versus Time of E. coli AB1157 Derivatives Versus WT Exposed to Benzyl Bromide (0.14 mg/mL) 100.000 AB1157 WT Delta ada
% Survival (Log10)
Delta recJ
Delta recN Delta rnt Delta ruvA
10.000
Delta sbmC Delta sulA Delta symE Delta umuC Delta uvrA Delta uvrD Delta uvrY
% Survival (log Scale)
Delta dinB
10
Delta umuD
WT
Delta umuDC
1.000
Delta dinB Delta umuDC
0
Exposure Time (Minutes)
(dG)
Frick, L.E.; Delaney, J.C.; Wong, C.; Drennan, C.L.; Essigmann, J.M. Allevia@on of 1,N6-‐ethanoadenine genotoxicity by the Escherichia coli adap@ve response protein AlkB Proc. Nat. Acad. Sci. USA 104(3) (2007) 755-‐76 New Research Direc@ons in DNA Repair, Chapter 5: “Direct Repair in Mammalian Cells” by S. L. Nay and T. R. O’Connor (edited by Clark Chen, ISBN 978-‐953-‐51-‐1114-‐6, Published: May 22, 2013) 123-‐162 Lehninger, et al. Principles of Biochemistry, 2nd Edi@on Quinlivan, E. P.; Gregory, J. F., 3rd, DNA diges@on to deoxyribonucleoside: a simplified one-‐step procedure. Analy@cal biochemistry 2008, 373 (2), 383-‐5.
ACKNOWLEDGEMENTS
Delta uvrC
Delta yebG
100
+
REFERENCES
100
50
2) Protein purifica@on
Delta ybfE
0
in vitro
f ybfE Characteriza0on o
% Survival Versus Time
0.1
Glycosylase
RESULTS
Delta recE
1
Dioxygenase
in vivo:
Delta recA
0 30 60 90 min.
AlkA
The normal survival assay will be conducted with the transformants from above and % survival will be examined to see if complementa@on was successful. The expected result is that WT will do the best, dele@on will do the worst with empty vector about the same and the dele@on strain with the plasmid+gene will do beher than the dele@on. This will confirm that the gene is cri@cal for repair of alkylated DNA
Delta mug
1. Centrifuge T0 3 min/6K rcf 2. Remove SN and wash/resuspend in 500 µL of 0.85% saline 3. Store on Ice 4. Repeat all steps for all timepoints
AidB AlkB
Several plasmids need to be transformed into strains of E. coli for complementa@on The transformants needed for complementa@on are as follows: a) WT b) Dele@on strain, e.g. ΔumuDC c) Dele@on strain transformed with gene-‐containing plasmid d) Dele@on strain transformed with emplty plasmid (no gene inserted)
Delta nfo
180 µL 0.85% saline into Rows C à G 100 µL washed RSP into Row B Serial Dilutions: 20 µL B à C; C à D…..F à G Plate 10 µL from G à B (bottom of plate à top)
aidB
• In vitro studies by LC-‐(ESI)-‐MS: React free nucleosides (dG; dA; dC; dT) with benzyl bromide and iden@fy/profile DNA adducts formed • In vivo studies by LC-‐(ESI)-‐MS: Analyze damaged DNA (DNA extrac@on; diges@on to nucleosides) from specific E. coli strain dele@ons showing high sensi@vity to benzyl bromide Examples: AB1157(ΔybfE and ΔrecA)
Part B. Survival Assays
To confirm that these genes are cri@cal for the repair of alkylated DNA and there isn’t some other factor involved, we have designed a protocol that allows the gene to be expressed in cells that have the gene deleted using plasmids
Delta mutS
1. 2. 3. 4.
alkA
Ada regulon
Delta dinG
S
alkB
COMPLEMENTATION STUDIES EXPERIMENTAL DESIGN
1. Centrifuge 10 min @ 3.8k rcf 2. Remove supernatant (SPN) 3. Resuspend pellet in 1 mL LB (New 15 mL snap caps) 4. 900 µL resuspension (RSP) à “S” eppie 5. 90 µL “S” RSP à T0 eppie 6. 90 µL AA into “S” eppies; mix
0
• Dele@on of recA and ybfE confers high sensi@vity styrene oxide, benzyl bromide, and chloroacetaldehyde and benzyl bromide • Complementa@on studies to confirm percent survival (are high sensi@vi@es (high cellular death) due to the specific gene dele@on?
Ada protein
Delta mutM
96-Well Plate Preparation: Using Multi-channel pipettes…...
• Strains are generally more resistant to styrene oxide and chloroacetaldehyde compared to benzyl bromide
Alkylating agent
SURVIVAL ASSAY EXPERIMENTAL DESIGN
CONCLUSIONS/FUTURE WORK
30
60
Exposure Time (minutes)
90
Delta alkB
Dr. Roger Giese, Barneh Ins@tute – Northeastern University Dr. April Gu, Civil and Environmental Engineering – Northeastern University Man Hu, Civil and Environmental Engineering – Northeastern University Dr. Keith McCarthy, Process R&D Rhodes Technologies Dr. Ewa Sokol, Process R&D Rhodes Technologies
