dam
Undergraduate Category: Physical and Life Sciences Degree Seeking: B.S. Biochemistry Abstract ID#1534
Determination of Escherichia coli Genes Important for DNA Repair Following Alkylation Christopher Joshi, Ariel Aiken, Emma Nash, Kelly Wong, Alyssa Carlson, Becky Leifer, Mark Muenter, Penny Beuning Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA
Background: Alkylating agents, common in industrial manufacturing and chemotherapy, can damage DNA by adding alkyl groups to nucleotides.
Data mfd, muH, and mutY strains with SO 1000
Direct base repair
•
Base excision repair
•
Nucleotide excision repair
Aim: better understand the mechanisms behind repair of alkylated DNA, first by confirming which genes are most critical.
% survival
DNA repair mechanisms can correct the lesions, including •
1000
wt+SO
100
mfd+SO mutH+SO
wt+CAA
100
mfd+CAA mutH+CAA
mutY+SO
Method: The survival of E. coli AB1157 strains, each with one of the 20 genes deleted, was measured upon exposure to one of two alkylating agents: styrene oxide (SO) or chloroacetaldehyde (CAA).
mutY+CAA
10
DNA alkylation:
0
Alkylating agents add carbon groups to the O- and N-atoms of DNA bases, causing incorrect base-pairing.
30 60 Exposure Time (min)
10
90
0
dam, nei, and nth strains with SO
30 60 Exposure Time (min)
90
dam, nei, and nth strains with CAA
1000
Alkylating agents studied:
Alkylating agent
mfd, muH, and mutY strains with CAA
% survival
Opportunity
1000
CAA
wt+SO
100
dam+SO nei+SO nth+SO
SO
Approach Bacterial strains were grown overnight
Colonies were counted and % survival was graphed vs. exposure time
10
Subcultures were incubated for ~40 min.
All serially diluted samples were plated on LB agar and grown overnight
Time point samples were serially diluted 1:10, 1:100, 1:1000, 1: 10,000, and 1:100,000
Alkylating chemicals (SO or CAA) were added to bacterial culture
Bacterial culture was sampled at 0 min, 30 min, 60 min, and 90 min. postexposure
Acknowledgements: Office of the Provost
dam+CAA nei+CAA
10
nth+CAA
30 60 Exposure Time (min)
90
0
30 60 Exposure Time (min)
90
Results • • • •
References:
1. Schlagman, S. L.; Hattman, S.; Marinus, M.G., Direct role of the Escherichia coli dam DNA methyltransferase in methylation-directed mismatch repair. J Bacteriol. 1985, 165 (3), 896-900. 2. Kropachev, K. Y.; Zharkov, D. O.; Grollman, A. P., Catalytic mechanism of Escherichia coli endonuclease VIII: roles of the intercalation loop and the zinc finger. Biochemistry 2006, 45 (39), 12039-49. 3. Mallik, R.; Carter, B. C.; Lex, S. A.; King, S. J.; Gross, S. P., Cytoplasmic dynein functions as a gear in response to load. Nature 2004, 427 (6975), 649-52. 4. Junop, M. S.; Yang, W.; Funchain, P.; Clendenin, W.; Miller, J. H., In vitro and in vivo studies of MutS, MutL and MutH mutants: correlation of mismatch repair and DNA recombination. DNA Repair 2003, 387-405.
wt+CAA
1 0
Subculture volumes were normalized to create comparable numbers of bacteria
% survival
% survival
100
The following knockout strains were more sensitive to the alkylating agent than the wild-type AB1157: dam+SO, dam+CAA, and nei+CAA. • Thus dam and nei probably play an important role in the DNA repair pathways. dam encodes DNA adenine methyltransferase, which methylates newly synthesized strands of DNA. dam knockouts have been shown to have increased rates of mutation (1). nei encodes endonuclease VIII, which is known to excise pyrimidines damaged by oxidation, as part of the base excision repair (BER) pathway (2). mutY and mutH knockout strains show modest increase in sensitivity to SO compared to wild-type AB1157. • mutY encodes adenine DNA glycosylase, which removes adenine as part of BER (3). • mutH encodes DNA mismatch repair protein, which cleaves the unmethylated DNA strand as part of mismatch repair (4).
Impact • • •
The survival assay used here quickly identifies genes that are important to DNA repair. Knowing the crucial genes will help elucidate the repair pathways. Better understanding repair pathways has implications in human oncology. • Similar genes to mutY, dam, and nei exist in humans
Determination of Escherichia coli Genes Important for DNA Repair Following Alkylation Christopher Joshi, Ariel Aiken, Emma Nash, Kelly Wong, Alyssa Carlson, Becky Leifer, Mark Muenter, Penny Beuning Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA
Background: Alkylating agents, common in industrial manufacturing and chemotherapy, can damage DNA by adding alkyl groups to nucleotides.
Data mfd, muH, and mutY strains with SO 1000
Direct base repair
•
Base excision repair
•
Nucleotide excision repair
Aim: better understand the mechanisms behind repair of alkylated DNA, first by confirming which genes are most critical.
% survival
DNA repair mechanisms can correct the lesions, including •
1000
wt+SO
100
mfd+SO mutH+SO
wt+CAA
100
mfd+CAA mutH+CAA
mutY+SO
Method: The survival of E. coli AB1157 strains, each with one of the 20 genes deleted, was measured upon exposure to one of two alkylating agents: styrene oxide (SO) or chloroacetaldehyde (CAA).
mutY+CAA
10
DNA alkylation:
0
Alkylating agents add carbon groups to the O- and N-atoms of DNA bases, causing incorrect base-pairing.
30 60 Exposure Time (min)
10
90
0
dam, nei, and nth strains with SO
30 60 Exposure Time (min)
90
dam, nei, and nth strains with CAA
1000
Alkylating agents studied:
Alkylating agent
mfd, muH, and mutY strains with CAA
% survival
Opportunity
1000
CAA
wt+SO
100
dam+SO nei+SO nth+SO
SO
Approach Bacterial strains were grown overnight
Colonies were counted and % survival was graphed vs. exposure time
10
Subcultures were incubated for ~40 min.
All serially diluted samples were plated on LB agar and grown overnight
Time point samples were serially diluted 1:10, 1:100, 1:1000, 1: 10,000, and 1:100,000
Alkylating chemicals (SO or CAA) were added to bacterial culture
Bacterial culture was sampled at 0 min, 30 min, 60 min, and 90 min. postexposure
Acknowledgements: Office of the Provost
dam+CAA nei+CAA
10
nth+CAA
30 60 Exposure Time (min)
90
0
30 60 Exposure Time (min)
90
Results • • • •
References:
1. Schlagman, S. L.; Hattman, S.; Marinus, M.G., Direct role of the Escherichia coli dam DNA methyltransferase in methylation-directed mismatch repair. J Bacteriol. 1985, 165 (3), 896-900. 2. Kropachev, K. Y.; Zharkov, D. O.; Grollman, A. P., Catalytic mechanism of Escherichia coli endonuclease VIII: roles of the intercalation loop and the zinc finger. Biochemistry 2006, 45 (39), 12039-49. 3. Mallik, R.; Carter, B. C.; Lex, S. A.; King, S. J.; Gross, S. P., Cytoplasmic dynein functions as a gear in response to load. Nature 2004, 427 (6975), 649-52. 4. Junop, M. S.; Yang, W.; Funchain, P.; Clendenin, W.; Miller, J. H., In vitro and in vivo studies of MutS, MutL and MutH mutants: correlation of mismatch repair and DNA recombination. DNA Repair 2003, 387-405.
wt+CAA
1 0
Subculture volumes were normalized to create comparable numbers of bacteria
% survival
% survival
100
The following knockout strains were more sensitive to the alkylating agent than the wild-type AB1157: dam+SO, dam+CAA, and nei+CAA. • Thus dam and nei probably play an important role in the DNA repair pathways. dam encodes DNA adenine methyltransferase, which methylates newly synthesized strands of DNA. dam knockouts have been shown to have increased rates of mutation (1). nei encodes endonuclease VIII, which is known to excise pyrimidines damaged by oxidation, as part of the base excision repair (BER) pathway (2). mutY and mutH knockout strains show modest increase in sensitivity to SO compared to wild-type AB1157. • mutY encodes adenine DNA glycosylase, which removes adenine as part of BER (3). • mutH encodes DNA mismatch repair protein, which cleaves the unmethylated DNA strand as part of mismatch repair (4).
Impact • • •
The survival assay used here quickly identifies genes that are important to DNA repair. Knowing the crucial genes will help elucidate the repair pathways. Better understanding repair pathways has implications in human oncology. • Similar genes to mutY, dam, and nei exist in humans
