presenter: cassandra lanette carr, claflin university mentor, dr. m. wyatt, coker life sciences...

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

DNA Double Helix

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

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

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

OH

PCNApolymerase

FEN1

DNA ligase

Short patch (major) Long patch (minor)

BER PathwayDNA glycosylase

AP endonucleaseOH

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

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

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)

.

Glycosylase-deficient cells are sensitiveto MMS on a gradient plate

- glycosylase+ glycosylase

MMS concentration

Data

Glucose Plates

•Control

•0.015% of MMS

•0.025% of MMS

ResultsMMS Gradient Plates

Wild typeE125QY165AL180S

Low concentration High concentration

Galactose Plates

•Control

•0.025% of MMS

•0.03% of MMS

More MMS Gradient Plates

Low concentration High concentration

Wild typeE125QY165AL180S

Chloroacetylaldehyde (CAA) introduces etheno-base damage.

AAG Substrates

1, -ethenoadenineN6

N

NN

N

N

R

N

NN

N

O

NH

R

1, -ethenoguanineN2

CCAA Gradient Plates

Glucose Plates

• Control

• 0.003% of CAA

• 0.006% of CAA

Wild typeE125QN169DN169S

Low concentration High concentration

CAA Gradient Plates

Galactose Plates

• Control

• 0.003% of CAA

• 0.006% of CAA

Wild type

Low concentration High concentration

E125QN169DN169S

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

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