Transcription-associated mutagenesis (TAM) refers to mutations that occur on the DNA strand being transcribed. The document discusses several causes and mechanisms of TAM, including DNA damage during transcription, cytosine deamination, and R-loop formation. It also covers TAM in yeast and E. coli, noting it increases under stress and affects both genomic and plasmid DNA. The roles of class switch recombination and somatic hypermutation in antibody gene diversification through TAM are also discussed.

1. MASTER’S SEMINAR –I
ON
SONAM MAHAWAR
PGS14AGR6524

2. OUTLINE
Introduction
Source of TAM
Types of mutagenesis
Detection of mutation
TAM in yeast
TAM in E.coli
Role of CSR and SHM
Future prospects
Conclusion

3. TRANSCRIPTION

4. Eukaryotes
Prokaryotes

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)

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

9. EFFECT OF TRANSCRIPTION ON DNA TEMPLATE
Mutagen

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

13. (Helmrich et al., 2013)

14. CYTOSINE DEAMINATION
Random incorporation of nucleotides leads further mutation
DC:DU mutation occurs at low level

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

17. (Stathaki and Proudfoot, 2015)

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.

23. Other repair for TAM
(Hendriks et al., 2010)

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…

35. DNA replication-independent production of erroneous
proteins
Damage
(Bregeon et al., 2011)

36. Transcriptional mutagenesis: causes and involvement
in tumor development
(Bregeon et al., 2011)

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

39. SHM AND CSR ANTIBODY MATURATION

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