presenter: cassandra lanette carr, claflin university mentor, dr. m. wyatt, coker life sciences...
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![Page 1: Presenter: Cassandra Lanette Carr, Claflin University Mentor, Dr. M. Wyatt, Coker Life Sciences (USC) The Cancer Research Training Program November 16,](https://reader036.vdocuments.us/reader036/viewer/2022062423/56649d425503460f94a1df4e/html5/thumbnails/1.jpg)
Presenter: Cassandra Lanette Carr, Claflin University
Mentor, Dr. M. Wyatt, Coker Life Sciences (USC)
The Cancer Research Training ProgramNovember 16, 2004
DNA REPAIR
Quality Control by DNA Repair
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DNA Double Helix
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N
N
N
N
N
R
H
H
N
N
N
NO
N
R
H
H
H
N
N
N
O R
H
H
NN
O
O
R
H
adenine
guanine cytosine
thymine
DNA Base Pairs
Acceptors Hydrogen donors Glycosidic bonds
CH3
Major Groove
Minor Groove
Major Groove
Minor Groove
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DNA base modifications can be toxic or mutagenic
N
N
N
NN
N
N
N
NOH
H
R
HR
CH3
+
• 3-Methyladenine is toxic because it blocks DNA polymerases
• Hypoxanthine (Hx, deaminated adenine) is mutagenic because DNA polymerases mis-insert cytosine
Background Information
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What causes DNA damage?
• Replication errors (base:base mismatches, insertion/deletion loops)
• Oxidative/hydrolytic damage (base damage, base loss)
• UV and x-rays• Carcinogens (alkylation damage)
– nitrosoamines, benzo[a]pyrene, aflatoxin • Most cancer chemotherapeutic drugs
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DNA Repair Systems
From Science, 1999, p. 1897
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OH
OH
PCNApolymerase
FEN1
DNA ligase
Short patch (major) Long patch (minor)
BER PathwayDNA glycosylase
AP endonucleaseOH
OH
polymerase
DNA ligase
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Nature 411,366-74
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• Substrates include:– incorrect bases (e.g., uracil in
DNA)– deaminated and oxidized bases– alkylated bases
DNA glycosylases remove incorrect or
damaged bases
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DNA glycosylase
DNA Glycosylase
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N
NN
N
NH2
CH3
+
N
NN
N
NH2
RR
Human 3-methyladenine DNA Glycosylase (AAG)
• Wide substrate range, removing a variety of damaged bases
• 3-methyladenine DNA glycosylases protect cells from methyl methanesulfonate (MMS) toxicity
adenine
methylation
3-methyladenine
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N
NN
N
NH2
CH3R
3-methyladenine 3-methylguanine 7-methylguanine
NH
NN
N
O
NH2
CH3R
NH
NN
N
O
NH2
H3C
R
N
NN
N
O
H
R
hypoxanthine xanthine
N
NN
N
O
O
H
H
R
AAG Substrates
(7-MeG)(3-MeA)
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.
Glycosylase-deficient cells are sensitiveto MMS on a gradient plate
- glycosylase+ glycosylase
MMS concentration
Data
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Glucose Plates
•Control
•0.015% of MMS
•0.025% of MMS
ResultsMMS Gradient Plates
Wild typeE125QY165AL180S
Low concentration High concentration
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Galactose Plates
•Control
•0.025% of MMS
•0.03% of MMS
More MMS Gradient Plates
Low concentration High concentration
Wild typeE125QY165AL180S
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Chloroacetylaldehyde (CAA) introduces etheno-base damage.
AAG Substrates
1, -ethenoadenineN6
N
NN
N
N
R
N
NN
N
O
NH
R
1, -ethenoguanineN2
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CCAA Gradient Plates
Glucose Plates
• Control
• 0.003% of CAA
• 0.006% of CAA
Wild typeE125QN169DN169S
Low concentration High concentration
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CAA Gradient Plates
Galactose Plates
• Control
• 0.003% of CAA
• 0.006% of CAA
Wild type
Low concentration High concentration
E125QN169DN169S
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The purpose of the gradient plate assay
• To qualitatively assess glycosylase activity by
measuring the survival of yeast when challenged with DNA damaging agents MMS and
CAA
What is different about CAA versus MMS
• MMS creates methylation damage
• 3-methyladenine is very toxic
– Wild-type 3-methyladenine DNA glycosylase protects the yeast from MMS toxicity
• CAA creates etheno-base damage
• etheno-adenine is toxic and very mutagenic
– Wild-type 3-methyladenine DNA glycosylase protects the yeast from CAA toxicity and mutagenicity
Conclusion
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Future Research
Future Research
•Finish more plates using CAA to gather further results
•Start working on a plasmid miniprep kit
This kit is designed to extract a DNA plasmid from a host cell