Catching RIP in the act.
Part I: A PCR assay to
detect DNA methylation
Paul Donegan
Freitag Lab
Biochemistry and Biophysics Department
Oregon 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
Identical Sequences
RIP
• RIP = Repeat Induced Point Mutation
• Genomic defense mechanism
– Silences repetitive DNA (no expression)
RIP triggered by repeated sequence
• Targets duplicated DNA segments
– linked or unlinked sequences
• Induces C to T transition mutations
Mutated Sequences
GCATATCAGTCATGCTCAGCGCACCTA
GCATATCAGTCATGCTCAGCGCACCTA
C to T point mutations induced by RIP
GTATATCAGTTATGTTCAGTGCACTTTA
GCATATTAGTTATGTTTAGCGCATTCTA
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
Neurospora
crassa
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Rosette of sexual spores,
nuclei labelled with GFP
Possible Mechanisms for C to T
Mutations caused by RIP (1)
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
H3C
Quic kTime™ and a
TIFF (Unc ompres sed) dec ompres sor
are needed to see this pic ture.
METHYLATION
C
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
DEAMINATION
CMe
T
Methyl Group Donor- S-adenosylmethionine (SAM)
• Methylation by a specific cytosine DNA
methyltransferase, followed by deamination
Possible Mechanisms for C to T
Mutations caused by RIP (2)
DEAMINATION
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
z
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Enz
C
Intermediate
U
• Cytosine is never methylated but instead deaminated
to uracil, which will be replaced with thymine by
DNA replication or repair
RIP timeline
FERTILIZATION
• RIP occurs during the
sexual cycle
• RIP occurs after
fertilization but before
karyogamy.
• ~10 mitotic divisions
while RIP can occur.
KARYOGAMY
RIP ZONE!
Image from: Shiu et al. (2001) Cell
Methylation Assay Timeline
• DNA was extracted during the expected RIP timeframe
• Methylation of interest should occur between fertilization and
karyogamy (nuclear fusion).
RIP ZONE (between fertilization and karyogamy)
Controls
Days of Interest
0
1
2
3
4
5
6
7
DAY
PCR after Digest
Digest with Sau3AI
Unmethylated site
Methylation-sensitive vs.
methylation-insensitive
restriction enzymes:
Sau3AI tests for cytosine
methylation, based on the
presence or absence of bands
GATCme
GATC
Digest
PCR
DpnII is not sensitive to
cytosine methylation:
-cuts regardless
-control (never amplifies)
Bands cannot
be amplified
when site is cut
Methylated site
Mutations in the RFP region
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
RFP amplification
Genomic DNA (Neurospora)
Plasmid DNA
RFP+
ABC
wild type
Primers: A B C
**
**
*
*
Primers:
*
*
1
**
*
Bands from 5/6 appear in all genomic
DNA’s but are absent in both plasmids
*
3
RFP region
**
Control
*
5
2
RFP+
ABC
*
Experimental
Primers 1+3 (A) and 2+3 (B) amplified RFP bands
only from RFP+ strain
Primers 5+6 (C) amplified control gene (hpo)
RFPABC
hpo
6
BUT: Assay never worked with positive
controls of methylated DNA

G
S
hpo
D
G
Positive control:
Methylated region
D
Negative control:
Unmethylated region
Expected band in S lane,
but no band in D lane
25 cycles
S
Expected no band in S or D lane
28 cycles
G = genomic DNA, no digest
S = Sau3AI, C-methylation sensitive
D = DpnII, C-methylation insensitive
31 cycles
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 tags
Tagging 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.
During RIP
GFP
DBD
BS
Protein
DNA
GFP
RFP
GFP
RFP
GFP
RFP
GFP
RFP
BS
DBD
RFP
Protein
DNA
Construction of protein tags
1
Amplified DBD from Aspergillus AflR and AlcR by PCR
2
Generated translational fusions by cloning into gfp and rfp plasmids
3
Transformed E. coli
4
Purified plamids, digested DNA and confirmed correct plasmids
5
Linearized plasmid and transformed into Neurospora his-3 mutant
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