DNA REPAIR

Ricardo R. Santos, MD
 LEARNING OBJECTIVES

1. Discuss the types of damage to DNA

2. Explain the different repair mechanisms

3. Enumerate some diseases associated with

damage to DNA

4. Differentiate mutation from polymorphism

 DNA molecules are irreplaceable

 Maintaining the integrity of the information

in DNA is imperative

 DNA integrity is supported by an elaborate

set of DNA repair systems
 DNA damage could be spontaneous or
triggered by environmental agents

 Unrepaired DNA damage results in a change

in the base sequence of the DNA

 Damaged DNA is then replicated and

transmitted to future generations of cells

 A permanent change in the nucleotide

sequence of DNA results (mutation)
TYPES OF DAMAGE TO DNA
I. Single-base alteration
A. Depurination
B. Deamination of cytosine to uracil
C. Deamination of adenine to hypoxanthine
D. Alkylation of base
E. Insertion or deletion of nucleotide
F. Base-analog incorporation

II. Two-base alteration

A. UV light-induced thymine-thymine dimer
B. Bifunctional alkylating agent cross-linkage
1. Depurination – loss of purine bases adenine &
guanine
- caused by chemical reaction with intermediate
metabolites
- results in formation of apurinic site
- repaired by base excision repair system
- can cause mutation

2. Alkylation of bases – caused by environmental agents

(methylating agents like nitrosamines, methylnitros-
urea) and akylating drugs (streptozotocin and
temozolamide)
- can cause mutation because of their miscoding
properties
- can also cause strand breaks
- repaired by base excision repair mechanism
3. Base analog incorporation
- can substitute for a normal nucleobase
- categorized into purine analogs and pyrimidine
analogs
- example is bromouracil which pair with adenine
- its isomer can pair with guanine
- results in transition point mutation
- repaired by base excision
TYPES OF DAMAGE TO DNA

III. Chain-breaks
A. Ionizing radiation
B. Radioactive disentegration of backbone
element
C. Oxidative free radical formation

IV. Cross-linkage
A. Between bases in same or opposite strands
B. Between DNA and protein molecules
 DNA REPAIR

• Cells have developed a number of systems

designed to repair DNA damage and correct
mutation.

• DNA repair is possible largely because the DNA

molecules consists of two complementary
strands

• DNA damage in one strand can be removed and

accurately replaced by using the undamaged
complementary strand as the template
 DNA REPAIR

• These mechanisms are not perfectly successful,

but without them mutation rates would be much
higher.

• Repair reduces the error rate from one in ten

million bases to one in a billion.
• Fewer than 1 in 1000 become a mutation.
(Leninger Principles of Biochemistry 6 th edition)
DNA REPAIR
corrects

caused by

by
for
for
TABLE1. DNA DAMAGE, CAUSES, & REPAIR
MECHANISM OF
DNA DAMAGE CAUSES
REPAIR
1. Mismatch of one to Replication error Methyl-directed
several bases escaped proofreading in mismatch repair
DNA synthesis
2. DNA polymerase Exposure of a cell to UV Nucleotide excision
cannot replicate DNA light covalently joins 2 repair
strand beyond the site adjacent pyrimidines
of dimer formation. (thymines) producing a
thymine dimer
3. Abnormal bases Cytosine deamination Base excision repair
or improper use of
dUTP instead of dTTP
during DNA synthesis
 TABLE1. DNA DAMAGE, CAUSES, & REPAIR
MECHANISM OF
DNA DAMAGE CAUSES
REPAIR
4.double-strand breaks High-energy radiation, Homologous
oxidative free radicals, recombination or Non-
or occur naturally homologous end-joining
during gene repair
rearrangements

References: Lippincott (5e) & Harper (27e)

 DNA DAMAGE: MISMATCH OF BASES

 Errors are a natural part of DNA replication

 Replication errors escape the proofreading

function during DNA synthesis, causing a
mismatch of one to several bases.
 METHYL DIRECTED MISMATCH REPAIR
STEPS
1. Identification of the
mismatched strand:
• by Mut proteins
(in E. coli)
• Discriminate
between the correct
strand and strand
with mismatch:
degree of
methylation

• GATC sequences
(1:1000
nucleotides) are
methylated on the
adenine residue
• Newly
synthesized DNA
is
hemimethylated:
 parental strand
= methylated,
correct
 daughter
strand = gets
repaired
IDENTIFICATION OF MISMATCHED STRAND

mut proteins must be able to discriminate between

the correct strand and the strand with mismatch

Discrimination = the degree of methylation

the GATC sequences (1: 1000 nucleotides)

, are methylated on adenine residue

methylated parental strand = assumed correct

daughter strand = repaired
REPAIR OF DAMAGED DNA
• Endonuclease nicks the strand
• Exonuclease removes mismatched nucleotide(s)
 (Additional nucleotides at the 5'- and 3'-ends of
the mismatch also removed)
• DNA polymerase fills gap: sister strand serves as
template
Polymerase fills the
gap and
ligase joins the
newly synthesized
DNA piece to the
original DNA strand.
• DNA ligase joins
 3'-hydroxyl: newly synthesized DNA
 5'-phosphate: original DNA strand
 DISEASES ASSOCIATED WITH MISMATCHED
BASES
• Endometrial Cancer
(Home reading assignment)
 DISEASES ASSOCIATED WITH MISMATCHED
BASES
Lynch Syndrome
(Home reading assignment)
 DISEASES ASSOCIATED WITH MISMATCHED
BASES
Colorectal Cancer
(Home reading assignment)
 DISEASES ASSOCIATED WITH MISMATCHED
BASES
Celiac Disease
(Home reading assignment)
B.DNA
REPAIR OF DAMAGE
DAMAGE: DAMAGECAUSED
CAUSEDBY
BY
ULTRAVIOLET (UV)LIGHT
ULTRAVIOLET LIGHT

• Exposure of a cell to UV
light
 Covalent joining of two
adjacent pyrimidines
(usually thymines),
producing a dimer.
 DNA polymerase
cannot replicate DNA
strand beyond site of
dimer formation.
 NUCLEOTIDE EXCISION REPAIR

• Bacteria: nucleotide excision repair

 Excision of thymine dimers
 by UvrABC proteins
• Humans
 Recognition and excision of dimers by UV specific
endonuclease
 Xeroderma pigmentosum proteins
RECOGNITION AND EXCISION OF DIMERS
BY UV SPECIFIC ENDONUCLEASE
Endonuclease recognizes the dimer and cleaves the
damaged strand on both 5' side and 3' side of the
dimer

A short oligonucleotide (containing the dimer)

is released

A gap (formerly containing dimer) in the DNA strand

is left
 After identification of strand with mismatch,
Endonuclease nicks the strand,

Exonuclease removes mismatched nucleotide

More at 5' and 3' ends of mismatch also removed

Gap left by removal of nucleotides filled by DNA

polymerase using sister strand as template

3'-hydroxy of the new DNA joined to the 5'-phosphate of the

remaining stretch of original DNA strand by DNA ligase
RECOGNITION &
EXCISION OF DIMERS
BY UV-SPECIFIC
ENDONUCLEASE:
First, a UV-specific
endonuclease (called
uvrABC excinuclease)
recognizes the dimer, and
cleaves the damaged strand
on both the 5'-side and 3'-
side of the dimer.
A short oligonucleotide
containing the dimer is
released, leaving a gap in
the DNA strand that formerly
contained the dimer.
This gap is filled in using
the same process described
previously.
DISEASES ASSOCIATED WITH DAMAGE CAUSED
BY UV LIGHT
Albinism
(Home reading assignment)
DISEASES ASSOCIATED WITH DAMAGE CAUSED
BY UV LIGHT
Xeroderma Pigmentosum
(Home reading assignment)
DISEASES ASSOCIATED WITH DAMAGE CAUSED
BY UV LIGHT
Sporadic Cancer
(Home reading assignment)
DISEASES ASSOCIATED WITH DAMAGE CAUSED
BY UV LIGHT
Lung Cancer
(Home reading assignment)
DISEASES ASSOCIATED WITH DAMAGE CAUSED
BY UV LIGHT
Breast Cancer
(Home reading assignment)
 DNA DAMAGE: ABNORMAL BASES

Cytosine deamination or improper use of dUTP

instead of dTTP during DNA synthesis
 DISEASES ASSOCIATED WITH BASE
EXCISION
• Alzheimer’s Disease
• Huntington’s Disease
• Leukemia
(Home reading assignment)
 BASE EXCISION REPAIR

• A cellular mechanism that repairs damaged DNA

throughout the cell cycle.
Removes small, non-helix-distorting base lesions
from the genome:
 nucleotide excision repair pathway repairs bulky
helix-distorting lesions.

1. Initiated by DNA glycosylases:

Recognize and remove specific damaged or
inappropriate bases
Forming AP sites
2. Then cleaved by AP endonuclease; resulting single-
strand break processed by:
• Short-patch  A single nucleotide is replaced, or
• Long-patch  2-10 new nucleotides are synthesized

The bases of DNA can be altered, either spontaneously

(e.g., cytosine),
Slowly undergoes deamination (loss of amino group) to
form uracil
Deaminating or Alkylating Compounds:
• Nitrous acid
 Precursors: nitrosamines, nitrites, nitrates
 Deaminates cytosine, adenine, guanine
• Spontaneous Loss: ~ 10,000 purine bases per
cell per day
 BASE EXCISION REPAIR
1. Removal of abnormal bases (e.g., uracil)

 Via cytosine deamination or improper use of

dUTP instead of dTTP during DNA synthesis.

 Specific glycosylases recognize them;

hydrolytically cleave them from the
deoxyribose–phosphate backbone of the
strand.

 This leaves an apyrimidinic/apurinic site.

 2. RECOGNITION AND REPAIR OF AN AP SITE

 Specific AP- endonuclease recognizes missing

base

 Initiate excision and gap-filling: an endonucleolytic

cut just to the 5'-side of the AP site.

 Deoxyribose phosphate lyase removes single,

base-free, sugar phosphate residue.

 DNA polymerase and DNA ligase complete repair

DOUBLE-STRAND BREAKS
DISEASES ASSOCIATED WITH DOUBLE-STRANDED
BREAKS
Bone Marrow Cancer
(Home reading assignment)
DISEASES ASSOCIATED WITH DOUBLE-STRANDED
BREAKS
Rothmund-Thomson
Syndrome
(Home reading assignment)
DISEASES ASSOCIATED WITH DOUBLE-STRANDED
BREAKS
Cerebellar Ataxia
(Home reading assignment)
DISEASES ASSOCIATED WITH DOUBLE-STRANDED
BREAKS
Lymphoma
(Home reading assignment)
DISEASES ASSOCIATED WITH DOUBLE-STRANDED
BREAKS
Myeloma
(Home reading assignment)
D. REPAIR OF DOUBLE-STRAND BREAKS
1. NON-HOMOLOGOUS END - JOINING REPAIR

 Two DNA fragment ends are brought together by

a group of proteins that effect their religation

 Some DNA is lost in the process.

 Error prone and mutagenic.

 Defects in this repair system are associated

with a predisposition to cancer and
immunodeficiency syndromes.
Homologous Proteins as present in Human

a. Identification of mismatched strand

b. Repair damage DNA
 Uses the enzymes
that normally
2. Homologous Recombination
perform genetic
recombination
between
homologous
chromosomes
during meiosis.
 Much less error-
prone than NHEJ
because any DNA
that was lost is
replaced using
homologous DNA
as a template.
 POLYMORPHISM VS MUTATION
 A mutation is defined as any change in a DNA
sequence away from normal.

 This implies there is a normal allele that is prevalent

in the population.

 Mutation changes this to a rare and abnormal

variant.

 In contrast, a polymorphism is a DNA sequence

variation that is common in the population. In this
case no single allele is regarded as the standard
sequence. Instead there are two or more equally
acceptable alternatives.
 POLYMORPHISM VS MUTATION

 DNA Polymorphism, is a variability in the sequence of

a certain allele among various members of a species.
It occurs in a greater incidence than mutations
and is considered to be normal.

 A mutation is a change in the DNA sequence that may

or may not result in alteration to the proteins produced
after DNA transcription and RNA translation. Most
mutations are harmful.
 POLYMORPHISM VS MUTATION

 A variation in the DNA sequence that occurs in a

population with a frequency of 1 % or higher is termed a
polymorphism. The higher incidence in the population
suggests that a polymorphism is naturally occurring.

 Any rare change in the nucleotide sequence, usually but

not always with a disease causing attribute, is termed a
“mutation” .
 The advent of new DNA sequencing technology results
in new knowledge about mutation and polymorphism

 Mutations that were thought to be rare in a population

have been found to exceed the frequency threshold set
at 1 %.

 Even more surprising, there is a lack of association of

some of these rare mutations with human diseases.

 When comparing populations separated by geographic

and physical barriers, a disease-causing mutation in
one population is found to be harmless in another, and
vice versa.
 The advent of new DNA sequencing technology results
in new knowledge about mutation and polymorphism

Example

 Sickle-cell anemia is caused by a nucleotide change

(SNP rs334) in a gene coding for the beta chain of the
hemoglobin protein.

 In fact, rs334 is classified as a SNP, since its minor

allele frequency in the population is >1 %.

The disease manifests in people who have two copies of

the mutated gene (rs334(T;T) genotype). Sickle cell
anemia is usually rare (<1 %) in the populations of
developed nations.
Example: Sickle cell anemia

 However, the heterozygous form of the gene (rs334(A;T)

genotype) is persistent in populations of Africa, India,
and other developing nations, where malaria is endemic

 In these geographic locations, heterozygote carriers of

rs334 have a survival advantage against the malaria
pathogen, and therefore this beneficial mutation is
passed through the offspring to succeeding
generations.

 Here, a rare variant, which in one population (developed

nations) causes a severe disease in homozygosis, can
persist in another population to confer a survival
advantage as a polymorphism in heterozygosis.
 Single Nucleotide Polymorphism (SNP)

 Polymorphisms can also be of one or more

nucleotide changes, just like mutations.

 The SNP exemplifies the commonest polymorphism,

thought to arise every 1,000 base pairs in the human
genome.

 Although thought to be naturally occurring, recent

research into SNPs has shown that they can be
associated with diseases like diabetes and cancers.

 At least 40 SNPs have been shown to associate with

type-2 diabetes alone.