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Flashcards in DNA Repair Stuff Deck (30):
1

Nucleic Acids

1) Stores and expresses genetic info
In cells it's carried by DNA
Some viruses carry it on RNA
2) Transmit info from generation to generation (basis for heredity)

2

Functions associated with DNA

1) Replication: copies genetic info and transmits it to each daughter cells
2) Transcription: express genetic info
3) Repair: deal with mutations/environmental damage and preserve DNA
4) Recombination: joins DNA molecules to form hybrid molecules. Ab synthesis or meiosis

3

Comparison between DNA and Proteins

Proteins: amino acid chains (1o structure), Beta and alpha helices (2o structure), 3-d conformations/globular domains (3o structures)

DNA: polynucleotide sequence (1o structure), DNA helix (2o structure), supercoiled DNA (3o structure)

4

Autinomycin D and some other anti-cancer drugs

Exert effect by intercalating into minor groove which interferes with DNA/RNA synthesis

5

Topologies of DNA helix

Relaxed: linear or circular b-form double helix
Supercoiled: double helix coils upon itself (helps to fit 6ft DNA in cell)

6

Negative supercoiling

DNA in cells is negatively supercoiled. Facilitate DNA separation in double helix, the energy for doing this is stored in the negative supercoils. These are energetically favorable. Negative supercoiling is spontaneous and right handed.

How are supercoils created?
When histones bind DNA, it wraps around the histone and that's a negative supercoil

7

DNA Topoisomerases

Relieve both positive supercoils (downstream) and excessive negative supercoils (upstream) of unwinding DNA. Have nuclease and Ligase activities: transiently break one or 2 strands, pass the strands through the break, reseal the break

Topoisomerase 1: cuts single strand of helix. Can introduce positive or negative supercoils.
Topoisomerase 2: cuts both strands. Can only introduce negative supercoils (only relax DNA )

Can exploit this to design anti-cancer drugs which inhibit topoisomerases

8

Bacterial DNA Gyrase

Can remove negative or positive supercoils
Facilitates bacterial DNA replication
Requires ATP
Can introduce negative supercoils into relaxed circular DNA
Antibacterial stop DNA Gyrase and no side affects on human

9

Chromatin

Nucleoprotein complex: DNA and histones
Histones are small, basic proteins full of arg and lys
5 classes: H1, H2A, H2B, H3, H4
Arranged in repeating units: nucleosomes

10

Nucleosomes Structure

Core with H2A/B, H3 and H4. 140 BP, 1.75 winds around histones
DNA spacer: 20-80 bp between nucleosomes bound to H1
H1 binding promotes packaging of nucleosomes into solenoid (helical tubular coil)

11

Further compaction during mitosis/meiosis

Solenoid loops around itself
Forms large DNA loops
DNA loops coil around protein scaffold
DNA loops radiate from scaffold: metaphase chromosomes
Classic 4 arm structure with a centromere holding the chromosome in place

12

Inhibitors of DNA Helicase-primase in Herpes Simplex Virus

Stabilize interaction of Helicase/primase with DNA to inhibit progression
Use this when virus is resistant to other drugs

13

Proofreading

3' --> 5' exonuclease activity of DNAPIII
If misplaced nucleotide gets away from DNAPIII, MMR can take over

14

Nucleosomes during replication

Displaced as the replication fork advances
Histones remain loosely associated with one parental strand
New histones are syn simultaneously
Nucleosomes reform behind advancing rep fork

15

Telomeres

Are the ends of linear chromosomes
Consist of short, non-coding G rich DNA repeats and associated proteins
Forms t-loops
Role is to:
1) protect ends of linear chromosomes from recognition as broken DNA and degradation, recombination, end-to-end fusion
2) prevent loss of important terminal sequences during replication
RNA primers needed for DNA replication; DNA is lost when primers are removed.

16

Telomerase

Ribonucleprotein complex (RNA + protein)
Adds short G-rich DNA repeats to ss 3'-ends of linear chromosomes
Uses RNA component as a means to synthesize telemoric repeats
Protein component has reverse transcriptase (h-TERT) activity
Terminal extension allows extra room for a primer to bind later-on and initiate lagging strand syn on the other strand. Prevents loss of seq and maintain appropriate length.

17

Telomerase Clinical Correlation

Telomerase is active in all cells before birth and remains active in stem/germ cells after birth
In somatic cells, telomeres shorten with each cell cycle, mitosis clock.
When telomeres decline to a critical point: end to end fusion of chromosomes could happen. To prevent this, DNA damage sensors induce growth-arrest
Cells use telomere length to keep track of mitosis cycles; mitotic clock
Telomerase is active in cancerous cells. Overrides all this regulation

18

Dyskeratosis congenita

Inherited; caused by reduced telomerase activity. Affects highly dividing cells such as hair follicles, oral squamous epithelium, bone marrow progenitor cells. Patients usually die from bone marrow failure due to loss of hematopoietic renewal

19

Hutchinson-Gilford Progeria

Rare and inherited; show accelerated telomeres shortening
Patients suffer from alopecia, aged skin appearance, short stature, accelerated arthereosclerosis. Patients die of myocardial infarction before age 20

20

Types of mutations

Replication errors: incorporation of wrong nucleo or an extra nucleo: MMR
Damage to single base: depurination, deamination, alkylation, oxidation: BER
Bulky DNA adducts: causes significant distortion in helix, induced by radiation: NER (GG or TC)

21

Other types of damage

SSB: induced by oxidative damage
DSBs: homologous recombination, non homologous end joining
Induced by ionization radiation, oxidizing agents, topoisomerase inhibitors

22

Defects in MMR

Predisposes you to cancer
Lynch syndrome (90% have mutations in MSH2 or MLH1)
Increased risk of colorectal, endometrial, skin, ovarian, gastric and renal cancers

23

Defects in BER

Mutation in DNA glycosylase MYH leads to very high risk for colon cancer (adenomatous colorectal polyposis syndrome)

24

Defects in RecQ family Helicase WRN

Werner Syndrome: rare autosomal recessive disorder (premature aging, cancer disposition, skin atrophy, hair loss and graying)
WRN Helicase: involved in BER, processing of telomeric DNA, activation of DNA damage responses.
Cells are sensitive to oxidation, show telomere shortening, chromosomal rearrangements, frequent telomere fusions

25

NER Clicinal correlations
Think cigarettes on the beach

UV induced TT dimers distort helix causes DNA frameshifts, can result in skin cancer without repair.

26

Xeroderma Pigmentosum

Hereditary resulting from defects in GG-NER
Defects in XPC/XPE (pathway specific to only GG-NER) and XPA/XPD (Helicase activity)
Extreme solar snesitivity
Increased risk of skin cancer
10-20 fold increase in internal cancers
May show neurological symptoms depending on which XP protein is affected

27

Cockayne Syndrome

Hereditary developmental/neurological disorder associated with TC-NER
Mutations in CSA/CSB (affects recognition of stalled RNAPII)
Growth and mental retardation
Neurological deficiency
Sun sensitive
NOT prone to skin cancer

28

Cisplatin

Used to treat cancerous cells with NER defects
Forms bulky adducts in DNA

29

Ataxia Oculomotor Apraxia

Autosomal recessive spinocerebellar ataxia syndrome
Uncoordinated movement and gait
Resembles ataxia telangiectasia but lacks non-neurological symptoms
Mutations in aprataxin gene (DNA end processor of 5' SSBs with hydroplane and transferase activities)

30

Ataxia Telangiectasia

Autosomal recessive
Hypersensitive to ionizing radiation
Mutations in ATM protein (signals cell cycle to slow down and recruits repair proteins, reponds to DNA breaks during B and T cell development)
Increased chromosomal abnormalities
Propensity to develop lymphoid cancer