M2C: Dna Damage And Repair

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Cancer

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Genomic instability

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Deamination

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Significant source of DNA damage. Due to loss of amine group on aa, normal base (a, g, c) to a non-standard base (u, HX, X). Causes transition mutation (pyrimidine to pyrimidine or purine to purine) in next round of replication. Deamination of C produces U which binds with A. This will replicate as an A-T pair rather than a C-G pair in the next round of replication.

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Base Loss

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~10,000 bps are spontaneously lost with each round of replication. Depurination happens more frequently than depyrimidination (higher rate).

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Reactive Oxygen Species

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ROS. Examples are hydrogen peroxide (H2O2) and super ozide anion (O2-). These species of Oxygen either have or easily create an oxygen with an unpaired electron (free radical). These naturally exist in the body as metabolic intermediates and in the immune system in phagocytosis, and are part of ionizing radiation. Damage DNA in a variety of ways. Can be removed by anti-oxidants or reducing factors.

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Ways ROS damage DNA

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1. Cleaving the deoxyribose sugar or the phosphate backbone leaving a strand break.
2. Damaging the base leaving a replication block or a misrepairing mutation after replication.

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Exogenous Sources of DNA Damage

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Radiation and carcinogens.

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Ionizating Radiation

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Gamma or X rays. Functions through radiolysis of water creating a free radical (H2O+ and e-). Ionizing radiation is rare and found most commonly in medicine. X-rays are low level and cause little damage. Radiation therapy has DNA damage as goal.

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Ultraviolet Radiation

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DNA absorbs UV light to 260nM. At or near this peak, light can photoactivate nucleotides causing distortion in the helix. The most reactive nucleotides are adjacent pyrimidines. They can covalently bond causing Pyrimidine Dimers. These prevent normal base-pairing during the next replication.

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Pyrimidine Dimers

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Covalently linked pyrimidines caused by exposure to peak levels of UV light. Two types possible:
1. Cyclobutane product. Usually T-T.
2. 6-4 product. Either 5'-T-C-3', or C-C.
Both prevent proper base pairing during next replication.

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Adduct Formation

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Chemicals cause covalent bonding to nucleotides. Usually bulky and simply block replication. Some can cause base substitution.

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Alkylating Agents

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Chemicals that can cause adduct formation with DNA. Electrophilic (electron loving) attach to electron-rich DNA. Often methyl, ethyl, or larger groups add to a base in DNA blocking further transcription. Benzo[a]pyrine is alkylating agent in cigarette smoke. Like others, must be activated upon generation of metabolic breakdown agent.

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Cross-linking agents

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Bifunctional chemicals that have two adduct forming groups. Can covalently bond with two different positions on DNA. Cause covalent bridges (cross-linkages) either intrabridges (same DNA) or interbridges (two DNA). Completely block transcription and replication.

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Errors in DNA replication

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Occur either by error of polymerase or microsatelite instability. Usually are repaired by mismatch repair.

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Polymerase Error

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Misincorporation of a nucleotide by polymerase. Happens on 1/10^6 bps. Usually arise because of existence of base tautomers in the cell. Tautomers base pair by different rules and will lead to substitution errors. Frequency reduced (1000 fold) by polymerase 3'-5' exonucleases. Frequency increased when there is an unbalanced base pool.

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Base Tautomers

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Alternate forms of standard bases. Often pair by different rules.

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G and T Tautomers

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G and T are usually found in keto form. Exist in enol tautomers. Enol T pairs with keto G instead of A and enol G pairs with keto T instead of C.

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A and C Tautomers

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A and C are normally in amino form. Exist in imino tautomers. Imino A pairs with amino C instead of T and imino C pairs with amino A instead of G.

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Repeat/microsatellite Instability

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Primer slips on template strand while transcribing highly repetitive sites. This can cause a bulge which can lead to a deletion or repeat sequence in daughter cell.

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How do cells respond to DNA damage

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1. Damage sensors. Recognize gaps, distortions, and breaks in the double helix.
2. Transducers: Usually protein kinases. Phosphorylate mediators.
3. Mediators: Tell cell what to do in responce to DNA damage. Activate or inhibit other gene products that lead to cell cycle checkpoints and DNA repair.

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Cell Cycle

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M: mitosis, S: synthesis (replication), G1: gap 1 (commitment to DNA replication), G2: gap 2 (preparation for mitosis).

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Why it is dangerous for a cell to undergo S or M phase with a damaged genome

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If double helix is intact, has to undergo error-prone post-replicational repair (translesion synthesis). If backbone is not intact, could result in double strand break, much more difficult and risky to repair. In mitosis, existance of strand breaks can cause aberrant chromosome segregation or improper fusions of chromosomes (abberations).

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DNA checkpoints

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Occurs during G1 and G2. Cell responds to DNA damage.

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Cell Cycle Arrest

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Responce of cell during checkpoint in presence of DNA damage. Gives cell more time to repair damage.

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Apoptosis

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Cell Death. Cell is removed by programmed cell death if checkpoint reveals accumulation of potentially cancer causing mutations in DNA.

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Cancer and loss of DNA prepair

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Mutations in genes that code for factors in DNA damage responce lead to tumorigenesis.

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p53

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p53 is a mediator in the response to DNA damage. It induces transcription of certain genes that code for repair mechanisms. It leads to cell cycle arrest OR apoptosis. p53 activity is lost in the majority of cancers. Example or inactivating mutations causing cancer (in comparison to oncogenes).

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Common chemotherapy for cancer cells

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Often include DNA damaging component. Includes alkylating agents, crosslinking agents, topoisomerase inhibitors, and ionizing radiation. High level side-effects exist because hard to specify target. Cancer cells are often quickly-replicating and therefore are more prone to checkpoint apoptosis. Can cause damage in previously healthy cells and in turn, cause therapy-induced tumors.

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Target: Deaminations, Depurinations

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Repair pathway: Base Excision Repair (BER)

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Target: Cross-linkages, Adducts, UV biproducts

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Repair pathway: Nucleotide Excision Repair (NER)

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Target: Bypass all of the above

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Repair pathway: Translesional Synthesis (TLS)

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Target: Replication Errors

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Repair pathway: Mismatch Repair (MMR)

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Target: Double strand breaks, Adducts, Cross-links

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Repair pathway: Homologous Recombination (HR)

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Target: Double Strand Breaks

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Repair pathway: End joining (EJ)

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Excision Repair

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Recognizes difference between mutated DNA and normal duplex DNA. Compiles multi-protein complex to excise and re-build DNA using un-mutated template strand.

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Base Excision Repair (BER)

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Uses DNA glycosylases to recognize and excise individual mutated bases. Not "flexible" because glycosylases are base-specific. Usually repair deaminations. When recognizes a mutation, excises the base and leaves an abasic (AP) site. Single nucleotide gap is filled by polymerase and nick is connected by ligase.

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Nucleotide Excision Repair (NER)

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Primary means to repair UV photoproducts. Not super specific. Uses multi-protein complex including XPA to recognize and excise mutations. NER cuts and removes 28bp strand (5 from 3' and 23 from 5'). Gap is filled and ligased. Is often used during transcription of exon genes. An RNA polymerase runs into mutation during transcription, calls for compilation of NER complex to fix problem.

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Mismatch Repair

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Usually used to fix mistakes made during transcription. Must recognize new strand from old in order to correct new strand. Often caused by primer problem (between okazaki fragments). Easier to fix if before nicks are sealed. Therefore, repair mechanisms must be linked to replication fork.

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How Mismatch Repair works

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Mismatch causes heteroduplex (bubble) region. Recognized by protein complex in mismatch repair pathway. Some factor then recognizes the most recently synthesized strand (we don't know why). An exonuclease then removes 300-500 nucleotides (from point of new strand recognition to mismatch). Fill-in and ligation uses intact template strand to complete repair.

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Hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch's Disease

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Colorectal cancer caused by mutation in gene coding for mismatch repair factors. Idividuals effected start with heterozygocity and then end up losing normal copy and lots more mutated copy. Then get consequent of buildup of secondary mutations in growth control genes causing cancer.

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Translesional Synthesis

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Refers to ability of replication fork to proceed through DNA damage. This synthesis often bypasses damage in a non-templated way. This is error-prone and often requires a special type of DNA polymerase.