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

Duplex DNADuplex DNA

Presented byBlake Miller

Department of Biochemistry and Biophysics

Dr. Christopher Mathews Laboratory

What is 5-Methylcytosine?What is 5-Methylcytosine?

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

Why Methylate DNA?Why Methylate DNA?

Methylation modifies nucleotides for Methylation modifies nucleotides for regulation of gene expression.regulation of gene expression.

Used as methyl tag in prokaryotes Used as methyl tag in prokaryotes for genomic stability (mismatch for genomic stability (mismatch repair).repair).

Protects DNA from restriction Protects DNA from restriction endonucleases.endonucleases.

Some Facts About Some Facts About 5-Methylcytosine 5-Methylcytosine

Represents about 2-3% of all Represents about 2-3% of all cytosines in the mammalian cytosines in the mammalian genomegenome

Represents <1% of all Represents <1% of all nucleotides in the genomenucleotides in the genome

Responsible for 30-40% of point Responsible for 30-40% of point mutations leading to human mutations leading to human genetic disorders or cancergenetic disorders or cancer

Flagging/Controlling with Flagging/Controlling with 5-Methylcytosine 5-Methylcytosine

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

What is X-inactivation?What is X-inactivation? Occurs only in female Occurs only in female

somatic cellssomatic cells

Dosage compensationDosage compensation

Random inactivationRandom inactivation

Gene RepressionGene Repression DNA methylation acts as gene regulator by DNA methylation acts as gene regulator by

inactivating specific genes.inactivating specific genes. Inactive genes are highly methylated in CpG Inactive genes are highly methylated in CpG

rich islands near promoter sequence.rich islands near promoter sequence.

Genetic Markers in BacteriaGenetic Markers in Bacteria During replication parent strand markedDuring replication parent strand marked Assists in replication fidelityAssists in replication fidelity

Restriction and ModificationRestriction and Modification

Endonuclease cleaves viral DNAEndonuclease cleaves viral DNA DNA methylation inhibits cleavageDNA methylation inhibits cleavage

DNA sequence in modifiedDNA sequence in modified Viral DNA progeny able to continueViral DNA progeny able to continue

Structural Similarities of Structural Similarities of PyrimidinesPyrimidines

Project SchemeProject Scheme

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

Use of the M13 PhagemidUse of the M13 Phagemid M13 plasmid is 6.4 kb in lengthM13 plasmid is 6.4 kb in length Exists as filamentous, single-stranded Exists as filamentous, single-stranded

phage DNA upon infection.phage DNA upon infection. Infects bacteria through sex pili coded by Infects bacteria through sex pili coded by

the F factor (JM105 and JM109 E. coli).the F factor (JM105 and JM109 E. coli). Host cell converts DNA to replicative form Host cell converts DNA to replicative form

(RF).(RF). Circularizes the filamentous DNACircularizes the filamentous DNA Converts to double-stranded DNAConverts to double-stranded DNA

MethodologyMethodology

Purification of RF M13 plasmid using Purification of RF M13 plasmid using Qiagen cellulose column.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.

Confirmation of MethylationConfirmation of Methylation

Hpa II methylase creates nucleotide sequence Hpa II methylase creates nucleotide sequence that is resistant to Hpa II endonuclease that is resistant to Hpa II endonuclease restriction.restriction.

Msp I methylase creates nucleotide sequence Msp I methylase creates nucleotide sequence that is resistant to Msp I endonuclease that is resistant to Msp I endonuclease restriction.restriction.

Methodology (continued)Methodology (continued)

Run restriction digest with MspI and HpaII Run restriction digest with MspI and HpaII endonucleases on the four samples.endonucleases on the four samples.

0.8% agarose gel:0.8% agarose gel:

Lane 1: W/T restricted with Hpa IILane 1: W/T restricted with Hpa II Lane 2: HpaII W/T restricted with HpaIILane 2: HpaII W/T restricted with HpaII Lane 3. W/T restricted with Msp ILane 3. W/T restricted with Msp I Lane 4: Msp I W/T restricted with Msp ILane 4: Msp I W/T restricted with Msp I Lane 5: Mut restricted with Msp ILane 5: Mut restricted with Msp I Lane 6: Msp I Mut restricted with Msp ILane 6: Msp I Mut restricted with Msp I Lane 7: Mut restricted with Hpa IILane 7: Mut restricted with Hpa II Lane 8: Hpa II Mut restricted with Hpa IILane 8: Hpa II Mut restricted with Hpa II

Cytosine Methylation Causes Cytosine Methylation Causes Structural Insult to B-form DNAStructural Insult to B-form DNA

Subtle structural modification from B-form Subtle structural modification from B-form DNA to rare E-DNA conformation.DNA to rare E-DNA conformation.

Exposes carbon #4 of cytosine base to Exposes carbon #4 of cytosine base to water to favor deamination.water to favor deamination.

Methylation results in a 21-fold faster Methylation results in a 21-fold faster mutation rate (demonstrated in previous mutation rate (demonstrated in previous experiment).experiment).

Structural or Chemical Basis Structural or Chemical Basis for Mutagenesis?for Mutagenesis?

Use M13 Construct (CCGG)Use M13 Construct (CCGG) Methylate outside cytosine using Msp1 Methylate outside cytosine using Msp1

methylasemethylase Methylate inside cytosine using HpaII Methylate inside cytosine using HpaII

methylasemethylase Observe mutation rates over 4 month Observe mutation rates over 4 month

periodperiod

Experiment from 1993Experiment from 1993

Studying mutation as a Studying mutation as a function of methylation.function of methylation.

Qualitative color assay Qualitative color assay using LacZusing LacZαα gene. gene.

Constructed gene Constructed gene unable to produce unable to produce color.color.

Both reversion Both reversion mechanisms produce mechanisms produce color.color.

Spontaneous DeaminationSpontaneous Deamination

Results from 1993 Results from 1993 ExperimentExperiment

Acknowledgements

Dr. Chris MathewsMathews’ Lab

HHMINSF