1. Assays for Determining
Lesion Bypass Efficiency
and Mutagenicity of
Site-Specific DNA
Lesions In Vivo
Paper by: James C. Delaney and John M. Essigmann
Presentation by: Patrick J. Dumas

2. DNA Lesions
 Unrepaired DNA damage may hinder translesion synthesis
 Mutagenicity
 Cytotoxicity
 Cells with defined lesions may be used as hosts for
replication
 Assays may identify specific enzymes/pathways in repair process
(AlkB)
 Differentiate between different mutations and repair processes

3. Genome Construction
 ss-M13 vector
 Treated with EcoRI
 2 Scaffolds are used on either side of lesion
 No base pair opposite lesion
 Equal Ligation efficiency
 Oligonucleotide ligation
 Scaffold/DNA polymerase removal
 Desalt
 Transfection

4. Genome Normalization
 Determine relative concentration of genome
 Incubations with HinFI – cleave 5’ & radiolabel P
HaeIII – cleave 3’
 Separate segments via PAGE
 Normalize to 34-mer insert

5. Lesion Bypass (CRAB) Assay
 Competitive Replication of Adduct Bypass
 Premix lesion vector & non-lesion vector, compare ratio
 Accounts for transfection inefficiencies
 Unbiased PCR amplification of lesion & non-lesion genome

6. CRAB Assay cont...
 BbsI enzyme cleaves at 5’ end of insert
 32P radiolabeling at exposed 5’ end
 HaeIII trims at 3’ end
 Creates: Fully ligated insert 34-mer, 21-mer competitor, 18-mer lesion signal

7. Lesion Mutagenesis (REAP) Assay
 Restriction Endonuclease and Postlabeling anaylsis
 Used to determine mutagenicity of several DNA lesions from entire output
 Radiolabel 5’ phosphate, separate hydrolyzed 5’-32P dNMPs on TLC plate
 Can also be used to verify site-specific incorporated DNA lesion post genome
construction

8. REAP Assay
 PCR primers selectively amplify lesion genome
 Polymerase is destroyed, and mixture desalted
 None of the possible mutation outcomes of the lesion sequence will create
cleavage sites for BbsI or HaeIII, ensuring proper representation
 Frameshift Mutations
 May occur because PCR primers do not anneal at 5-base region containing the
lesion
 Accounted for by carrying a 17-mer oligonucleotide w/o lesion as -1 marker control

9. REAP Cont...
 PAGE analysis
 Radiolabeled 18-mer develops as well-defined band on gel
 (14-mer counterpart is dissolved in 20% denaturing gel)
 Band containing 18-mer isolated by “crush and soak”
 TLC analysis
 Isolated 5’-dNMPs transferred to TLC plate
 Treated with variety of buffers, result analyzed

10. Conclusion/future
 Describes latest version of lesion bypass and mutagenesis
assays
 REAP assay has been used with 50 lesions including
oxidative, alklyative and non-natural atomically mutated
base analog damage
 Tested in E. coli and cells of higher organisms
 Great potential in Human chromosomal damage
evaluation