DNA repair system lecture that were prepered by Ph.D. students Mohammed Mohsen and Aliaa Hashim at microbiology department / college of medicine / babylon university.

1. :

2. • DNA repair is a collection of processes by which a cell identifies and corrects damage
to the DNA molecules that encode its genome. In human cells, both normal metabolic
activities and environmental factors such as radiation can cause DNA damage, resulting
in as many as 1 million individual molecular lesions per cell per day.
• Many of these lesions cause structural damage to the DNA molecule and can alter or
eliminate the cell's ability to transcribe the gene that the affected DNA encodes.
• When normal repair processes fail, and when cellular apoptosis does not occur,
irreparable DNA damage may occur, including double-strand breaks and DNA cross
linkages (interstrand crosslinks or ICLs)

3. 1. Direct reversal of the chemical reaction responsible for DNA damage, and
2. Removal of the damaged bases followed by their replacement with newly
synthesized DNA. Where DNA repair fails
The mechanisms of DNA repair can be divided into two general classes:

4. Mechanisms of DNA Repair
1. Direct Reversal of DNA Damage
• Only a few types of DNA damage are repaired in this way, particularly pyrimidine
dimers resulting from exposure to ultraviolet (UV) light and alkylated guanine
residues that have been modified by the addition of methyl (CH3) or ethyl (CH2-
CH3) groups at the O6 position of the purine ring.
• The major type of damage induced by UV light is the formation of pyrimidine
dimers, in which adjacent pyrimidines on the same strand of DNA are joined by
the formation of a cyclobutane ring resulting from saturation of the double bonds
between carbons 5 and 6.

5. • The formation of such dimers distorts:-
1. the structure of the DNA chain.
2. blocks transcription or replication past the site of damage, so their repair is
closely correlated with the ability of cells to survive UV irradiation.
• One mechanism of repairing UV-induced pyrimidine dimers is direct reversal of
the dimerization reaction. The process is called photoreactivation because energy
derived from visible light is utilized to break the cyclobutane ring structure.

6. Direct repair of thymine dimers. UV-induced thymine dimers can be repaired by photoreactivation, in which
energy from visible light is used to split the bonds forming the cyclobutane ring (double bond).

7. • The second form of direct repair deals with damage resulting from the reaction between
alkylating agents and DNA.
• Alkylating agents are reactive compounds that can transfer methyl or ethyl groups to a
DNA base, thereby chemically modifying the base.
• The principle of damage process?
• Methylation of o6 position of Guanine O6 methyl Guanine form complementary
base pairing with T instead of C
• Rapier mechanism by:-
• Enzyme called O6 methylguanine methyltransferase transfer methyl group from o6
methylguanine to Cysteine residues in its active site.
• As a result the original guanine is restored.

8. CH3
O6 methylguanine
methyltransferase

9. Single strand damage
• In excision repair, the damaged DNA is recognized and removed, either as free bases or
as nucleotides.
• The resulting gap is then filled in by synthesis of a new DNA strand, using the
undamaged complementary strand as a template.
• Three types of excision repair:-
1. Base -excision repair.
2. nucleotide-excision repair.
3. mismatch repair.
• Enable cells to cope with a variety of different kinds of DNA damage.

10. • The uracil-containing DNA can be repaired by base-excision repair, in which single
damaged bases are recognized and removed from the DNA molecule . Uracil can arise
in DNA by two mechanisms:-
• (1) Uracil (as dUTP [deoxyuridine triphosphate]) is occasionally incorporated in place
of thymine during DNA synthesis.
• (2) uracil can be formed in DNA by the deamination of cytosine (result in formation of
Uracil )
• The second mechanism is of much greater biological significance because it alters the
normal pattern of complementary base pairing and thus represents a mutagenic event
A- Base-excision repair

11. DNA glycosylase
DNA polymerase
Deoxyribose phosphodiesterase
AP endonuclease
Ligase
1. DNA glycosylase cleave the bond ( glycosidic bond) that
link U to deoxyribose of DNA backbone
2. This yield apyrimidinic site (AP site) its mean sugar without base attach + free Uracil
3. AP endonuclease recognized AP site and repairing it by cleaved adjacent to AP site
4. Deoxyribose phosphodiesterase remove remaining deoxyribose
5. Gap filled by DNA polymerase and sealed by the action of ligase
6. The result is incorporating of correct base C opposite to G
DNA containing U formed by deamination of C
AP Site

13. B- nucleotide-excision repair
Recognize a wide variety of damaged bases that distort the DNA molecule,
including UV-induced pyrimidine dimers and bulky groups added to DNA bases
as a result of the reaction of many carcinogens with DNA
• Why it is named by this name ?
• because the damaged bases (e.g., a thymine dimer) are removed as part of an
oligonucleotide containing the lesion.

14. DNA Polymerase
oligonucleotide
Damage recognition
nucleotide cleavage
Helicase Excised
Ligase
1. UV - induced pyramiding dimer
2. Damaged DNA is recognized and then cleaved on
both sides of a thymine dimer by 3′ and 5′ nucleases
3. Unwinding by a helicase results in excision of oligonucleotide
containing the damaged bases
4. The result gap is filled by DNA polymerase and sealed by ligase

15. 1. uvr A recognize damage site recruit uvr B+uvr C.
2. uvr B+uvr C cleave on 3 and 5 side of damaged site and the (Uvr B+C)
Excising oligonucleotide consisting 12 or 13 bases.
3. Helicase (uvr D) required to remove damaged
containing oligonucleotide from ds DNA molecules
4. Gap filled by DNA polymerase I and sealed by Ligase
 In E. coli, nucleotide-excision repair is catalyzed by the products of three
genes (uvrA, B, and C)
uvrA
uvrB uvrC

16. • Nucleotide-excision repair systems have also been studied extensively in
eukaryotes, particularly in yeasts and in humans. In yeasts, as in E. coli, several
genes involved in DNA repair (called RAD genes for radiation sensitivity) have
been identified by the isolation of mutants with increased sensitivity to UV light.
• In humans, DNA repair genes have been identified largely by studies of
individuals suffering from inherited diseases resulting from deficiencies in the
ability to repair DNA damage. The most extensively studied of these diseases is
xeroderma pigmentosum (XP), a rare genetic disorder that affects approximately
one in 250,000 people. Individuals with this disease are extremely sensitive to
UV light and develop multiple skin cancers on the regions of their bodies that are
exposed to sunlight.

19. C- Mismatch repair system

20.  In E. coli, the ability of the mismatch repair system to distinguish
between parental DNA and newly synthesized DNA is based on the fact
that DNA of this bacterium is modified by the methylation of adenine
residues within the sequence GATC to form 6- methyladenine
 Since methylation occurs after replication, newly synthesized DNA
strands are not methylated and thus can be specifically recognized by
the mismatch repair enzymes
 Mismatch repair is initiated by the protein MutS, which recognizes
the mismatch and forms a complex with two other proteins called
MutL and MutH
 The MutH endonuclease then cleaves the unmethylated DNA strand at
a GATC sequence
 MutL and MutS then act together with an exonuclease and a helicase
(uvr D) to excise the DNA between the strand break and the mismatch
 Resulting gap being filled by DNA polymerase and sealed by ligase.

22. Mismatch repair in mammalian cells

23.  In mammalian cells, it appears that the strand-specificity of mismatch
repair is determined by the presence of single-strand breaks (which
would be present in newly replicated DNA) in the strand to be repaired
 The eukaryotic homologs of MutS and MutL then bind to the
mismatched base and direct excision of the DNA between the strand
break and the mismatch, as in E. coli
 The importance of this repair system is dramatically illustrated by the
fact that mutations in the human homologs of MutS and MutL are
responsible for a common type of inherited colon cancer (hereditary
nonpolyposis colorectal cancer, or HNPCC)
 HNPCC is one of the most common inherited diseases; it affects as
many as one in 200 people and is responsible for about 15% of all
colorectal cancers in this country

25.  The repair of damage to both DNA strands is particularly important in maintaining genomic integrity.
 There are two main mechanisms for repairing double strand breaks: homologous recombination (HR) and classical
nonhomologous end joining(NHEJ).
 HR relies on undamaged template DNA as reference to repair the DSB, resulting in the restoration of the original sequence.
 NHEJ modifies and ligates the damaged ends regardless of homology
 DSB can occur naturally due to the presence of reactive species generated by metabolism, and various external factors (e.g.
ionizing radiation or chemotherapeutic drugs).
 In mammalian cells, there are numerous cellular processes that induce DSB :-
 Firstly, DNA topological strain from topoisomerase during normal cell growth can cause the majority a cell’s DSB.
 Secondly, cellular processes such as meiosis and the maturation of antibodies can cause nuclease-induced DSB.
 Thirdly, the cleavage of different DNA structures such as reversed or blocked DNA replication forks, and DNA interstrand
crosslinks (ICLs) can also cause DSB
DNA double strand break repair

26. Homologous recombination (HR)

27.  The chromosome formation occurs when the cell in
dividing state in S/G2 phase
 These 3 proteins (RAD 50+MRE11) come in contact with 5’
end .The function of MRN complex in DNA end resection ,pick
5’ end and make resection of thousand nucleotide leaving 3’
overhang
 The next protein RAD51 take help of BRCA 2 and
replace RPA on strand, function is help in search for
homologous DNA and help in process of invasion
 Small different proteins that can cover single strand DNA are
called as Replication protein A(RPA), The function of RPA is
to protect ssDNA from nucleases and to prevent them from
coiling back again.

28.  The invasion process lead to formation of D- loop
 The 3’ end use the strand as a template, once the
replication process complete, the termination
process start
 There are two option
first called Non- cross over HR repair :- all
homologous chromosomes will be like they were
before.
Or, they exchanged part with each other and called
cross- over HR repair

30.  KU- 70/80 detect or recognize the broken DNA ends, protect them from
nuclease activity and bind to DSB and recruit DNA-Pkcs
 DNA-Pkcs undergo auto-phosphorylation favoring the process of DNA
ends by Artemis which trims single strand overhangs due to it has
(exonuclease activity).
 Where DNA-PKcs refer to (Protein Kinase Catalytic Subunit).
 DNA ligase IV + XRCC4 + XLF complex ligate the
processed DNA ends
 XLF : stimulate Ligase IV activity, aiding in DSB by
formation of filament between XRCC4 and XLF.
 XRCC4: DNA repair protein and stabilize Ligase IV.
 This entire complex lead to fixation of dsDNA breaks.
 DNA Ligase IV help in repair ds breaks during lymphocyte
receptor development.
 Other factors (AFLP+PNKP) help in fixation
Non-homologous end joining

35. SOS Repair
SOS repair or “bypass” or “Emergency” repair is one of the DNA repair mechanisms.
SOS repair primarily recovers the DNA damage caused due to environmental stresses.
It serves as a regulatory system, which comprises many complex inducer proteins that
repair the damaged DNA
SOS system also includes a repressor protein, namely LexA. The RecA protein floats
around the cell, which regulates the activity of LexA protein.
The RecA regulatory protein mediates the repression or expression of LexA repressor
“SOS response system” refers to the mechanism in which an organism initiates the
production of activator protein (RecA), which results in the dissociation of LexA
repressor and activates the SOS inducer proteins.

36. It stands for “Save Our Soul”. The SOS system remains repressed until the conversion of RecA protein into RecA
protease.
It does not repair the DNA damage completely but provides tolerance ability to the affected organism.
 In normal DNA, LexA acts as a repressor protein that
binds to the particular site of DNA or SOS box. The
binding will repress the activity of SOS genes.
 But in mutated DNA, RecA acts as an activator of SOS
genes in the SOS system, which causes proteolysis of
the repressor protein and allows the SOS genes
expression into different DNA repairing inducer
proteins.

37.  Mechanism of SOS Repair 1. In case of excessive DNA damage, stress conditions etc., a cell
responds by activating signal or RecA protein. It floats in the vicinity
of the cell in search of any damage in the DNA.
2. A RecA protein specifically binds to the single stranded DNA. On
binding with the single stranded DNA fragments, RecA forms a
filament like structure around the DNA.
3. Then, a LexA repressor comes in contact with the nucleoprotein
filament assembled by the RecA protein. When RecA interacts with
the repressor protein, it converts into RecA protease.
4. The formation of RecA protease causes autocatalytic proteolysis of
LexA repressor protein. Thus, a LexA protein could not bind with the
SOS operator.
5. Inactivation of LexA protein activates the inducer proteins that
repair the DNA damage but alters the DNA sequence.
6. After DNA repair, the RecA protein loses its efficiency to cause
proteolysis, and the LexA protein will again bind to the SOS operator
or switch off the SOS system.

38.  SOS system only activates in case of excessive DNA damage, leading to single strand breakage at the replication fork. This DNA
damage activates the RecA regulatory protein that links with the single stranded DNA through the cellular energy ATP.
 RecA protein and ssDNA attachment will give rise to a right-handed nucleoprotein complex or = “RecA + ssDNA filament”.
The interaction of LexA repressor with the nucleoprotein complex causes proteolytic cleavage of LexA dimer.
 Proteolytic cleavage is due to the conversion of RecA protein into protease, which suppresses LexA protein’s activity.
 The SOS box genes will now express into different inducer proteins to recover the damaged DNA.
 The expression of inducer proteins will not occur all at once but express relatively to the type of DNA damage. Therefore, an
SOS system switches on and off in the presence and absence of activator RecA protein, respectively.
SOS conclusion
Rec A ssDNA
Nucleoprotein
filament
LexA
repressor
proteolytic
cleavage
of LexA
RecA
protease

39. SOS Inactivation
 An SOS system always switches off when a DNA is healthy. The
LexA promoter produces LexA repressor protein. The association
of LexA repressor to the consensus sequence (having 20 base
pairs of the SOS-box) suppresses the functioning of the SOS
system. Thus, LexA blocks the SOS box, which arrests the activity
of SOS genes that participate in the recovery of damaged DNA.
SOS activation
 The SOS repair system comes into action when the DNA is not
normal, and all the repair system fails. The organisms activate
the SOS system by themselves in response to the damage against
UV-light or any other factors

40. THANK YOU