2. Summary of last lecture
• DNA damage can occur naturally in vivo and also due to
exogenous sources
• Bases or side chains can undergo hydrolysis (depurination,
deamination) resulting in deletions or transversions
• Base analogs, intercalating reagents
• UV and Ionizing radiations
• Repaired directly or excision repair
• Base, nucleotide and mismatch repair
• Double strand breaks are produced by accident or ionizing
radiations
3. Double strand break repair
Double strand breaks are repaired by either
• Nonhomologous end-joining
Ends are degraded (some sequence is lost) and ends are joined by
DNA Ligase IV
Or by
• Homologous end-joining
The DNA sequence from the other chromosome is used to restore the
sequence and then it is ligated to restore normal sequence (uses
enzymes and process as in recombination)
5. NHEJ and ZFN gene engineering
(EXTRA)
• Artificially generated Zinc finger nucleases specifically target gene
sequences
• Bind to DNA at specific sequences
• Nuclease activity induces double stranded breaks
• NHEJ by DNA ligase IV
• Results in mutations
6. Translesion synthesis
• For AP sites and thymine dimers
• DNA polymerase IV or V, Y
• Large active sites that can facilitate the insertion of bases opposite
damaged nucleotides
• Can be error prone but some times accurate
• Polymerase Pol η (eta), Pol ζ (zeta) and many other enzymes and
proteins
• Pol ζ can extend terminal mismatches, others polymerases cannot
7. What do cells do if damage is extreme?
• Cells exposed to large amounts of ionizing radiation, ultraviolet
light or chemicals
• Accumulation of damage, specifically double strand breaks or
stalled replication forks, signal a global response to DNA damage
• Undergo apoptosis and die, or survive at the cost of living with
a modified genome
• SOS response in bacteria
8. SOS response in bacteria
Global response
SOS system is regulated by two key proteins: LexA and RecA
LexA homodimer is a transcriptional repressor that binds to operator
sequences of genes at SOS boxes
SOS activated by single stranded DNA (ssDNA), or double strand
breaks
RecA when bound with ssDNA activates autoprotease activity of LexA
which results in LexA dimer cleavage and degradation
and forms DNA polymerase V together with some subunits from
DNA polymerase III and repairs the defect
Error prone
9. Response in eukaryotes
• Simple Eukaryotic cells--same global response
• Complex eukaryotic organisms: different cells respond to damage
differently
• Yeast Rev1 and human polymerase η are DNA polymerases
present during global response to DNA damage
• Error prone replication
10. Repair defects
In humans Xeroderma Pigmentosa
• XP patients are extremely sensitive to UV light and develop multiple
skin cancers on sunlight exposure
• Deficient in the ability to carry out nucleotide-excision repair
• Molecular cloning has now identified seven different repair genes
(XPA through XPG)
11. DNA damage and mutation
• DNA damage and mutation, the two major types of error in DNA
• DNA damages and mutation are different
• Damages are physical abnormalities in the DNA
• DNA damages can be recognized and repaired
• A mutation is a change in the base sequence of the DNA
• If the base change is present in both DNA strands, a mutation
cannot be repaired
• DNA damages can result in mutations if they are not repaired
15. DNA methylation
• In bacteria GATC is methylated by dam (dna methylases)
• In vertebrates CpG islands upstream many eukaryotic genes are
methylated
• Excessive methylation of these islands can result in gene silencing
• Degree of methylation can also have an effect on to the extent a
gene will be repressed
16. Mechanism
• DNA methyl transferase (DNMT) transfers methyl groups to
selected C residues in CpG islands
• Methylation inhibits transcription of these genes via the action of a
protein, MeCP2 (methyl CpG-binding protein 2)
• MeCP2 specifically binds to methylated DNA and represses
transcription
• MeCP2 functions as a complex with histone deacetylase, linking
DNA methylation to alterations in histone acetylation and
nucleosome structure
18. Methylome
• DNA methylation can alter gene expression without changing the
DNA sequence
• Each cell has its own specific methylation pattern
• In humans there are 100-214 cell types
• Assume 400,000 methylated cytosines
• 40-80 million methylation events will have to be studied
• NIH project to map methyl sites in the genome (Roadmap
Epigenomics project)-Papers published in 2015
20. Epigenetic gene regulation comprises the heritable changes in gene
expression that occur in the absence of changes to the DNA sequence
Heritable over rounds of cell division and sometimes to next
generation
Involve
DNA modification
DNA modification involved in epigenetics
Methylation
Paula Dominguez-Salas et al. 2014 Maternal nutrition at conception
modulates DNA methylation of human metastable epialleles NATURE
COMMUNICATIONS
21. An example
Agouti viable yellow mice
• Have a retrotransposon inserted at the 5’ end of the agouti locus
which derives pigment production in mice (Avy)
• The methylation status of the retrotransposon determines
expression of the alleles at this locus (Yellow or agouti mice)
Hypothesis
• As diet-derived methyl donors and cofactors are necessary for the
synthesis of S-adenosylmethionine, required for CpG methylation
early nutrition may influence adult phenotype via DNA methylation
(shown to be true)
23. RNA modification
• Adenine is methylated by m6A methyltransferase
• N6-Methyladenosine (m6A)
• In many mRNA of viruses, yeast and most eukaryotes including
mammals, insects, and plants
• Also found in noncoding RNA, tRNA, rRNA, SnRNA and XIST
24. Special case of DNA modification
X-inactivation
(Lyonization)
25. TSIX (noncoding RNA) is expressed from both X chromosomes but its
expression becomes confined to the active chromosome
When TSIX is low, XIST is high
XIST (a noncoding RNA-X-inactivation specific transcript) coats
one X
XIST is only expressed by the inactive chromosome
YY1 tethers XIST RNA to the inactive X nucleation center Cell. 2011 146:119-133
XIST recruits other proteins
Silences by methylation of DNA and histone modification
XIST and TSIX are overlapping genes and TSIX is antisense to XIST
XIST is around 17kb while TSIX is about 40kb
X-inactivation is random in every cell in most mammals, so females
are a mosaic for expression of genes on X-Chromosome
26. XIST is only expressed from the inactive X
RNA from cell lines
One X chromosome is selected to remain active
(some autosomal factor protects it from inactivation?)
28. Types of X-inactivation
1. Random X inactivation
2. Imprinted X inactivation (preferential X chromosome)
Barr body
X-inactivation is reversed in the female germline, so that all oocytes
contain an active X chromosome
• Dosage compensation (different mechanism in flies, worms and
mammals)
Some genes can still be expressed on the inactive X chromosome
Pseudoautosomal regions
Inactivation is usually after blastocysts form in humans occurring at
about the time of gastrulation in the epiblast
30. Involves paternal or maternal gene expression or suppression
(Parent-of-Origin-Specific gene expression)
Methylation
Results in silencing of genes on that chromosomal region
Maternally imprinted genes are expressed only from the father’s
chromosome
Paternally imprinted genes are expressed only from the mother’s
chromosome
Imprinting is only present in insects, plants, placental mammals (and
not in monotremes)
Is set in each gamete, so after fertilization, the imprint is present in
the zygote
32. Imprinting, methylation and cancer
1. Loss of imprinting of normally imprinted gene
• Igf2 loss of imprinting results in predisposition to colorectal
cancer
With loss of imprinting, IGF2 is overexpressed resulting in over growth
2. Methylation of genes which are normally unmethylated
• MLH1 methylation can also cause a form of colorectal cancer
MLH1 is a tumour repressor gene and its methylation results in gene
silencing
33. Other modifications
• A group of prokarytoic genes, known as the dnd gene cluster,
gives the ability to add Sulfur to the sugar-phosphate DNA
backbone as a phosphorothioate
• S in place of non-bridging Oxygen
• Sequence- and stereo-specific manner
• Phosphorothioate is present about every several thousand base
pairs in these genomes
• Many different strains of prokaryotes
• Gene cluster dndA-E, can be passed between prokaryotes, much
like genes for antibiotic resistance
• Role: Modification against restriction?
34.
35. 1. Jia G, Fu Y, Zhao X, Dai Q, Zheng G, Yang Y, Yi C, Lindahl T, Pan T, Yang
YG, He C. N6-methyladenosine in nuclear RNA is a major substrate of the obesity-
associated FTO. Nat. Chem. Biol. 2011 7: 885–887.
2. Meyer KD, Saletore Y, Zumbo P, Elemento O, Mason CE, Jaffrey SR
Comprehensive Analysis of mRNA Methylation Reveals Enrichment in 3' UTRs
and near Stop Codons. Cell. 2012 149: 1635–1646.
3.Dad’s Diet Shapes Children’s Chromatin
http://www.biotechniques.com/news/Dads-Diet-Shapes-Childrens-Chromatin/biotechniques
-356406.html#.VWLUTE-qqko
Ost, A., Lempradl, A., Casas, E., et al (2014). Paternal Diet Defines Offspring
Chromatin State and Intergenerational Obesity. Cell 159(6): 1352–1364.
4.The Living, Breathing Human Genome
http://www.biotechniques.com/news/The-Living-Breathing-Human-Genome/
biotechniques-356943.html#.VWLT_k-qqko
5. DNA methylation age of blood predicts all-cause mortality in later life.
http://www.biotechniques.com/news/DNA-Methylation-Predicts-Death/
biotechniques-356521.html#.VWLjgk-qqko
Genome Biol. 2015 Jan 30;16(1):25.
EXTRA
