Presenter: Cassandra Lanette Carr, Claflin University Mentor, Dr. M. Wyatt, Coker Life Sciences (USC) The Cancer Research Training Program November 16, 2004 DNA REPAIR Quality Control by DNA Repair

DNA Double Helix

DNA Base Pairs AcceptorsHydrogen donorsGlycosidic bonds CH 3 Major Groove Minor Groove Major Groove Minor Groove

DNA base modifications can be toxic or mutagenic 3-Methyladenine is toxic because it blocks DNA polymerases Hypoxanthine (Hx, deaminated adenine) is mutagenic because DNA polymerases mis-insert cytosine Background Information

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

DNA Repair Systems From Science, 1999, p. 1897

OH PCNA polymerase  FEN1 DNA ligase Short patch (major)Long patch (minor) BER Pathway DNA glycosylase AP endonuclease OH polymerase  DNA ligase

Nature 411,366-74

Substrates include: –incorrect bases (e.g., uracil in DNA) –deaminated and oxidized bases –alkylated bases DNA glycosylases remove incorrect or damaged bases

DNA glycosylase DNA Glycosylase

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

AAG Substrates (7-MeG)(3-MeA)

Data

Glucose Plates Control 0.015% of MMS 0.025% of MMS Results MMS Gradient Plates Wild type E125Q Y165A L180S Low concentration High concentration

Galactose Plates Control 0.025% of MMS 0.03% of MMS More MMS Gradient Plates Low concentrationHigh concentration Wild type E125Q Y165A L180S

Chloroacetylaldehyde (CAA) introduces etheno-base damage. AAG Substrates

CCAA Gradient Plates Glucose Plates Control 0.003% of CAA 0.006% of CAA Wild type E125Q N169D N169S Low concentrationHigh concentration

CAA Gradient Plates Galactose Plates Control 0.003% of CAA 0.006% of CAA Wild type Low concentrationHigh concentration E125Q N169D N169S

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

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