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31 Cards in this Set
- Front
- Back
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Nucleosome
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-Composed of 8 histones (146 bp)
-Basic subunit of chromatin. |
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Deoxyribonucleosides vs. Deoxyribonucleotides
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Deoxyribonucleosides: Base + Sugar
Deoxyribonucleotides: Base + Sugar + Phosphate |
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General concepts about Replication
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SEMI-Conservative
- Substrates dATP, dGTP, dCTP, dTTP - Template: DNA double helix - DNA Polymerase Activity - 5' -> 3' DNA Polymerase activity - 3' -> 5' Exonuclease activity (proofreading) |
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Initiation of DNA Replication
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Origin of replication is where replication begins at.
DNA replication requires careful orchestration of events and strong correlations between G-C content, gene density, transcriptional activity, and replication timing. |
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Complex Usage of Human Origins
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Forks are where replication begins at, but forks have different rates. Fast fork rates for DNA replication have less frequent initiation because the protein required is delivered while forks with slower rates have more frequent initiation because the protein required is not delivered yet. This may not always be the case. If a fast fork approaches a stop, more initiations will be fired off to compensate.
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Lagging and Leading strand
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Leading strand replicates in a continuous matter.
Lagging strand replicates in Okazaki fragments because polymerase works from 5' -> 3'. It is important to not, among other things, that Okazaki fragments replicate in 100-200bp in Eukaryotes, but in Prokaryotes the fragments are much larger; 1-2 kb. |
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Processivity
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Measure of the number of nucleotides that are copied by the polymerase prior to dissociation from the template.
Proofreading mechanism by polymerase decreases errors from 10^-4 to 10^-8 |
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How is the RNA primer removed during DNA replication in human cells?
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DNA polymerase delta and helicase lift that primer from the succeeding okazaki fragment and Flap endonuclease (FEN1) cleaves the primer at the branch point created by polymerase delta and helicase.
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Helicase
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Separates two strands of double helix. 95 in humans, 64 RNA and 31 DNA.
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Topisomerase I
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Relieve torsional stress. Cleaves one strand, pass the other through then reseals the cut strand.
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Replication Protein A
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Single stranded binding protein whose responsibility is to keep DNA strands separated.
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Irinotecan
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Topoisomerase Inhibitors
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5-Fluorouracil
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Anti-metabolite
-Effective against colorectal cancer. We see a 10-15% response rate when used as a first- line treatment against advanced colorectal cancer. -Combination with new chemotherapies like Irinotecan and oxaliplatin have an improved response rate of 40-50% against advanced colorectal cancer. |
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Oxaliplatin
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DNA damaging agent
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Paclitaxel
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Microtubule Inhibitor
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Telomerase
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Complex of RNA and protein that copies RNA template into DNA, through the actions of Reverse Transcriptase.
-Useful because Lagging strand can not be fully replicated -Active in reproductive and stem cells to maintain integrity of genetic code. |
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Shelterin
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Protein complex that helps prevent abnormal fusion events
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Rare Telomere Diseases
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Dyskeratosis congenita (DKC) : Bone marrow failure in first or second decade due to the result from inherited defects in any of several different genes important for the repair or protection of Telomeres.
- aplastic anemia is usually fatal - increased risk of cancer Characterized by: - dystrophic nails - patchy skin hyperpigmentation - oral leukoplakia |
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Telomeres and their effects on common diseases
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- Telomerase is active in many cancer cells, but short telomeres are still present.
- Strong evidence that telomere dysfunction plays a critical role in prostate cancer initiation and progression. - Telomere shortening and risk of heart disease is speculated -Telomeres naturally shorten as we age, but telomere shortening is not thought to be the cause of aging for two reasons: 1) Dyskeratosis congenita patients do not prematurely age 2) Not all cell types show significant age related telomeric shortening. |
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Melanoma
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Melanocytes create melanin to protect skin cells from damaging effects of UV rays. Mutations in the promoter of the TERT gene (which encodes a subunit of Telomerase) introduce a new regulatory element (ETS Factor).
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Endogenous Sources of DNA Damage
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DNA damage caused from within
-DNA replication errors: misincorporation, polymerase slippage - DNA metabolism error: topoisomerase error - Deamination: Cytosine -> Uracil (Enzymatic or spontaneous) -Depurination: Loss of guanine or adenine spontaneously |
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Environmental DNA Damage soures
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-Ionizing radiation
- Ultraviolet radiation - Chemical mutagens - reactive oxygen species |
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Types of Repair
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Direct Repair - Replacement of damaged nucleotide
Excision repair - ( Base excision repair or nucleotide excision repair) damaged region is excised and DNA is resynthesized in that region. Mismatch Repair - Excised and repaired Nonhomologous end-joining: joining of strands after a double strand break |
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Clinical Correlate of Direct Repair?
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-MGMT protein repairs O^6-methylguanine lesions
-Loss of MGMT by somatic mutations or epigenic silencing is associated with increased cancer risk and sensitivity to methylating agents -However, in the case of cancer, the loss of a repair protein can prove useful because it makes the tumors susceptible to certain chemotherapies or other treatments. |
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Clinical Correlate of Base Excision Repair (Excision repair I)
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Familial Adenomatous Polyposis 2 (FAP2): Autosomal recessive disorder that confers an increased risk of developing colorectal cancer. (Mutations in MYH gene)
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Clinical correlate of nucleotide excision repair (Excision repair II)
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Xeroderma pigmentosum: autosomal recessive XPC, ERCC2, or POLH mutations.
-Sun sensitivity, elevated risk of cancer Cockayne Syndrome: Autosomal Recessive ERCC6 or ERCC8 mutations -Sun sensitiy, mental and growth retardation. |
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Mismatch Repair Clinical correlates
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Hereditary Non-polyposis Colorectal Cancer (HNPCC) or Lynch Syndrome
Sporadic Microsatellite-instabiity colorectal cancers |
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Hereditary Non-polyposis Colorectal Cancer (HNPCC) or Lynch Syndrome
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Autosomal Dominant germline mutations in any of a small number of genes.
-increased risk of cancer. Mismatch repair correlate |
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Sporadic Microsatellite-instabiity colorectal cancers
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Repair genes are inactivated by somatic mutations (acquired mutations, not inherited mutations)
Microsatellite sequences are repetitive DNA sequences usually several base pairs in length mismatch repair correlate To determine if a patient has Sporadic microsatellite instability colorectal cancer, samples from tumor undergo PCR amplification and there fragments are analyzed on gel electrophoresis. Microsatellite instability will lead to shorter fragments because the mutations in the strands will cause the primers to bind to other areas so some of the strands will be shorter. (SLIDE 38) |
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Nonhomologous end-joining repair process
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Can repair a double stranded break at any time during cell cycle and does not require sequence homology.
Clinical Correlation: NHEJ is responsible for chromosomal aberrations of some cancer cells. This includes translocations, inversions, and deletions. |
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NHEJ Clinical Correlate
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Fanconi Anemia: Autosomal recessive disorder caused by genomic instability
-Developmental abnormalities in major organs -upper limb malformations -early onset bone marrow failure -increased cancer predisposition |
