DNA Repair Mechanisms

• A mutation is a change in the nucleotide sequence of a short region

of a genome Many mutations are point mutations that replace one
nucleotide with another; others involve insertion or deletion of one
or a few nucleotides.

• Mutations result either from errors in DNA replication or from the

damaging effects of mutagens, such as chemicals and radiation,
which react with DNA and change the structures of individual
nucleotides.
• All cells possess DNA-repair enzymes that attempt to minimize the
number of mutations that occur.

•These enzymes work in two ways. Some are pre-replicative and

search the DNA for nucleotides with unusual structures, these being
replaced before replication occurs; others are post-replicative and
check newly synthesized DNA for errors, correcting any errors that
they find.

•A possible definition of mutation is therefore a deficiency in DNA

repair.
Single base changes
 Deamination

Deamination of cytosine to uracil (spontaneously or by chemical

mutagen) creates a mismatched U-G pair.
Replication errors
• Replication error might insert adenine instead of cytosine to
create an A-G pair.

• Similar consequences could result from covalent addition of a

small group to a base that modifies its ability to base pair.

• These changes may result in very minor structural distortion (as

in the case of a U-G pair) or quite significant change (as in the case
of an A-G pair), but the common feature is that the mismatch
persists only until the next replication.

• So only limited time is available to repair the damage before it is

fixed by replication
Structural distortions
Structural distortions may provide a physical impediment to replication
or transcription.

Introduction of covalent links between bases on one strand of DNA or

between bases on opposite strands inhibits replication and transcription.
Ultraviolet irradiation
Addition of a bulky adduct
• A single-strand nick or the removal of a base, prevents a strand from
serving as a proper template for synthesis of RNA or DNA.

• The common feature in all these changes is that the damaged adduct
remains in the DNA, continuing to cause structural problems and/or
induce mutations, until it is removed.

• Injury to DNA is minimized by systems that recognize and correct

the damage.

• The repair systems are as complex as the replication apparatus itself,

which indicates their importance for the survival of the cell.

• When a repair system reverses a change to DNA, there is no

consequence.

• But a mutation may result when it fails to do so.

Repair systems often can recognize a range of distortions in DNA
as signals of action, and a cell is likely to have several systems able
to deal with DNA damage.

Importance of DNA repair in Eukaryotes is indicated by the

identification of more than 130 genes in human genome.
Repair systems can be divided into several general types

• Some enzymes directly reverse specific sorts of damage to DNA

• There are pathways for base excision repair, nucleotide excision

repair and mismatch repair, all of which function by removing and
replacing material

• There are systems that function by using recombination to retrieve

an undamaged copy that is then used to replace a damaged duplex
sequence.

• The nonhomologous end-joining pathways rejoins broken double

stranded ends.

• Several different DNA polymerases can resynthesize stretches of

replacement DNA
Direct repair is rare and involves the reversal or simple removal
of the damage.

Photoreactivation of pyrimidine dimers, in which the offending

covalent bonds are reversed by a light-dependent enzyme is a
good example. This system is widespread in nature, and appears
to be especially important in plants.
• Mismatches between the strands of DNA are one of the major targets
for repair systems.

• Mismatch repair is accomplished by scrutinizing DNA for apposed

bases that do not pair properly.

• Mismatches that arise during replication are corrected by

distinguishing between the "new" and "old" strands and preferentially
correcting the sequence of the newly synthesized strand.

• Mismatches are usually corrected by excision repair, which is

initiated by a recognition enzyme that sees an actual damaged base or
a change in the spatial path of DNA.
There are two types of excision repair system.

• Base excision repair systems directly remove the damaged base and
replace it in DNA. A good example is DNA uracil glycolase, which
removes uracils that are mispaired with guanines.

• Nucleotide excision repair systems excise a sequence that includes

the damaged base(s); then a new stretch of DNA is synthesized to
replace the excised material.

There are often multiple excision repair systems in a single cell type.
Excision repair systems in E. coli
Excision repair
• In the incision step, the damaged structure is recognized by an
endonuclease that cleaves the DNA strand on both sides of the
damage.

• In the excision step, a 5'-3' exonuclease removes a stretch of the

damaged strand.

• In the synthesis step, the resulting single-stranded region serves as

a template for a DNA polymerase to synthesize a replacement for
the excised sequence.

• Finally, DNA ligase covalently links the 3' end of the new
material to the old material.
The uvr system of excision repair includes three genes, uvrA,B,C,
that code for the components of a repair endonuclease.

First, a UvrAB combination recognizes pyrimidine dimers and other

bulky lesions

Then UvrA dissociates (this requires ATP), and UvrC joins UvrB.

The UvrBC combination makes an incision on each side, one 7

nucleotides from the 5' side of the damaged site, and the other 3-4
nucleotides away from the 3' side.

UvrD is a helicase that helps to unwind the DNA to allow release of

the single strand between the two cuts.

The enzyme that excises the damaged strand is DNA polymerase I.

The enzyme involved in the repair synthesis probably also is DNA
polymerase I.
DNA mismatch repair system
Principle of Mismatch Repair Methylated Bases—Chemical Structure
Hemimethylated DNA: Old Strands Versus New
The MutSHL Mismatch Repair System
• MutS recognizes a mismatch shortly after DNA replication.

• MutS recruits MutL and two MutH proteins to the mismatch.

• MutH locates the nearest GATC of the new strand by identifying

the methyl group attached to the “mother” strand.

• MutH cleaves the non-methylated strand and the DNA between

the cut and the mismatch is degraded.

• The region is replaced and the mismatch is corrected.

Excision repair system Also known as “cut and patch” repair.A
DNA repair system that recognizes bulges in the DNA double helix,
removes the damaged strand and replaces it

Mismatch repair system DNA repair system that recognizes

mispaired bases and cuts out part of the DNA strand containing the
wrong base

DNA adenine methylase (Dam) A bacterial enzyme that methylates

adenine in the sequence GATC

DNA cytosine methylase (Dcm) A bacterial enzyme that methylates

cytosine in the sequences CCAGG and CCTGG

hemi-methylated Methylated on only one strand

DNA glycosylase Enzyme that breaks the bond between a base and
the deoxyribose of the DNA backbone
Eukaryotic Nucleotide excision Repair
pathways
Model for the core NER reaction.
(A)Bulky DNA lesions that destabilize duplex DNA are induced by a
number of damaging agents.

(B)In global genome NER, strongly distorting lesions are directly

recognized by XPC-RAD23B, which binds the non-damaged strand
opposite the lesion.

(C)TFIIH interacts with XPCRAD23B and pries the DNA open with
its XPB subunit allowing XPD to track along DNA until stalls at the
damage and verifies the chemical modification (bulkiness) of the
lesion.
(D) Stalling of XPD at the lesion allows for the formation of the
preincision complex by recruitment of XPA, RPA, and XPG. The
endonuclease XPG does not make an incision at this point.

(E) Recruitment of ERCC1-XPF by interaction with XPA to the

complex leads to incision 5’ to the lesion.

(F) Initiation of repair synthesis by Pol d and Pol k or Pol 1 and

associated factors, followed by 3’ incision by XPG.

(G) Completion of repair synthesis and sealing of the nick by DNA

ligase IIIa/XRCC1 or DNA ligase I completes the process.
The NER pathway is involved in the removal of a wide range of
DNA damage, including that caused by photoproducts and by
chemicals that give rise to bulky DNA adducts and DNA cross links.

Damage in DNA that is actively transcribed by RNA polymerase II

(RNAPII) is repaired more rapidly and more completely than is
DNA damage in the genome overall.

These two different sub-pathways are known as transcription

coupled repair (TCR) and global genome repair (GGR),
respectively.

All the genes involved in XP (XPA–XPG), with the notable

exception of the XPC gene (and possibly XPE), contribute to both
sub-pathways of NER.

The XPC gene product appears to be essential in the recognition

(and repair) of DNA damage in the genome overall and in the non-
transcribed strand of active genes.
A model for the mechanism of mammalian nucleotide excision
repair (NER) that shows the two sub-pathways of NER.

Left, the global genome repair (GGR) pathway that deals with lesions in

the genome overall; right, the transcription
coupled repair (TCR) pathway that is involved in the repair of DNA
lesions in the transcribed strand of active genes.

(a) Damage recognition. In GGR, the initial recognition of DNA damage

could occur through the binding of the XPC protein at the site of the
lesion in complex with the human homolog of the yeast RAD23B
protein (HHR23B).

Binding of the XPC–HHR23B protein complex to damaged DNA could

induce a conformational change.
In TCR, RNA polymerase II (RNAPII) stalls at a DNA lesion in an
actively transcribed gene, and the Cockayne syndrome
complementation group A (CSA) and CSB protein complexes change
the conformation of the stalled RNAPII to facilitate the binding of
DNA-unwinding proteins.

(b) DNA unwinding. In GGR and TCR, the basal transcription

initiation factor IIH (TFIIH) and the XPA protein are recruited to the
complex.

These two complexes could initiate the local opening of double-

stranded DNA around the lesion.

The XPA protein, in complex with replication protein A (RPA),

forms a ‘pre-incision complex’.
The function of the XPA–RPA protein complex is unclear but it could
confer NER substrate specificity.

(c) Incision. The endonuclease, XPG, cuts the DNA five to

six nucleotides downstream of the DNA lesion.

XPF, in complex with the excision repair cross-complementing 1

(ERCC1) protein, cleaves the strand 20–22nucleotides upstream of the
lesion.

(d) DNA excision. The damaged oligomer is released and the gap is

filled with DNA polymerase (DNA pol) δ and/or ϵ.

Other proteins involved in replication repair include proliferating cell

nuclear antigen (PCNA), RPA and replication factor C (RF-C).

The 3′ nick is closed by DNA ligase I. XPD and XPB are two subunits of

TFIIH.
SOS repair system
Damage to DNA results in the induction of a variety of genes whose
products help to minimize the effects of the damage.

Some of these are repair enzymes and others delay cell division until
the damage has been repaired. Yet others provide a pathway of last
resort—they allow DNA replication to proceed through severely
damaged zones, even at the cost of introducing mutations, a process
known as error-prone repair.

The SOS system of E. coli was so named because it responds to

severe and potentially lethal DNA damage.

Many single-stranded regions of DNA are generated by damage,

partly due to excision repair and to stalled replication reinitiating
beyond the damage.
Since single-stranded DNA activates RecA, severe DNA damage
activates many RecA proteins.

Activated RecA induces the SOS system by activating LexA, a

transcriptional repressor of the SOS genes, by inducing its self
cleavage.

Once cleaved, LexA no longer blocks transcription of SOS genes.

The SOS proteins combat the DNA damage.

Among the genes induced by the SOS response two are of special
note. These are umuC and umuD (umu = ultraviolet mutagenesis),
which encode DNA polymerase V.

This polymerase lacks a proofreading subunit so it can replicate past

pyrimidine dimers and missing bases (i.e. AP-sites). Pol V makes
mistakes when passing damaged DNA
The PolV subunits, UmuC and UmuD, form a complex containing a
dimer of UmuD plus one UmuC protein.

However, when first made, UmuD2C does not act as a polymerase

but delays normal DNA replication in order to allow time for repair.

Activated RecA then induces UmuD to undergo self-cleavage to

UmuD’. When a dimer of UmuD’ combines with one UmuC, the
error-prone PolV (UmuD’2C) is formed.

Like E. coli, yeast, flies and humans all have error-prone DNA
polymerases that respond to DNA damage and can replicate past
damaged regions.

In higher organisms these repair enzymes appear to be more

specialized and less error-prone. For example, when human
Polymerase Eta passes a symmetrical thymine dimer it usually
puts in AA.
The seven types of proteins that participate
in controlling cell growth

I) Growth factors ,

II) Growth factor receptors

III) Signal-transduction proteins 

IV) Transcription factors

V) Pro- or anti-apoptotic proteins

VI) Cell cycle control proteins and

VII) DNA repair proteins

The genes that have been implicated in carcinogenesis are divided
into two broad categories: tumor-suppressor genes and Oncogenes.

Tumor-suppressor genes act as a cell’s brakes; they encode

proteins that restrain cell growth and prevent cells from becoming
malignant.

Oncogenes, on the other hand, encode proteins that promote the loss
of growth control and the conversion of a cell to a malignant state
Mutations changing the structure or expression of proteins in
classes I – IV generally give rise to dominantly active oncogenes.

The class VI proteins mainly act as tumor suppressors; mutations in

the genes encoding these proteins act recessively to release cells
from control and surveillance, greatly increasing the probability that
the mutant cells will become tumor cells.

Class VII mutations greatly increase the probability of mutations in

the other classes. Virus-encoded proteins that activate growth-factor
receptors (Ia) also can induce cancer.
 Gain-of-Function Mutations Convert
Proto-Oncogenes into Oncogenes

Oncogene is any gene that encodes a protein able to transform cells in

culture or to induce cancer in animals.

Of the many known oncogenes, all but a few are derived from normal
cellular genes (i.e., proto-oncogenes) whose products participate in
cellular growth-controlling pathways.

Because most proto-oncogenes are basic to animal life, they have

been highly conserved over eons of evolutionary time.
Loss of Function Mutations –
Loss-of-function mutations, also called inactivating mutations,
result in the gene product having less or no function (being partially
or wholly inactivated).

When the allele has a complete loss of function (null allele), it is

often called an amorph or amorphic mutation in the Muller's
morphs schema.

Phenotypes associated with such mutations are most often

recessive.

Exceptions are when the organism is haploid, or when the reduced

dosage of a normal gene product is not enough for a normal
phenotype (this is called haploinsufficiency).
Contrasting effects of mutations in tumorsuppressor genes (a) and oncogenes (b).
Whereas a mutation in one of the two copies (alleles) of an oncogene
may be sufficient to cause a cell to lose growth control, both copies
of a tumor-suppressor gene must be knocked out to induce the same
effect.

Oncogenes arise from proto-oncogenes as the result of gain-of-

function mutations, that is, mutations that cause the gene product to
exhibit new functions that lead to malignancy.

Tumor-suppressor genes, in contrast, suffer loss-of-function

mutations and/or epigenetic inactivation that render them unable to
restrain cell growth
Activation of a proto-oncogene to an oncogene.
Activation can be accomplished in several ways as indicated in this
figure.

In pathway a), a mutation in the gene alters the structure and

function of the encoded protein.

In pathway b), gene amplification results in overexpression of the

gene.

In pathway c), a rearrangement of the DNA brings a new DNA

segment into the vicinity or up against the gene, altering either its
expression or the structure of the encoded protein.
Most tumors contain alterations in both tumor-suppressor genes and
oncogenes, suggesting that the loss of a tumor-suppressor function
within a cell must be accompanied by the conversion of a proto
oncogene into an oncogene before the cell becomes fully malignant.

Even then, the cell may not exhibit all of the properties required to
invade surrounding tissues or to form secondary colonies by
metastasis.

Mutations in additional genes, such as those encoding cell-adhesion

molecules or extracellular proteases, may be required before these cells
acquire the full life-threatening phenotype.