5. MUTATION
Mutation refers to any change in the genetic material (DNA) that
is heritable
Types of mutation:
Single base substitution
Transition
Transversion
Frameshift mutation
Rearrangement classes
Deletion
Inversion
Translocation
Duplication
(Ennis D.G., 2001)
6.
7. CAUSES OF MUTATION
DNA fails to copy accurately
External features can create mutations
Chemical or radiation break down in DNA
Error in DNA repair
8. TRANSCRIPTION ASSOCIATED MUTAGENESIS
Transcription copies only one DNA strand other remain single
stranded
Bacteria- relatively straight forward process
10. Collision of RNAP and replisome
Co directional collision occur in leading strand
Head on collision takes place in lagging strand
TRANSCRPTION – REPLICATION INTERACTION
11. COLLISION OF RNA POLYMERASE AND
REPLISOME
Leading strand
Lagging strand
12. SOURCE OF TAM
DNA damage- Alter specificity of codon- anticodon
Nascent mRNA by RNA poly.
Cytosine deamination on transcribed and non transcribed strand
R-loop
15. TRANSCRIPTION MEDIATED R-LOOP
R loop structure was first characterized by Thomas et al., 1976
and R loop exists in vivo is demonstrated by Crouch and
colleagues in 1995
R loop formation occur in bacterial cell and is consequences of
transcription process
(Drolet et al., 1995)
16. R-LOOPS
R loop is a 3 strand nucleic acid structure formed by RNA :DNA
hybrid plus a displaced DNA strand (ssDNA) identical to the RNA
molecule.
2 models:-
RNA-DNA hybrid model- not accepted
Threading back model
Depends on 3 features:
A. high G density
B. Negative supercoiling
C. DNA nicks
Roy et al., 2010
18. IMPACT OF REPAIR ON TAM
Transcription-associated mutations occurs asymmetrically on the
transcribed and non transcribed strands.
When all the transcription associated mutations that occur in a
cell are not repaired, its two daughter cells have different
genomic DNA.
19. DNA REPAIR
Three major DNA repairing mechanisms:
1.Base excision repair
2.Nucleotide excision repair
3.Mismatch repair
Friedberg et al.,1996
20. 1. DNA glycosylase
recognizes a damaged
base and cleaves between
the base
2. AP endonuclease cleaves
the phosphodiester
backbone near the AP site
3. DNA polymerase I re
synthesized the missing
part
4. The nick remaining after
DNA polymerase I is
sealed by DNA ligase
BASE EXCISION
21. NUCLEOTIDE EXCISION
1.Three exonucleases bind DNA at
the site of bulky lesion in which
UvrA bind first
2.UvrB binds to the UvrA-DNA
complex and increases
specificity of complex for
irradiated DNA.
3.UvrC remove DNA 8 bases
upstream and 4 or 5 bases
downstream of dimer.
4. The gap is filled by DNA
polymerase I
5. The remaining nick is sealed with
DNA ligase.
22. MISMATCH REPAIR
1. Repair process begins with the
protein MUT S which bind to
mismatch base repair.
2. MUT L form a complex and
activates MUTH which bind to
GATC sequences.
3. Activation of MUT H cleaves the
unmethylated strand at the
GATC site.
4.Subsequently the segment from the
cleavage site to the mismatch is
removed by exonuclease.
5. Gap is filled by DNA pol III and
DNA ligase.
24. TYPES OF MUTAGENESIS
PCR Based Methods
Site-directed mutagenesis
Mismatched mutagenesis
Insertional Mutagenesis
Transposon mutagenesis
In vivo Mutagenesis
Direct Mutagenesis
25. SITE DIRECTED MUTAGENESIS
Where a specific site in a cloned DNA needs to be altered in a
precise, pre-determined way
Can be designed to create specific nucleotide substitutions,
deletions, and so on
26. MISMATCHED MUTAGENESIS
Can create a desired point mutation at a unique predetermined
site within a cloned DNA molecule
At the intended mutation site it bears a base that is
complementary
27. DIRECT MUTAGENESIS
A largely discredited hypothesis proposing that organisms can
respond to environmental stresses through directing mutations to
certain genes or areas of the genome
28. DETECTION OF MUTATION
Two types:
Forward mutagenesis assay- inactivate a functional gene
Reversion mutagenesis assay, is essentially the reciprocal event, in
which a mutation restores the normal function
Reversion assay:
It is test for detection of mutagen which cause mutation
Reversion by frame-shift mutation
29. AMES test:
o Mutant strains of the bacteria Salmonella tymphimurium
o Mutation genotype His‾
o Plating His- S. typhimurium onto media with trace amounts
histidine and adding chemicals to be tested for mutagenicity
o Secondary mutations occur at a low spontaneous rate;
o Grow in media lacking histidine, these mutants are called
revertants
o The number of colonies growing on the plate indicates the
number of revertants
30. Mechanism of TAM in yeast
1. The location of the reporter on plasmid versus the chromosome.
2. The orientation of reporter relative to replication fork movement.
3. The specific growth condition used.
31. TAM in yeast is directly proportional to the level of gene
expression and influenced by the direction of DNA replication
Tetracycline regulated LYS 2 reporter system was developed to
modulate the transcriptional level over a broad range in S.
cerevisae.
(Kim et al., 2007)
32. Same orientation
Opposite orientation
(Kim et al., 2007)
Transcription impairs replication fork progression in a directional manner
Replication originDirection of transcription
33. TAM UNDER STRESS IN E. coli
Transcription associated mutations are considered to occur
regardless of specific secondary structures.
Transcription-associated mutagenesis becomes active under stress
and occurs in both genomic DNA and plasmid DNA in E.coli .
Transcription associated mutagenesis is considered to be an
intrinsic source of mutations.
(Kim et al., 2010)
34. The mutation rate is one base pair per genome per replication per
TAM .
TAM not affect on other genomic strand.
TAM can be considered a safe way for dividing cells to rapidly
increase the sequence diversity of the next generation.
Nondividing- nonrevertant, but engineered E.coli. for dividing
cell- stress applied- detect colonies of mutation .
(Kim et al., 2010)
Contd…
37. CSR AND SHM IN TRANSCRIPTION
ASSOCIATED MUTAGENESIS
Vertebrate antibody gene undergo 3 genetic alterations
CSR and SHM regulated by different mechanisms
RNA editing enzyme, activation-induced cytidine deaminase
(AID), regulates both in mouse and human
Regulation of two different types of genetic alteration
mechanism by AID indicates that mammals are equipped with
surprisingly sophisticated and complex layers of the genetic
alteration mechanisms to diversify our genomic information
(Honjo et al. , 2002)
38. Contd…
Mutation in Ig genes start from 150bp downstream of Ig promoter
AID –B cell specific deaminase converts cytosine to uracil in
ssDNA –initiates SHM and CSR
SHM- GC>TA transitions
CSR-double strand breaks
AID interact with DNA in stalled transcription bubble
40. Gene transcription increases DNA damage induced mutagenesis in
mammalian stem cells
In mammalian stem cell ,mutation in transcribed gene underlie genetic
diseases including cancer
The RNA polymerase stalls at UV lesions, forming a potential block for
replication fork
In vitro, stalled RNA polymerases do not pose any barrier for DNA
replication
Transcription affects DNA damage induced gene mutations
Double strand DNA breaks are generated when replication forks
collide with transcription complexes stalled at DNA lesions
(Hendriks et al., 2008)
41. PCR analysis of genomic
DNA from untreated (−UV)
and UV-irradiated Hprt-mg2
ES cells (+UV) to identify
intragenic deletions of the
Hprt minigene.
Cells were irradiated in the
absence of transcription (6-
TGR clones A–E) or
presence of transcription (6-
TGR clones F–J)
(Hendriks et al., 2008)
Effect of UV on transcription
42. APPLICATION OF MUTAGENESIS
Gene inactivation methods for genome-scale analysis include
transposon mutagenesis and gene disruption through allelic
exchange
High throughput mutagenesis techniques such as mRNA
expression inhibition and signature-tagged mutagenesis were
designed to efficiently extract novel biological information
relevant to an organism’s survival
43. Function of a particular gene is a multistep process ,
1. Using bioinformatics' tools to predict gene function
2. Measure gene and protein expression patterns
3. Functional analysis involves system perturbation where the
gene in question is inactivated
44. FUTURE PROSPECTS
Mutagenesis can be used as a powerful genetic tool:
By inducing mutations in specific ways and then observing the
phenotype of the organism the function of genes and even individual
nucleotides can be determined
Identifying molecular mechanism in which R loops promote
termination provide new insights to regulates gene expression at
transcription level