Mechanisms of DNA Damage and Repair - PowerPoint Presentation

Slide1

Mechanisms of DNA Damage and Repair

Principles of Molecular BiologySlide2

Group Members

Maira Aleem

Bilal Naveed

Tanzeela Raza

Maheen

Malik

Zaigham

AbbasSlide3

DNA Structure

DNA = Deoxyribose nucleic

acid.

Four Nucleotides

- A

denine

,

Cytosine

,

Thymine

, or

Guanine

.

The amounts of A = T, G = C, and purines = pyrimidines [Chargaff’s Rule]. Slide4

DNA Structure

Double h

elix

with antiparallel strands

Discovered

in 1953 by James Watson and Francis

Crick.

Bases on opposite strands are linked by hydrogen bonding: A with T, and G with C

.

Nucleotides in each strand are linked by 5’-3’

phosphodiester

bondsSlide5
Slide6

“

Volumes

of history is written in the ancient alphabet of

G and C

and

A and T

.”

Sy

MontgomerySlide7

DNA Damage

Alteration

in

C

hemical

structure of DNA, such as a break in a strand of DNA, a base missing from the backbone of

DNA or,

Chemically changed baseSlide8

DNA Damage vs Mutations

Both

are types of error in DNA.

DNA

damage

is an abnormal chemical structure in

DNA.

Mutation

is a change in the sequence of standard base pairs.Slide9

Continued…

Body repairs the damage caused to DNA.

Not 100% efficient.

2

points of error:

Replication of past damages in the template strand of DNA, or

D

uring repair

of DNA damagesSlide10

DNA Repair

Various

checkpoints

in the body.

Involved

in

protein synthesis

that can only prevent the transduction of mutations to daughter cells by means of efficient DNA damage repair machinery

. Slide11

Different ways of DNA Damage

Bilal

NaveedSlide12

Factors mainly responsible for DNA Damage

Radiation Hydrolysis

Alkylation OxidationSlide13

Radiation

High-powered particles, transmitted via X rays, alpha, beta, gamma rays etc.

X rays and gamma rays are electromagnetic waves like light, energy high and shorter wavelength

UV light is a radiation of intermediate energy that can damage cells (sunburn).Slide14

Radiation

Breaking the long string of letters and this can happen to one or both of the strands.

Cells are good in fixing one broken strand but both broken strands are really an issue for the cell. Slide15

Radiation

Double strand breaks doesn't get fixed, then part of DNA can be deleted, duplicated.

Any of these problem cause genetic disordersSlide16

Radiation

UV radiation in sunlight can damage DNA by messing up the base pairing

UV light will make two T's that are next to each other stick together making something called a

dimerSlide17

Radiation

Some cells with lots of thymine

dimers

will die E.g. Skin peeled after a sunburn. Slide18

Hydrolysis

Deamination

of cytosine is most frequent and important kind of hydrolytic damage

It is the removal of an amine group from a molecule

Deaminases

enzyme

The

deamination

of “C” to “U”

In this “U” will cause “A” to be inserted opposite it and cause a C:G to T:A transition when the DNA is replicated.

Deamination

converts adenine to hypoxanthine and guanine is converted in to

xanthine

, which continues to pair with

cytosin

, though with only two hydrogen bonds.Slide19

Hydrolysis Slide20

Hydrolysis

Depurination

 by spontaneous hydrolysis of the N-

glycosyl

linkage

In DNA it is a chemical reaction of

purine

in which the β-N-

glycosidic

bond is hydrolytically cleaved releasing a nucleic baseSlide21

Alkylation

Alkylation

 is the transfer of an alkyl group from one molecule to another.

methyl or ethyl groups are transferred to reactive sites on the bases and to phosphates in DNA backbone.

It does not immediately leading to mispairing but they do make the bond between sugar and base more labile, or more apt to break.

It leaves an

apurinic

site, a sugar without its

purine

.

This obviously cannot be replicated properly

If they do, they frequently insert the wrong base across from an

apurinic

site, and this generates a mutationSlide22

Alkylation

Ethylmethane

sulfonate

, which transfers ethyl groups to DNA.

O6-rthylgaunine, often

mispairs

with thymine, resulting in the change of G:C base pair into an A:T base pair when the damaged DNA is replicated.Slide23

Oxidation

DNA oxidation

 is the process of oxidative damage on Deoxyribonucleic Acid

It occurs most readily at guanine residues due to the high oxidation potential of this base relative to cytosine, thymine, and adenine.

Reactive oxygen species (O2-, H2O2, and OH) are generated by ionizing radiation and by chemicals agents that generate free radicals.

An important oxidation product is 8-hydroxyguanine, which

mispairs

with adenine, resulting in G:C to T:A

transversions

.Slide24

OxidationSlide25

Others ways of Damage

DNA damage

may also result from exposure to polycyclic aromatic hydrocarbons (PAHs). 

PAHs are atmospheric pollutants commonly associated with oil, coal, cigarette smoke, and automobile exhaust fumes.

A common marker for DNA damage due to PAHs is 

Benzo

(a)

pyrene

diol

epoxide

It is found to be very reactive, and known to bind covalently to proteins, lipids, and guanine residues of DNA to produce BPDE adducts.

If left unrepaired,  may lead to permanent mutations and finally leading to tumor development.Slide26

DNA Repair Mechanisms

By: Tanzeela RazaSlide27

DNA Repair

MechanismsSlide28

Direct enzymatic Repair

1.

Photoreactivation

It is a enzymatic cleavage of thymine dimers activated by visible light.

It is only present in prokaryotes (e.g.

E.col

i

)

Mechanism

Enzyme photolyase (encoded by

phr

gene) binds to a pyrimidine dimer.

Visible light shines on cell then

FADH absorbs

that light and release electron.

Electron interact with dimer.

Then splitting of

cyclobutane

ring in dimer due to electron interaction.

Finally, enzyme leaves the DNA and the DNA structure returned to its prior state.Slide29

PhotoreactivationSlide30

Direct enzymatic Repair

2.Removal of methyl groups

.

Another example of Direct enzymatic repair.

MechanismSlide31

Removal of methyl groupsSlide32

Excision

Repair

A general mechanism of DNA repair.

Various enzymes are involved that can sense DNA damage

During excision repair bases and nucleotides are removed from damaged strands

Gap is then patched using complementarity with the remaining strand.

Excision repair is broadly categorized into

Base excision repair

Nucleotide excision repair

Mismatch repairSlide33

Base Excision Repair (BER)

Base

f

rom

a nucleotide within DNA can be removed in several ways such

as

:

It repairs

DNA bases damaged by:

Slide34

Mechanism of Base excision repair by using Enzyme

Uracil-DNA glycosylase enzyme recognizes uracil within DNA and cleavages it out at the base sugar (

glycosidic

) bond. The resulting site is called an AP (

apurinic

-

apyrimidinic

) site, because of lack of purine and pyrimidine.

AP endonucleases then sense the minor distortion of the DNA double helix and initiate excision of single AP nucleotide. AP endonucleases class I nick at 3' side of AP site and class II nick at 5' side of AP site.

DNA polymerase then inserts a nucleotide at the AP site

.

Lyase

or phosphodiesterase then removes the base free nucleotide.

DNA ligase then close the Nick.Slide35

b) Eukaryotic BERSlide36

Nucleotide excision repair (NER)

NER

pathway can recognize and remove a wide variety of bulky, helix-distorting lesions from DNA.

It repairs

DNA damaged which are produced by the UV component of

sunlight.

While

mechanistically similar to BER, the NER pathway is more complex, requiring some thirty different proteins to carry out a multi-step ‘cut-and-patch’-like mechanism

.

General steps of NER pathwaySlide37

Importance

The biological importance of NER is

that

defects in NER cause several human genetic disorders, including

X

eroderma

pigmentosum

Cockayne

syndrome

Trichothiodystrophy

These all are characterized

by extreme sun sensitivity.

In

addition, these diseases demonstrate overlapping symptoms associated with cancer, developmental delay, immunological defects, neurodegeneration, and premature aging Slide38

Mechanism of Nucleotide excision repair

ABC excinuclease

(composed of subunits coded by 

uvrA

, 

uvrB

 and 

uvrC

 genes) moves along DNA and can detect Thymine dimers and

for

excision endonuclease.

UvrA and UvrB complex attach on distortion site then UvrA will dissociates.

UvrB

attracts UvrC and nicks 5 nucleotides at 3’ side of DNA while 8 nucleotides nicks at 5’ side of DNA will be produced by UvrC subunit. 

UvrD

(DNA helicase II) removes 12

oligonucleotides.

DNA

polymerase I now fills in gap in 5'>3' direction

DNA ligase seals the gaps.Slide39
Slide40

NER in Eukaryotes

NER in ProkaryotesSlide41
Slide42
Slide43

Mismatch

repair (MMR)

The MMR system plays an essential role in post-replication repair of

misincorporated

bases that have escaped the proofreading activity of replication polymerases.

Accounts for 99% of all repairs 

The

MMR pathway can be divided into three principle steps:

A recognition step where

mispaired

bases are recognized by

MutS

,

MutL

complexes.

An excision step where the error-containing strand is degraded resulting in a gap (

MutH

nicks progeny DNA strands

).

A repair synthesis step, where the gap is filled by the DNA

resynthesis

.Slide44
Slide45
Slide46

Double Strand break, causes and repairing pathways

MAHEEN MALIKSlide47

What is double strand break?

The type of damage in which both strands of DNA are broken

DNA gets damaged by any source and both strands of DNA are brokenSlide48

Sources

Sources that are responsible causing the

Dsb

are

uv

radiation, chemical agents, and ionizing radiationSlide49

DNA damage

The genome of cell is continuously damaged.

It is inevitable because DNA damage arises as a result of normal cellular processes

e.G.

Ros

Damage can cause lesions that block the replication ultimately leading to

double strand breakSlide50

Mechanism

Our body has evolved check point mechanism that activate the repair pathways.

These checkpoints are proteins that search the whole genome that if there is any damage to the

DNA.

When encounter the damage, then they turn on the mechanisms that are involved in

repairing.Slide51

What if damage is not repaired?

If the damage is not repaired than it may result in cell death.

If not repaired correctly than it leads it to the genomic rearrangements found in many cancerous cells.Slide52

Pathways involved in DNA repair

Two types of pathways are involved in DNA repair mechanism

Homologous recombination repair pathway (HR)

Non homologous end joining (NHEJ)Slide53
Slide54
Slide55

Non homologous end joining (NHEJ)

Non-homologous

end joining (NHEJ) functions in all kinds of cells, from bacteria to man, and is involved in

DNA repair.

More common

pathway

Occurs at all the stages of cell cycleSlide56
Slide57

Mechanism

NHEJ is initiated by the recognition and binding of the Ku protein to the broken DNA

ends.

Ku is a heterodimer of Ku70 and Ku80 that forms the DNA-binding component of DNA-dependent protein kinase (DNA-PK). 

It encircles the

DNA

and forms a bridge between the broken ends of

DNA.

It then recruits DNA-

PKcs

that is the catalytic subunit of protein

kinase.Slide58

Continue…

NHEJ requires the blunt ends for joining and these blunt ends are then created by

artemis

proteins that cut the sticky

ends.

These blunt ends are then ligated by using ligase

enzyme.

The

DNA

that is repaired is not exactly the same as damaged one because few nucleotides are missing.Slide59
Slide60

SOS response

The SOS-response has been found in many bacterial species, but not in eukaryotic cells.Slide61
Slide62

Mechanism

The term SOS response refers to a set of co-regulated

genes.

 SOS system consists of more than 40 genes and is

regulated

by the

LexA

repressor

protein.Slide63

Continued…

Following a single-stranded DNA (

ssDNA

) or double-stranded DNA (dsDNA) break, activated

RecA

promoters assemble into filaments on chromosomal sites.

Interactions

between activated

RecA

promoters and the

LexA

repressor induce the auto cleavage of

LexA

, which causes it to dissociate from the DNA, thereby relieving repression of the SOS regulon.Slide64
Slide65

Diseases caused by DNA damage and repair

ZAIGHUM ABBASSlide66

Factors that cause damage

Our body consist over 12 trillion cells and contains over 3 billion nucleotides in the

DNA.

DNA is constantly challenged by natural and man-made chemicals, natural and man-made forms of radiation, and even by endogenous metabolism.Slide67

Diseases

DNA damage can lead to many different disease processes,

including

Cancer

Aging

Neuro-degeneration

Cardiovascular disease

Tissue toxicities.Slide68
Slide69

Cancer

Cells have acquired mutations, they become immortal and reproduce indefinitely as tumor cells

.

Cancer-causing DNA mutations also include the loss of genes that act as tumor suppressors and the activation of oncogenes, which promote cancer

.

The abnormal insertion of methyl groups into DNA at the regulatory portion of certain genes has been noted in many

cancers.Slide70

Cancer

Sometimes people inherit a susceptibility to damage by certain environmental agents. For example, albinos have inherited an absence of skin and hair pigments, which cause DNA damage

.

Most cancers arise from the accumulated mutations in our somatic cells caused by years of exposure to external toxins.

E.g

smokingSlide71

Aging

A

ccumulation

of uncorrected DNA damage over years is a major cause of aging. Their

reason is:

Animals with the fastest rates of DNA repair generally have the longest life spans.

Humans who have genetic diseases resulting in greater spontaneous DNA damage or inefficient DNA repair often show signs of premature aging.

Exposure to external causes of DNA damage (ultraviolet light, tobacco) decreases life span.Slide72
Slide73
Slide74

Neuro-degeneration

It is main feature of many nervous system and aging diseases such

as

Hungtington

disease

Alzheimer

diseases

Parkinson’s diseaseSlide75
Slide76
Slide77

References

Asimuddin

, M., & Jamil, K. (2012). Insight into the DNA repair mechanism operating during cell cycle checkpoints in eukaryotic cells. 

Biology and Medicine

, 

4

(4), 147.

Boboye

, B., &

Alao

, A. (2008). Effect of mutation on

Trehalose

-catabolic-enzyme synthesized by a tropical Rhizobium species F1. 

Research Journal of Microbiology

, 

3

(4), 269-275.

Mathews, L. A., &

Cabarcas

, S. M. (2013). 

DNA repair of cancer stem cells

. E. M. Hurt (Ed.). Springer.

Stivers

, J. T., & Jiang, Y. L. (2003). A mechanistic perspective on the chemistry of DNA repair glycosylases. 

Chemical reviews

, 

103

(7), 2729-2760.

Boiteux

, S., &

Jinks

-Robertson, S. (2013). DNA repair mechanisms and the bypass of DNA damage in Saccharomyces cerevisiae. 

Genetics

, 

193

(4), 1025-1064.

Altieri, F., et al., DNA damage and repair: from molecular mechanisms to health implications.(Comprehensive Invited Review). Antioxidants & Redox Signaling, 2008. 10(5): p. 891(47).

de

Laat

, W.L., N.G.J. Jaspers, and J.H.J.

Hoeijmakers

, Molecular mechanism of nucleotide excision repair. Genes & Development, 1999. 13(7): p. 768-785

.Slide78

Berndt, S.I., et al., Genetic variation in the nucleotide excision repair pathway and colorectal cancer risk. Cancer

Epidemiol

Biomarkers

Prev

, 2006. 15(11): p. 2263-2269.

Tubbs, J. L.,

Latypov

, V.,

Kanugula

, S., Butt, A.,

Melikishvili

, M.,

Kraehenbuehl

, R., ... &

McGown

, G. (2009). Flipping of alkylated DNA damage bridges base and nucleotide excision repair. 

Nature

, 

459

(7248), 808-813.

Youds

, J. L., Barber, L. J., Ward, J. D., Collis, S. J., O'Neil, N. J.,

Boulton

, S. J., & Rose, A. M. (2008). DOG-1 is the Caenorhabditis

elegans

BRIP1/FANCJ homologue and functions in

interstrand

cross-link repair. 

Molecular and cellular biology

, 

28

(5), 1470-1479.