DNA Repair System & Its mechanisms(3)

DNA repair and cancer

- At least 34 Inherited human DNA repair gene mutations that increase cancer risk - Many of these mutations cause DNA repair to be less effective than normal - In particular, Hereditary non-polyposis colorectal cancer (HNPCC) is strongly associated with specific mutations in the DNA mismatch repair pathway. - BRCA1 and BRCA2 mutations confer 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.

A cell that has accumulated a large amount of DNA damage, or one that no longer effectively repairs damage incurred to its DNA, can enter one of three possible states:

1. an irreversible state of dormancy, known as senescence 2. cell suicide, also known as apoptosis (programmed cell death) 3. unregulated cell division, which can lead to the formation of a tumor that is cancerous The DNA repair ability of a cell is vital to the integrity of its genome and thus to the normal functionality of that organism.

DNA in cells requires constant maintenance to repair damage and correct errors

The agents that damage DNA can either be external to the cell or they may arise as undesired effects of internal cell chemistry.

1. Direct reversal of the DNA damage

• Damage can occur in only one of the four bases • Do not involve breakage of the phosphodiester backbone • Three human genes have been implicated in this mechanism • The best-characterized encodes the 0-6-methylguanine-DNA methyttransferase, which is able to remove methyl groups from guanines that have been incorrectly methylated. • UV radiation-produced thymine dimers can be directly resolved by the enzyme photolyase using the energy of visible light (photoreactivation). • Although mammals possess enzymes related to photolyase, they use them for a quite different purpose, to control their circadian clock. • Photolyase, an old enzyme present in bacteria, fungi, and most animals no longer functions in humans, who instead use nucleotide excision repair to repair damage from UV irradiation.

The effects of DNA damage

- Cytotoxic: when the DNA is replicated, many types of damage will cause the replication fork to stall. - Even outside S phase of the cell cycle, during transcription RNA polymerase stalls at lesions in the DNA, preventing expression of the damaged gene. - These problems are potentially lethal to the cell. Special multiprotein machines constantly patrol the DNA of a cell detecting and responding to damage. - Progression through the cell cycle is delayed until the damage has been repaired, and irreparable damage triggers apoptosis (programmed cell death). - Malfunctions of the systems for detecting DNA damage and coordinating the cellular response have major roles in the development of cancer. - Even if a cell is able to survive with damaged DNA, unrepaired damage is likely to be mutagenic. - If mutations occur in germ cells, they can give rise to new variants in the population that provide much of the raw material for evolution.

Non-homologous end joining (NHEJ)

- DNA Ligase IV, that forms a complex with the cofactor XRCC4, directly joins the two e - To guide accurate repair, NHEJ relies on short homologous sequences called micro-homologies present on the single- - If these overhangs are compatible, repair is usually accurate. - NHEJ can also introduce mutations during repair. - Loss of damaged nucleotides at the break site can lead to deletions, and joining of non-matching termini forms insertions or translocations. - NHEJ is especially important before the cell has replicated its DNA, since there is no template available for repair by homologous recombination. - There are "backup" NHEJ pathways in higher eukaryotes. Besides its role as a genome caretaker, NHEJ is required for joining hairpincapped double-strand breaks induced during V(D)J recombination, the process that generates diversity in B- cell and T-cell receptors in the vertebrate immune s

Genes required for MMEJ

- FEN1, LigaseIII, MRE11, NBS1, PARP1 and XRCC1 All six of these genes are up-regulated in one or more cancers. - MMEJ in cancer 1. FEN1 is over-expressed in the majority of cancers of the breast, prostate, stomach, neuroblastomas, pancreatic, and lung. 2. Ligase III is upregulated in chronic myeloid leukemia, multiple myeloma, and breast cancer. 3. MRE11 is over-expressed in breast cancers. 4. NBS1 is over-expressed in some prostate cancers, in head and neck cancer, and in squamous cell carcinoma of the oral cavity. 5. PARP1 is over-expressed in tyrosine kinase-activated leukemias, in neuroblastoma, in testicular and other germ cell tumors,] and in Ewing's sarcoma. 6. XRCC1 is over-expressed in non-small-cell lung carcinoma (NSCLC), and at an even higher level in metastatic lymph nodes of NSCLC

repair mechanisms

- In response to these various forms of damage, cells deploy a whole range of repair mechanisms. - Different mechanisms correct different types of lesion. The importance of effective DNA repair systems is highlighted by the ~130 human genes participating in DNA repair, and by the severe diseases that affect people with deficient repair systems

Microhomology-mediated end joining (MMEJ)

- MMEJ starts with short-range end resection by MRE11 nuclease on either side of a DSB to reveal microhomology regions - Poly (ADP-ribose) polymerase 1 (PARP1) is required and may be an early step in MMEJ. - There is pairing of microhomology regions followed by recruitment of flap structure-specific endonuclease 1 (FEN1) to remove overhanging flaps. - This is followed by recruitment of XRCC1-LIG3 to the site for ligating the DNA ends, leading to an intact DNA. - MMEJ is always accompanied by a deletion, so that MMEJ is a mutagenic pathway for DNA repair.

Homologous Recombination (HR)

- 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 or a homologous chromosome as a template. - DSBs caused by the replication machinery attempting to synthesize across a single-strand break or unrepaired lesion cause collapse of the replication fork and are typically repaired by recombination

Mismatch repair systems

- are present in essentially all cells to correct errors that are not corrected by proofreading. - These systems consist of at least two proteins. 1. detects the mismatch, 2. recruits an endonuclease that cleaves the newly synthesized DNA strand close to the region of damage.

Mechanisms of DNA Repair in Human Cells

- ~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. - Others induce potentially harmful mutations in the cell's genome. - As a consequence, the DNA repair process is constantly active as it responds to damage in the DNA structure. - When normal repair processes fail, and when cellular apoptosis does not occur, this can eventually lead to cancer

3. Double-Strand Breaks (DSBs)

-both strands in the double helix are severed, are particularly hazardous to the cell because they can lead to genome rearrangements. -The cell will die in the next mitosis or in some rare instances, mutate. -Three mechanisms exist to repair DSBs; 1. non-homologous end joining (NHEJ), 2. homologous recombination (HR), 3. microhomology-mediated end joining (MMEJ).

The major threats to the stability of a cell's DNA come from internal chemical events

1. Depurination 2. Deamination 3. Attack by reactive oxygen species 4. Non-enzymatic methylation

The molecular mechanisms of DNA repair processes.

1. Direct reversal of the DNA damage 2. Single-strand damage 3. Double-Strand Breaks (DSBs)

Damage recognition

1. Global genomic NER (GG-NER) - DNA-damage binding (DDB) and XPC-Rad23B complexes scan and recognize the damage . - Associated disease: Xeroderma pigmentosum (XP), severe photosensitivity, high cancer rates in areas of the body exposed to the sun (e.g. skin). 2. Transcription coupled repair (TC-NER) - RNA polymarase recognize the damage - Associated disease: 1- Trichothiodystrophy (TTD), some individuals are photosensitive, ichthyosis, mental/physical retardation 2-Cockayne syndrome (CS), photosensitivity, mental retardation, progeria-like features, microcephaly

3 main external agents likely to cause DNA damage

1. Ionizing radiation: gamma rays and X-rays can cause single-strand or double-strand breaks in the sugar-phosphate backbone. 2. Ultraviolet radiation: UV-C rays (with a wavelength of about 260 nm) are especially damaging, but the major source of UV damage in humans is from the UV-B rays (260-315 nm) in sunlight that can penetrate the ozone layer. UV radiation causes cross-linking between adjacent pyrimidines on a DNA strand to form cyclobutane pyrimidine dimers and other abnormal photoproducts. 3.Environmental chemicals: These include hydrocarbons (ex; in cigarette smoke), some plant and microbial products such as the aflatoxins, and chemicals used in cancer chemotherapy. Alkylating agents can transfer a methyl or other alkyl group onto DNA bases and can cause cross-linking between bases within a strand or between different DNA strands

Non-enzymatic methylation

Accidental non-enzymatic DNA methylation by S-adenosyl methionine produces about 300 molecules per cell per day of the cytotoxic base 3- methyl adenine, plus a quantity of the less harmful 7 -methyl guanine. This is quite distinct from the enzymatic methylation of cytosine to produce 5-methyl cytosine, which cells use as a major method of controlling gene expression. The methylated adenine or guanine bases distort the double-helix and interfere with vital DNA-protein interactions. Additonally, errors might arise during normal DNA metabolism. A certain error rate is inevitable during DNA replication. Proofreading mechanisms correct the great majority of the resulting mismatches, but a few may persist, producing sequence variants. Failure of the proofreading mechanism in somatic cells is one cause of cancer. In addition, occasional errors in replication or recombination leave strand breaks in the DNA, which must be repaired if the cell is to survive.

DNA variations

DNA variations can occur 1. DNA replication 2. Recombination 3. A failure to repair DNA damage Chemical attack by exogenous or endogenous agents, and errors arising during its normal functioning, pose constant threats to the integrity of the genome.

Attack by reactive oxygen species

Highly reactive superoxide anions (02 - ) and related molecules are generated as a by-product of oxidative metabolism in mitochondria. They can also be produced by the impact of ionizing radiation on cellular constituents. These reactive oxygen species attack purine and pyrimidine rings. Mechanisms of DNA damage by reactive oxygen species (ROS) ROS are generated in cells both endogenously (oxygen metabolism) and exogenously (drugs, radiation).

The rate of DNA repair is dependent on many factors

a) including the cell type, b) the age of the cell, c) the extracellular environment.

2. Single-strand damage

p 17 - 18- 19

Deamination

~100 cytosines each day in each nucleated human cell are spontaneously deaminated to produce uracil. Less frequently, spontaneous deamination of adenine produces hypoxanthine.

Depurination

~5000 adenine or guanine bases are lost every day from each nucleated human cell by spontaneous hydrolysis of the base-sugar link.