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5-Methylcytosine as Mutagenic “Hot Spot” in Duplex DNA

5-Methylcytosine as Mutagenic “Hot Spot” in Duplex DNA. Presented by Blake Miller Department of Biochemistry and Biophysics. Dr. Christopher Mathews Laboratory. What is 5-Methylcytosine?. Modified nucleobase similar to cytosine but takes on different biochemical properties.

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5-Methylcytosine as Mutagenic “Hot Spot” in Duplex DNA

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  1. 5-Methylcytosine as Mutagenic “Hot Spot” in Duplex DNA Presented byBlake 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.

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

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

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

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

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

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

  9. Restriction and Modification • Endonuclease cleaves viral DNA • DNA methylation inhibits cleavage • DNA sequence in modified • 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?

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

  13. Methodology • Purification of RF M13 plasmid using Qiagen cellulose column. • Methylate four separate samples. • 1 sample W/T with Msp I methylase. • 1 sample W/T with Hpa II methylase. • 1 sample Mut with Msp I 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. • 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. • 0.8% agarose gel: • Lane 1: W/T restricted with Hpa II • Lane 2: HpaII W/T restricted with HpaII • Lane 3. W/T restricted with Msp I • Lane 4: Msp I W/T restricted with Msp I • Lane 5: Mut restricted with Msp I • Lane 6: Msp I Mut restricted with Msp I • Lane 7: 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. • Exposes carbon #4 of cytosine base to water to favor deamination. • Methylation results in a 21-fold faster mutation rate (demonstrated in previous experiment).

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

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

  19. Spontaneous Deamination

  20. Results from 1993 Experiment

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

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