Catching RIP in the act.Catching RIP in the act. Part I: A PCR assay to Part I: A PCR assay to detect DNA methylationdetect DNA methylation

Paul DoneganPaul Donegan

Freitag LabFreitag Lab

Biochemistry and Biophysics DepartmentBiochemistry and Biophysics Department

Oregon State UniversityOregon State University

Background

• MUTAGENESIS: Mutations of base pairs in genetic material– Induced by UV, X-ray, viruses, etc.– Spontaneous occurrence – triggers DNA repair

• Hypermutagenesis– Induced and controlled by cells– Not spontaneous

--AID deaminase--ApoBec (HIV)

--RIP

R I P

• RIP = Repeat Induced Point Mutation

• Genomic defense mechanism– Silences repetitive DNA (no expression)

• Targets duplicated DNA segments – linked or unlinked sequences

• Induces C to T transition mutations

RIP triggered by repeated sequence

Identical Sequences

Mutated Sequences

C to T point mutations induced by RIP

GCATATTAGTTATGTTTAGCGCATTCTAGTATATCAGTTATGTTCAGTGCACTTTA

GCATATCAGTCATGCTCAGCGCACCTAGCATATCAGTCATGCTCAGCGCACCTA

Relevance

We are interested in RIP because we want to:

– gain insights into evolutionary mechanisms that shape genomes.

– understand genome defense mechanisms and mutagenesis.

Summer Research Objective• To differentiate between two possible molecular

mechanism that can explain RIP

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Neurosporacrassa

Rosette of sexual spores,nuclei labelled with GFP

Possible Mechanisms for C to T Mutations caused by RIP (1)

• Methylation by a specific cytosine DNA methyltransferase, followed by deamination

Methyl Group Donor- S-adenosylmethionine (SAM)

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C CMe T

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H3C

METHYLATION DEAMINATION

Possible Mechanisms for C to T Mutations caused by RIP (2)

• Cytosine is never methylated but instead deaminated to uracil, which will be replaced with thymine by DNA replication or repair

z

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C U

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Enz

DEAMINATION

Intermediate

Image from: Shiu et al. (2001) Cell

RIP timeline

• RIP occurs during the sexual cycle

• RIP occurs after fertilization but before karyogamy.

• ~10 mitotic divisions while RIP can occur.

FERTILIZATION

KARYOGAMY RIP ZONE!

• DNA was extracted during the expected RIP timeframe

• Methylation of interest should occur between fertilization and karyogamy (nuclear fusion).

0 1 2 3 4 5 6 7

DAY

RIP ZONE (between fertilization and karyogamy)

Methylation Assay Timeline

Controls

Days of Interest

PCR after Digest

Methylation-sensitive vs. methylation-insensitive restriction enzymes:

Sau3AI tests for cytosine methylation, based on the presence or absence of bands

Methylated site

Digest

PCR

GATCme

Digest with Sau3AI

DpnII is not sensitive to cytosine methylation:

-cuts regardless-control (never

amplifies)

Unmethylated site

Bands cannot be amplified when

site is cut

GATC

RFP

• ‘tdimerRed’ has two identical segments that trigger RIP• integrated into the Neurospora genome (not in WT)• here, we look for DNA methylation induced by RIP

• EVIDENCE OF METHYLATION SUGGESTS MECHANISM 1

Mutations in the RFP region

RFP amplification

Primers 1+3 (A) and 2+3 (B) amplified RFP bands only from RFP+ strainPrimers 5+6 (C) amplified control gene (hpo)

*

RFP region

1

2 3hpo5

6*

Bands from 5/6 appear in all genomic DNA’s but are absent in both plasmids

Genomic DNA (Neurospora) Plasmid DNA

wild typePrimers: A B C A B C

*

RFP- RFP+

**

* **

*

*

*

Experimental Control

RFP+

**

Primers: A B C A B C

**

*

BUT: Assay never worked with positive controls of methylated DNA

25 cycles 28 cycles 31 cycles

G S D G S D

hpo

G = genomic DNA, no digestS = Sau3AI, C-methylation sensitiveD = DpnII, C-methylation insensitive

Positive control:Methylated region

Negative control:Unmethylated region

Expected band in S lane,but no band in D lane

Expected no band in S or D lane

Catching RIP in the act.Part II: Tagging of duplicated DNA with fluorescent DNA

binding proteins

Goals• Tag DNA of Neurospora crassa with fluorescent proteins:

– to visualize pairing of duplications during RIP;

– to track chromosome territory movement (e.g., centromeres, telomeres, nucleolar DNA, specific genes)

– to track movements of DNA binding proteins from nucleus to nucleus

– to target enzymes to specific regions on chromosomes

Protein tagsTagging with RFP or GFP

Specific DNA binding proteins recognize target sequences (binding sites, BS).

Tag = translational fusion of a DNA binding domain (DBD) to RFP or GFP.

Binding sites recruit DBD-GFP or DBD-RFP fusion; co-localization = yellow.

GFP RFPBSDBD

BSDBD

DNA DNA

Protein Protein

During RIP

GFP RFP

GFP RFP

GFP RFP

GFP RFP

Construction of protein tags

3 Transformed E. coli

4 Purified plamids, digested DNA and confirmed correct plasmids

5 Linearized plasmid and transformed into Neurospora his-3 mutant

1 Amplified DBD from Aspergillus AflR and AlcR by PCR

2 Generated translational fusions by cloning into gfp and rfp plasmids

6 Selected His+ Neurospora transformants that showed fluorescence

AlcR-RFP AflR-GFP

Fusion proteins localized in nuclei

Construction of DNA binding sites

2 Binding site: DNA sequences specifically recognized by AflR or AlcR

AflR:TCGNNNNNCGA AlcR: GCGGRRCCGC

Need 200+ copies of recognized sequence to bind enough fluorescent protein for visibility.

Summary

1 PCR assay: Did not work in many attempts. We need a new approach.

2 DNA tagging: The protein tags are expressed, binding sites still needed.

Acknowledgements

• HHMI (Howard Hughes Medical Institute)• URISC (Undergraduate Research,

Innovation,Scholarship & Creativity)• Kevin Ahern• Michael Freitag • Kristina Smith• Freitag Lab