1. 5-Methylcytosine as Mutagenic “Hot Spot” in Duplex DNA Presented by Blake Miller Department of Biochemistry and Biophysics Dr. Christopher Mathews Laboratory

2. What is 5-Methylcytosine? Modified nucleobase similar to cytosine but takes on different biochemical properties. Modified nucleobase similar to cytosine but takes on different biochemical properties.

3. Why Methylate DNA? Methylation modifies nucleotides for regulation of gene expression. Methylation modifies nucleotides for regulation of gene expression. Used as methyl tag in prokaryotes for genomic stability (mismatch repair). Used as methyl tag in prokaryotes for genomic stability (mismatch repair). Protects DNA from restriction endonucleases. Protects DNA from restriction endonucleases.

4. Some Facts About 5-Methylcytosine Represents about 2-3% of all cytosines in the mammalian genome Represents about 2-3% of all cytosines in the mammalian genome Represents <1% of all nucleotides in the genome Represents <1% of all nucleotides in the genome Responsible for 30-40% of point mutations leading to human genetic disorders or cancer Responsible for 30-40% of point mutations leading to human genetic disorders or cancer

5. Flagging/Controlling with 5-Methylcytosine X-inactivation X-inactivation Gene repression Gene repression Markers (bacteria) Markers (bacteria) Restriction and modification Restriction and modification

6. What is X-inactivation? Occurs only in female somatic cells Occurs only in female somatic cells Dosage compensation Dosage compensation Random inactivation Random inactivation

7. Gene Repression DNA methylation acts as gene regulator by inactivating specific genes. DNA methylation acts as gene regulator by inactivating specific genes. Inactive genes are highly methylated in CpG rich islands near promoter sequence. Inactive genes are highly methylated in CpG rich islands near promoter sequence.

8. Genetic Markers in Bacteria During replication parent strand marked During replication parent strand marked Assists in replication fidelity Assists in replication fidelity

9. Restriction and Modification Endonuclease cleaves viral DNA Endonuclease cleaves viral DNA DNA methylation inhibits cleavage DNA methylation inhibits cleavage DNA sequence in modified DNA sequence in modified Viral DNA progeny able to continue Viral DNA progeny able to continue

10. Structural Similarities of Pyrimidines

11. Project Scheme Transition mutagenesis is far more likely to originate at a mC-G base pair than a C-G base pair. Why? Transition mutagenesis is far more likely to originate at a mC-G base pair than a C-G base pair. Why?

12. Use of the M13 Phagemid M13 plasmid is 6.4 kb in length M13 plasmid is 6.4 kb in length Exists as filamentous, single-stranded phage DNA upon infection. Exists as filamentous, single-stranded phage DNA upon infection. Infects bacteria through sex pili coded by the F factor (JM105 and JM109 E. coli). Infects bacteria through sex pili coded by the F factor (JM105 and JM109 E. coli). Host cell converts DNA to replicative form (RF). Host cell converts DNA to replicative form (RF). Circularizes the filamentous DNA Circularizes the filamentous DNA Converts to double-stranded DNA Converts to double-stranded DNA

13. Methodology Purification of RF M13 plasmid using Qiagen cellulose column. Purification of RF M13 plasmid using Qiagen cellulose column. Methylate four separate samples. Methylate four separate samples. 1 sample W/T with Msp I methylase. 1 sample W/T with Msp I methylase. 1 sample W/T with Hpa II methylase. 1 sample W/T with Hpa II methylase. 1 sample Mut with Msp I methylase. 1 sample Mut with Msp I methylase. 1 sample Mut with Hpa II methylase. 1 sample Mut with Hpa II methylase.

14. Confirmation of Methylation Hpa II methylase creates nucleotide sequence that is resistant to Hpa II endonuclease restriction. Hpa II methylase creates nucleotide sequence that is resistant to Hpa II endonuclease restriction. Msp I methylase creates nucleotide sequence that is resistant to Msp I endonuclease restriction. Msp I methylase creates nucleotide sequence that is resistant to Msp I endonuclease restriction.

15. Methodology (continued) Run restriction digest with MspI and HpaII endonucleases on the four samples. Run restriction digest with MspI and HpaII endonucleases on the four samples. 0.8% agarose gel: 0.8% agarose gel: Lane 1: W/T restricted with Hpa II Lane 1: W/T restricted with Hpa II Lane 2: HpaII W/T restricted with HpaII Lane 2: HpaII W/T restricted with HpaII Lane 3. W/T restricted with Msp I Lane 3. W/T restricted with Msp I Lane 4: Msp I W/T restricted with Msp I Lane 4: Msp I W/T restricted with Msp I Lane 5: Mut restricted with Msp I Lane 5: Mut restricted with Msp I Lane 6: Msp I Mut restricted with Msp I Lane 6: Msp I Mut restricted with Msp I Lane 7: Mut restricted with Hpa II Lane 7: Mut restricted with Hpa II Lane 8: Hpa II Mut restricted with Hpa II Lane 8: Hpa II Mut restricted with Hpa II

16. Cytosine Methylation Causes Structural Insult to B-form DNA Subtle structural modification from B-form DNA to rare E-DNA conformation. Subtle structural modification from B-form DNA to rare E-DNA conformation. Exposes carbon #4 of cytosine base to water to favor deamination. Exposes carbon #4 of cytosine base to water to favor deamination. Methylation results in a 21-fold faster mutation rate (demonstrated in previous experiment). Methylation results in a 21-fold faster mutation rate (demonstrated in previous experiment).

17. Structural or Chemical Basis for Mutagenesis? Use M13 Construct (CCGG) Use M13 Construct (CCGG) Methylate outside cytosine using Msp1 methylase Methylate outside cytosine using Msp1 methylase Methylate inside cytosine using HpaII methylase Methylate inside cytosine using HpaII methylase Observe mutation rates over 4 month period Observe mutation rates over 4 month period

18. Experiment from 1993 Studying mutation as a function of methylation. Studying mutation as a function of methylation. Qualitative color assay using LacZα gene. Qualitative color assay using LacZα gene. Constructed gene unable to produce color. Constructed gene unable to produce color. Both reversion mechanisms produce color. Both reversion mechanisms produce color.

19. Spontaneous Deamination

20. Results from 1993 Experiment

22. Acknowledgements Dr. Chris Mathews Mathews’ Lab HHMI NSF