Various causes of DNA damage, Methods of DNA repair for the Damage to the DNA structure, Gene regulation and Gene Expression in eukaryotes and Prokaryotes.

1. Molecular Biology – DNA Repair,
Gene Mutation, Gene Expression
Dr Anurag Yadav
MBBS, MD
Assistant Professor
Department of Biochemistry
Instagram page –biochem365
YouTube – Dr Biochem365
Email: dranurag.y.m@gmail.com
MNR MEDICAL COLLEGE & HOSPITAL

2. Content
DNA Damage
DNA repair Mechanism
Mutation
Gene Expression

4. DNA Damage & Repair
• Being the carrier of genetic information, the cellular
DNA must be replicated (duplicated), maintained, and
passed down to the daughter cells accurately.
• In general, the accuracy of replication is extremely high.
• However, there do occur replication errors.
• The cells do posses the capability to repair damages
done to DNA to a large extent.

5. Causes of DNA Damage
• Misincorporation of deoxynucleotides during
replication
• By spontaneous deamination of bases during normal
genetic functions
• From x-radiation that cause “nicks” in the DNA
• From UV irradiation that causes thymine dimer
formation
• From various chemicals that interact with DNA.

7. MUTATIONS
Mutation: Change in the DNA structure of a
gene.
Mutagens: are Substance which bring about
(induce) mutation are collectively.
Changes that occur in DNA on mutation are
reflected in replication, transcription and
translation.

8. Types of Mutation
Two major
types
Point
mutation
Frameshift
mutation

9. 1. Point Mutation
• The replacement of one base pair by another
results in point mutation.
– Transition: a purine (or pyrimidine) is replaced by
another.
– Transversion: characterised by replacement by a
purine by a pyrimidine or vice versa.

10. 2. Frameshift Mutation
• Occur when one or more base pairs are
inserted in or deleted from the DNA.
• Causing insertion or deletion mutation.

11. Consequences of Point Mutation
1. Silent Mutation: There is no detectable
effects.
Eg: UCA code is changed to UCU, but still codes
for Serine.
This is due to degeneracy of the genetic code.

12. Consequences of Point Mutation
2. Missense mutation: the changed base may code
for an different amino acid.
Eg: UCA codes for serine, replaced to ACA which
codes for threonine.
Amino acid may be acceptable, or Partially
acceptable or Unacceptable.
Eg: sickle cell anemia.

13. Consequences of Point Mutation
3. Nonsense mutation: sometime, the code with
the altered base may become a termination
codon.
Eg: second base change in UCA of serine to UAA -
which is a termination codon.
Causing premature termination of the protein
synthesis.

14. Consequences of Frameshift
mutations
• The insertion or deletion of a base in a gene
results in an altered reading frame of the mRNA.
• The translation continues with new codon
inserted.
• Result in protein with several altered aminoacids
or premature termination of protein synthesis.

15. REPAIR OF DNA
Cell possess the inbuilt system to repair
the damaged DNA.
• Base excision repair
• Nucleotide excision repair
• Mismatch repair
• Doubled strand break repair
Achieved by
4 distinct
mechanisms.

16. 1. BASE EXCISION REPAIR
• The bases cytosine, adenine and guanine can
undergo spontaneous depurination to
respectively form uracil, hypoxanthine and
xanthine.
• These altered bases do not exist in the normal
DNA, and therefore need to be removed.
• This is carried out by base excision repair

18. 2. NUCLEOTIDE EXCISION REPAIR
• The DNA damage due to ultraviolet light, ionizing
radiation and other environmental factors often
results in the modification of certain bases,
strand breaks, cross-linkages etc.
• Nucleotide excision-repair is ideally suited for
such large-scale defects in DNA.

19. 2. NUCLEOTIDE EXCISION REPAIR
• After the identification of the defective piece of the
DNA, the DNA double helix is unwound to expose
thedamaged part.
• An excision nuclease (exinuclease) cuts the DNA on
either side (upstream and downstream) of the
damaged DNA. This defective piece is degraded.
• The gap created by the nucleotide excision is filled up
by DNA polymerase which gets ligated by DNA ligase

21. • Xeroderma pigmentosum (XP) is a rare
autosomal recessive disease. The affected
patients are photosensitive and susceptible to
skin cancers. It is now recognized that XP is
due to a defect in the nucleotide excision
repair of the damaged DNA.

22. 3. MISMATCH REPAIR
• Despite high accuracy in replication, defects
do occur when the DNA is copied.
• Cytosine (instead of thymine) could be
incorporated opposite to adenine.
• Mismatch repair corrects a single mismatch
base pair e.g. C to A, instead of T to A.

23. Template strand
of the DNA
exists in a
methylated
form,
while the newly
synthesized
strand is not
methylated.

24. • Hereditary nonpolyposis colon cancer
(HNPCC) is one of the most common
inherited cancers. This cancer is now linked
with faulty mismatch repair of defective DNA.

25. 4. DOUBLE-STRAND BREAK REPAIR
• Double-strand breaks (DSBs) in DNA are
dangerous.
• They result in genetic recombination which may
lead to chromosomal translocation, broken
chromosomes, and finally cell death.
• DSBs can be repaired by homologous
recombination or non-homologous end joining

28. GENE EXPRESSION AND REGULATION
• Organisms adapt to environmental changes by
altering gene expression.
• The regulation of the expression of genes is
necessary for the growth, development,
differentiation and the very existence of the
organism.

29. TYPES OF GENE EXPRESSION
TWO MAIN
TYPES
POSITIVE
REGULATION
NEGATIVE
REGULATION

30. 1. POSITIVE REGULATION
• When the expression of genetic information is
quantitatively increased by the presence of
specific regulatory element, it is called as
positive regulation.
• The element or molecule mediating positive
regulation is called positive regulator.

31. 2. NEGATIVE REGULATION
• When the expression of genetic information is
decreased by the presence of a specific
regulatory element, it is called as negative
regulation.
• The element or molecule mediating the negative
regulation is called a negative regulator.

32. ONE CISTRON-ONE SUBUNIT
CONCEPT
• Earlier hypothesis proposed that one gene
produces one enzyme or protein and “one gene-
one enzyme” concept was introduced.
• It is now known that some enzymes and protein
molecules are composed of two or more
nonidentical subunits, which cannot be explained
by “one gene-one enzyme” theory and so it is not
valid.

33. • The cistron is now considered as the genetic unit
coding for the structure of the subunit of an
enzyme or protein molecule, acting as it does as
the smallest unit of genetic expression.
• Hence, the “one gene-one enzyme” idea might
be more accurately regarded as “one cistron-one
subunit concept”.

34. THE OPERON CONCEPT
• The Operon is the coordinated unit of genetic
expression in bacteria.
• The concept of Operon was introduced by
Jacob and Monod in 1961.
• regulation of lactose metabolism in E. coli.
This is popularly known as LAC OPERON.

35. Operon is defined as a segment
of a DNA strand consisting of:
•Structural genes
•Operator gene
•Regulator gene
•P site (Promoter site)

36. Lac-operon Structure
consists of a regulatory gene (I; I for inhibition),
operator gene (O) and
three structural genes (Z, Y, A).
Besides these genes, there is a promoter site (P), next to the
operator gene, where the enzyme RNA polymerase binds.
The structural genes Z, Y and A respectively, code for the
enzymes β-galactosidase, galactoside permease and galactoside
acetylase.

37. • β-Galactosidase hydrolyses lactose (β-
galactoside) to galactose and glucose while
permease is responsible for the transport of
lactose into the cell.

39. Repression of Lac operon
• The regulatory gene (I) is constitutive. It is
expressed at a constant rate leading to the
synthesis of lac repressor.
• lac repressor binds to operator gene (O).
• Prevents binding of enzyme RNA polymerase to
promoter site (P).
• There by blocking the transcription of Structural
gene.

40. DEREPRESSION OF LAC OPERON
• In the presence of lactose (inducer) in the
medium, a small amount of it can enter the E.
coli cells. The repressor molecules have a high
affinity for lactose.
• The lactose molecules bind and induce a
conformational change in the repressor.

41. • Repressor gets inactivated and cannot bind to
operator gene (O).
• RNA polymerase attaches to DNA at promoter
site and transcription proceeds.
• Leading to formation of polycistronic mRNA
and finally 3 enzymes are formed.
• Thus lactose induce synthesis of 3 enzymes.

44. GENE EXPRESSION IN EUKARYOTES
• Each cell of the higher organism contains the
entire genome. As in prokaryotes, gene
expression in eukaryotes is regulated to
provide the appropriate response to biological
needs.
• This may occur in the following ways

45. • Expression of certain genes (housekeeping
genes) in most of the cells.
• l Activation of selected genes upon demand.
• l Permanent inactivation of several genes in all
but a few types

46. • Histone acetylation and deacetylation
• Methylation of DNA and inactivation of genes

48. Important Q
• Mutation
• Types of Mutation
• Clinical consequence of Mutation
• Causes of DNA damage
• DNA repair mechanisms
• Gene Expression and Regulation
• Types of Gene regulation