5-Methylcytosine as Mutagenic

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5-Methylcytosine as Mutagenic Hot Spot in
Duplex DNA
Presented byBlake Miller Department of
Biochemistry and Biophysics
Dr. Christopher Mathews Laboratory
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What is 5-Methylcytosine?

• Modified nucleobase similar to cytosine but takes
  on different biochemical properties.

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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.

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Some Facts About
5-Methylcytosine

• Represents about 2-3 of all cytosines in the
  mammalian genome
• Represents lt1 of all nucleotides in the genome
• Responsible for 30-40 of point mutations leading
  to human genetic disorders or cancer

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Flagging/Controlling with
5-Methylcytosine

• X-inactivation
• Gene repression
• Markers (bacteria)
• Restriction and modification

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What is X-inactivation?

• Occurs only in female somatic cells
• Dosage compensation
• Random inactivation

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Gene Repression

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

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Genetic Markers in Bacteria

• During replication parent strand marked
• Assists in replication fidelity

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Restriction and Modification

• Endonuclease cleaves viral DNA
• DNA methylation inhibits cleavage
• DNA sequence in modified
• Viral DNA progeny able to continue

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Structural Similarities of Pyrimidines
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Project Scheme

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

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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

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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.

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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.

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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

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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).

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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

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Experiment from 1993

• Studying mutation as a function of methylation.
• Qualitative color assay using LacZa gene.
• Constructed gene unable to produce color.
• Both reversion mechanisms produce color.

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Spontaneous Deamination
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Results from 1993 Experiment
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Acknowledgements
Dr. Chris Mathews Mathews Lab HHMI NSF