dna damage cellular sensing responding and repair n.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
DNA damage, cellular sensing/responding, and repair PowerPoint Presentation
Download Presentation
DNA damage, cellular sensing/responding, and repair

Loading in 2 Seconds...

play fullscreen
1 / 95

DNA damage, cellular sensing/responding, and repair - PowerPoint PPT Presentation


  • 318 Views
  • Uploaded on

DNA damage, cellular sensing/responding, and repair. Rebecca Fry, Ph.D. DNA damage. DNA damage, due to environmental factors and normal metabolic processes inside the cell, occurs at a rate of 1,000 to 1,000,000 molecular lesions per cell per day. . DNA Damage.

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'DNA damage, cellular sensing/responding, and repair' - kaya


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
dna damage
DNA damage

DNA damage, due to environmental factors and normal metabolic processes inside the cell, occurs at a rate of 1,000 to 1,000,000 molecular lesions per cell per day.

slide3

DNA Damage

While this constitutes only 0.000165% of the human genome's approximately 6 billion bases (3 billion base pairs)…

unrepaired lesions in critical genes (such as tumor suppressor genes) can impede a cell's ability to carry out its function and appreciably increase the likelihood of tumor formation.

slide4

Failure to repair DNA lesions may result in blockages of transcription and replication, mutagenesis, and/or cellular cytotoxicity.

In humans, DNA damage has been shown to be involved in a variety of genetically inherited disorders, in aging, and in carcinogenesis.

slide5

Sources of DNA Damage

DNA damage can be subdivided into two main types:

ENDOGENOUS and EXOGENOUS

slide6

Endogenous sources of DNA Damage

endogenous damage such as attack by reactive oxygen species produced from normal metabolic byproducts

slide7

Types of Damage

The main types of damage to DNA due to endogenous cellular processes:

oxidation of bases [e.g. 8-oxo-7,8-dihydroguanine (8-oxoG)] In living cells ROS are formed continuously as a consequence of metabolic and other biochemical reactions . These ROS include superoxide (O2–·), hydrogen peroxide (H2O2), hydroxyl radicals (OH·) and singlet oxygen (1O2)

alkylationof bases (usually methylation), such as formation of 7-methylguanine, 1-methyladenine, O6 methylguanine

3. hydrolysis of bases, such as deamination, depurination and depyrimidination.

slide8

4. "bulky adduct formation" (i.e. benzo[a]pyrenediolepoxide-dG adduct).

5. mismatch of bases, due to errors in DNA replication, in which the wrong DNA base is stitched into place in a newly forming DNA strand, or a DNA base is skipped over or mistakenly inserted.

slide9

Exogenous sources of DNA damage

  • caused by external agents such as
  • ultraviolet [UV 200-300nm] radiation from the sun
  • other radiation frequencies, including x-rays and gamma rays
  • human-made mutagenic chemicals
  • cancer chemotherapy and radiotherapy
slide11

It is important to distinguish between DNA damage and mutation, the two major types of error in DNA.

DNA damage and mutation are fundamentally different.

Damage is a physical abnormality in the DNA, such as single and double strand breaks, 8-hydroxydeoxyguanosine residues and polycyclic aromatic hydrocarbon adducts.

slide12

In contrast to DNA damage, a mutation is a change in the base sequence of the DNA. A mutation cannot be recognized by enzymes once the base change is present in both DNA strands, and thus a mutation cannot be repaired.

At the cellular level, mutations can cause alterations in protein function and regulation.

Mutations are replicated when the cell replicates. In a population of cells, mutant cells will increase or decrease in frequency according to the effects of the mutation on the ability of the cell to survive and reproduce.

what are the types of dna damage

What are the types of DNA damage??

Base loss

Base modification

Replication errors

Inter-strand X-links

DNA-protein X-links

Strand Breaks

1 base loss
1. Base loss

The bond linking DNA bases with deoxyribose is labile under physiological conditions.

Within a typical mammalian cell, several thousand purines and several hundred pyrimidines are spontaneously lost per diploid genome per day.

Loss of a purine or pyrimidine base creates an apurinic/apyrimidinic (AP) site (also called an abasic site):

slide16
2. Base modification

2a. Deamination

The primary amino groups of nucleic acid bases are somewhat unstable. The amino group is removed from the amino acid and converted to ammonia.

In a typical mammalian cell, about 100 uracils are generated per haploid genome per day in this fashion.

Other deamination reactions include conversion of adenine to hypoxanthine, guanine to xanthine, and 5-methyl cytosine to thymine.

slide17

Example: cytosine deamination

Spontaneous deamination is the hydrolysis reaction of cytosine into uracil, releasing ammonia in the process.

slide18
2. Base modification

2b. Chemical modification

The nucleic acid bases are susceptible to numerous modifications by a wide variety of chemical agents.

For example, several types of hyper-reactive oxygen (singlet oxygen, peroxide radicals, hydrogen peroxide and hydroxyl radicals) are generated as byproducts during normal oxidative metabolism and also by ionizing radiation (X-rays, gamma rays). These are frequently called Reactive Oxygen Species (ROS). ROS can modify DNA bases. A common product of thymine oxidation is thymine glycol:

another type of chemical modification methylation alkylation
Another type of chemical modification: methylation/alkylation
  • Many environmental chemicals, including "natural" ones (frequently in the food we eat) can also modify DNA bases, frequently by addition of a methyl or other alkyl group (alkylation).
  • In addition, normal metabolism frequently leads to alkylation.
  • It has been shown that S-adenosylmethionine, the normal biological methyl group donor, reacts accidentally with DNA to produce alkylated bases like 3-methyladenine at a rate of several thousand per day per mammalian diploid genome.
slide21
2. Base modification

2b. Photodamage

Ultraviolet light is absorbed by the nucleic acid bases, and the resulting influx of energy can induce chemical changes.

  • The most frequent photoproducts are the consequences of bond formation between adjacent pyrimidines within one strand, and, of these, the most frequent are cyclobutanepyrimidinedimers (CPDs).
slide22

Ultraviolet light induces the formation of covalent linkages by reactions localized on the C=C double bonds

slide23
3. Replication errors

Another major source of potential alterations in DNA is the generation of mismatches or small insertions or deletions during DNA replication.

Although DNA polymerases are moderately accurate, and most of their mistakes are immediately corrected by polymerase-associated proofreading exonucleases, nevertheless the replication machinery is not perfect.

slide24
4. Inter-strand crosslinks

By attaching to bases on both strands, bifunctionalalkylating agents such as the psoralens can cross-link both strands.

Cross-links can also be generated by UV and ionizing radiation.

slide26
5. DNA-protein crosslinks

DNA topoisomerases generate covalent links between themselves and their DNA substrates during the course of their enzymatic action.

Usually these crosslinks are transient and are reversed as the topoisomerase action is completed. Occasionally something interferes with reversal, and a stable topoisomerase-DNA bond is established.

Bifunctionalalkylating agents and radiation can also create crosslinks between DNA and protein molecules. All of these lesions must be repaired.

slide27
6. Strand breaks

Single-strand and double-strand breaks are produced at low frequency during normal DNA metabolism by topoisomerases, nucleases, replication fork "collapse", and repair processes.

Breaks are also produced by ionizing radiation.

slide32

DNA damage recognition

DNA damage is recognized by sensor proteins that then initiate a network of signal transduction pathways.

This ultimately results in the activation of effector proteins that execute the functions of the DNA damage response, including recruitment of DNA repair proteins, cell cycle arrest, damage induced transcription, or the induction of apopotosis.

an option dna damage checkpoints
An option: DNA damage checkpoints
  • After DNA damage, cell cyclecheckpoints are activated.
  • Checkpoint activation pauses the cell cycle and gives the cell time to repair the damage before continuing to divide.
cell cycle
Cell Cycle
  • The cell cycle of eukaryotic cells can be divided into four successive phases:
  • M phase (mitosis), in which the nucleus and the cytoplasm divide;
  • S phase (DNA synthesis), in which the DNA in the nucleus is replicated,
  • two gap phases, G1 and G2.
  • The G1 phase is a critical stage, allowing responses to extracellular cues that induce either commitment to a further round of cell division or withdrawal from the cell cycle (G0) to embark on a differentiation pathway.
slide36

The transition from one phase of the cell cycle to the next is controlled by cyclin–CDK (cyclin-dependent kinase) complexes which ensure that all phases of the cell cycle are executed in the correct order.

slide37
DNA damage checkpoints occur at the G1/S and G2/M boundaries. An intra-S checkpoint also exists.
  • Checkpoint activation is controlled by two master kinases, ATM and ATR.
  • ATM responds to DNA double-strand breaks and disruptions in chromatin structure, whereas ATR primarily responds to stalled replication forks.
  • These kinasesphosphorylate downstream targets in a signal transduction cascade, eventually leading to cell cycle arrest.
dna damage checkpoints
DNA damage checkpoints
  • A class of checkpoint mediator proteins including BRCA1, MDC1, and 53BP1 has also been identified.
  • These proteins seem to be required for transmitting the checkpoint activation signal to downstream proteins.
  • p53 is an important downstream target of ATM and ATR, as it is required for inducing apoptosis following DNA damage.
what happens if we have defective atm

What happens if we have defective ATM??

ataxia telangiectasia mutated

slide41

Disease: Ataxia-telangiectasia

Ataxia-telangiectasia is a rare, childhood neurological disorder that causes degeneration in the part of the brain that controls motor movements and speech.

Its most unusual symptom is an acute sensitivity to ionizing radiation, such as X-rays or gamma-rays. The first signs of the disease, which include delayed development of motor skills, poor balance, and slurred speech, usually occur during the first decade of life.

slide42

Telangiectasias (tiny, red "spider" veins), which appear in the corners of the eyes or on the surface of the ears and cheeks, are characteristic of the disease, but are not always present and generally do not appear in the first years of life.

slide43

About 20% of those with A-T develop cancer, most frequently acute lymphocytic leukemia or lymphoma.

Many individuals with A-T have a weakened immune system, making them susceptible to recurrent respiratory infections.

atm mutations are associated with breast cancer
ATM mutations are associated with breast cancer
  • Researchers have found that having a mutation in one copy of the ATM gene in each cell (particularly in people who have at least one family member with ataxia-telangiectasia) is associated with an increased risk of developing breast cancer.
  • About 1 percent of the United States population carries one mutated copy of the ATM gene in each cell. These genetic changes prevent many of the body's cells from correctly repairing damaged DNA.
slide46

DNA repair refers to 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 UV light and 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.

slide47

DNA repair mechanisms

Cells cannot function if DNA damage corrupts the integrity and accessibility of essential information in the genome (but cells remain superficially functional when so-called "non-essential" genes are missing or damaged).

Depending on the type of damage inflicted on the DNA's double helical structure, a variety of repair strategies have evolved to restore lost information.

slide49

Direct reversal

Cells are known to eliminate damage to their DNA by chemically reversing it.

These mechanisms do not require a template, since the types of damage they counteract can only occur in one of the four bases.

Such direct reversal mechanisms are specific to the type of damage incurred and do not involve breakage of the phosphodiester backbone.

slide50

An example:

methylation of guanine bases, is directly reversed by the protein methyl guanine methyl transferase (MGMT), the bacterial equivalent of which is called as ogt.

This is an expensive process because each MGMT molecule can only be used once; that is, the reaction is stoichiometric rather than catalytic.

exposure to alkylating agents
Exposure to alkylating agents!

Damage induced mimics

some chemotherapeutics

X

guanine

cytosine

Damage induced mimics

environmental exposures

thymine

O6-meG can mispair with thymine

G/C to A/T transitions

Can be cytotoxic or mutagenic lesion

using genomics to predict population responses to exposures
Using genomics to PREDICT population responses to exposures

Can we use gene expression levels

to predict responses to

DNA alkylating agents?

Fry et al, Genes and Development 2008

slide55

NIH PDR Cell Lines Represent Healthy Genetically Diverse Population

450 healthy, unrelated individuals

24 lymphoblastoid cell lines

slide56

Establish Range of Sensitivity in Cells Exposed to MNNG (0.5 ug/ml)

Sensitive

Resistant

Training Population

slide57

Establish training population based on extreme

responders

Sensitive

Resistant

Training Population

slide58

Alkylation-Sensitivity-Associated Gene Sets Identified

Sensitive

Resistant

Sensitive

Resistant

Expression intensity

% Control growth

Statistically significant association (p<0.01) of % control growth and expression

Statistically Significant Differential Expression

1.5 FC , p-value < 0.05

slide59

Apply two-class prediction algorithm:SVM to 16 cell lines of test population

Sensitivity

94% accuracy

Basal

Treated

Ratio

48 genes

121 genes

39 genes

Resistance

high expression in mnng resistant cells
High expression in MNNG resistant cells

Sensitive

Resistant

2 genes

48 genes

Low

High

slide66

Base excision repair (BER), which repairs damage to a single base caused by oxidation, alkylation, hydrolysis, or deamination.

  • The damaged base is removed by a DNA glycosylase, resynthesized by a DNA polymerase, and a DNA ligase performs the final nick-sealing step.
slide67

These hydrolyze the N-glycosylic bond between the base and the deoxyribose, as illustrated here by the action of uracil DNA N-glycosylase:

slide70

2. Nucleotide excision repair (NER), which recognizes bulky, helix-distorting lesions such as pyrimidinedimers and 6,4 photoproducts.

A specialized form of NER known as transcription-coupled repair deploys NER enzymes to genes that are being actively transcribed.

ner involves the following steps
NER involves the following steps:
  • Damage recognition
  • Binding of a multi-protein complex at the damaged site
  • Double incision of the damaged strand several nucleotides away from the damaged site, on both the 5' and 3' sides
  • Removal of the damage-containing oligonucleotide from between the two nicks
  • Filling in of the resulting gap by a DNA polymerase
  • Ligation
slide75

Xeroderma pigmentosum (XP)

Xerodermapigmentosa, or XP, is an autosommalressessive genetic disorder of DNA repair in which the ability to repair damage caused by ultraviolet (UV) light is deficient (NER deficiency).

This disorder leads to multiple basal cell carcinomas (basaliomas) and other skin malignancies at a young age.

slide76

In severe cases, it is necessary to avoid sunlight completely.

The two most common causes of death for XP victims are metastatic malignant melanoma and squamous cell carcinoma.

slide77

Cockayne syndromeis a rare autosomal recessivecongenital disorder characterized by growth failure, impaired development of the nervous system, abnormal sensitivity to sunlight (photosensitivity), and premature aging.

slide78

3. Mismatch repair (MMR), which corrects errors of DNA replication and recombination that result in mispaired (but undamaged) nucleotides.

slide82

Double-strand breaks

Double-strand breaks (DSBs), in which both strands in the double helix are severed, are particularly hazardous to the cell because they can lead to genome rearrangements.

Various mechanisms exist to repair DSBs:

non-homologous end joining (NHEJ),

recombinational repair (also known as template-assisted repair or homologous recombination repair

slide84

In NHEJ

DNA Ligase IV, a specialized DNA Ligase that forms a complex with the cofactor XRCC4, directly joins the two ends.

slide85

DNA ligase, shown above repairing chromosomal damage, is an enzyme that joins broken nucleotides together by catalyzing the formation of an internucleotide ester bond between the phosphate backbone and the deoxyribose nucleotides.

slide86

Recombinational Repair

Recombinational repair requires the presence of an identical or nearly identical sequence to be used as a template for repair of the break.

The enzymatic machinery responsible for this repair process is nearly identical to the machinery responsible for chromosomal crossover during meiosis.

This pathway allows a damaged chromosome to be repaired using a sister chromatid (available in G2 after DNA replication) or a homologous chromosome as a template.

slide89

Translesion synthesis

Translesion synthesis is a DNA damage tolerance process that allows the DNA replication machinery to replicate past DNA lesions such as thymine dimers or AP sites.

It involves switching out regular DNA polymerases for specialized translesion polymerases (e.g. DNA polymerase V), often with larger active sites that can facilitate the insertion of bases opposite damaged nucleotides.

slide90

The polymerase switching is thought to be mediated by, among other factors, the post-translational modification of the replication processivity factor PCNA.

Translesionsynthesis polymerases often have low fidelity (high propensity to insert wrong bases) relative to regular polymerases.

PCNA

Proliferating Cell Nuclear Antigen

slide91

DNA repair and cancer

Inherited mutations that affect DNA repair genes are strongly associated with high cancer risks in humans.

Hereditary nonpolyposis colorectal cancer (HNPCC) is strongly associated with specific mutations in the DNA mismatch repair pathway.

BRCA1 and BRCA2, two famous mutations conferring a hugely increased risk of breast cancer on carriers, are both associated with a large number of DNA repair pathways, especially NHEJ and homologous recombination.

slide92

Modern cancer treatments attempt to localize the DNA damage to cells and tissues only associated with cancer, either by physical means (concentrating the therapeutic agent in the region of the tumor) or by biochemical means (exploiting a feature unique to cancer cells in the body).

slide93

Cancer Chemotherapy

  • The hallmark of all cancers is continuous cell division.
  • Each division requires both
    • the replication of the cell's DNA (in S phase) and
    • transcription and translation of many genes needed for continued growth.
  • So, any chemical that damages DNA has the potential to inhibit the spread of a cancer.
  • Many (but not all) drugs used for cancer therapy do their work by damaging DNA.
slide94

The table lists (by trade name as well as generic name) some of the anticancer drugs that specifically target DNA.

some questions
Some questions
  • How many DNA repair proteins are there in humans?
  • What about conservation across species??