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.
Why Methylate DNA?
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Methylation modifies nucleotides for
regulation of gene expression.
Used as methyl tag in prokaryotes for
genomic stability (mismatch repair).
Protects DNA from restriction
endonucleases.
Some Facts About
5-Methylcytosine

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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
Flagging/Controlling with
5-Methylcytosine
•
•
•
•
X-inactivation
Gene repression
Markers (bacteria)
Restriction and modification
What is X-inactivation?

Occurs only in female
somatic cells

Dosage compensation

Random inactivation
Gene Repression

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DNA methylation acts as gene regulator by
inactivating specific genes.
Inactive genes are highly methylated in CpG rich
islands near promoter sequence.
Genetic Markers in Bacteria

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During replication parent strand marked
Assists in replication fidelity
Restriction and Modification

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Endonuclease cleaves viral DNA
DNA methylation inhibits cleavage

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DNA sequence in modified
Viral DNA progeny able to continue
Structural Similarities of Pyrimidines
Project Scheme

Transition
mutagenesis is far
more likely to
originate at a mC-G
base pair than a
C-G base pair. Why?
Use of the M13 Phagemid

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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).

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Circularizes the filamentous DNA
Converts to double-stranded DNA
Methodology
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Purification of RF M13 plasmid using
Qiagen cellulose column.
Methylate four separate samples.

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1
1
1
1
sample
sample
sample
sample
W/T with Msp I methylase.
W/T with Hpa II methylase.
Mut with Msp I methylase.
Mut with Hpa II methylase.
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.
Methodology (continued)
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Run restriction digest with MspI and HpaII
endonucleases on the four samples.
0.8% agarose gel:
Lane
Lane
Lane
Lane
Lane
Lane
Lane
Lane
1: W/T restricted with Hpa II
2: HpaII W/T restricted with HpaII
3. W/T restricted with Msp I
4: Msp I W/T restricted with Msp I
5: Mut restricted with Msp I
6: Msp I Mut restricted with Msp I
7: Mut restricted with Hpa II
8: Hpa II Mut restricted with Hpa II
Cytosine Methylation Causes
Structural Insult to B-form DNA
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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).
Structural or Chemical Basis for
Mutagenesis?

Use M13 Construct (CCGG)
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Methylate outside cytosine using Msp1
methylase
Methylate inside cytosine using HpaII
methylase
Observe mutation rates over 4 month period
Experiment from 1993
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Studying mutation as a
function of methylation.
Qualitative color assay
using LacZα gene.
Constructed gene
unable to produce color.
Both reversion
mechanisms produce
color.
Spontaneous Deamination
Results from 1993 Experiment
Acknowledgements
Dr. Chris Mathews
Mathews’ Lab
HHMI
NSF