Qiao1-Replication

Question 1

What can the cell do to preserve genome integrity?

Answer 1

-If insults are excessive, DNA damage check point is activated to halt cell cycle progression

Question 2

Why is cancer said to be a genomic disorder?

Answer 2

• Somatic mutations activate oncogenes or inactivate tumor suppressors:
  1) Overactivity mutation (gain of function): single mutation activates oncogene that promotes cell transformation.
  2) Underactivity mutation (loss of function): two mutation events to inactivate the two gene copies of tumor suppressor genes.

Question 3

What are caretaker and gatekeeper genes?

Answer 3

*Caretaker genes: for DNA repair or fidelity of chromosome segregation. Control the stability of the genome & prevent accumulation of mutations.

*Gatekeeper genes: regulate growth of tumor by inhibiting growth or by promoting death.

Question 4

What are some characteristics of DNA?

Answer 4

• Polymer of deoxyribo-nt monophosphates linked by 3’->5’ phosphodiester bonds
• Chains are antiparallel
• Hydrophilic phosphate backbone
• Hydrophobic bases stacked inside
• High AT content DNA: Tm of 65C
• High GC content DNA: Tm of 92C

Question 5

How does Dactinomycin work?

Answer 5

• Cytotoxic effect by intercalating into the narrow groove of the DNA helix interfering with DNA/RNA synthesis

Question 6

What are some general characteristics of DNA replication?

Answer 6

1. Semi-conservative
2. Specialized DNA Pols
3. DNA Pol I has 3 activities
4. High fidelity
5. Bidirectional (P & E)
6. DNA replication fork is asymmetrical
7. Substrate needed for new DNA synthesis

Question 7

What is the Meselson-Stahl experiment?

Answer 7

Experiment proved that DNA replication is semiconservative:

1- E.Coli was grown in 15N-labeled medium

2- Centrifugation showed 1 band: 15N

3- 15N-labeled E.Coli was added to 14N-medium

4- After replication: 1 band 14N/15N

5- After 2nd replication: 14N/14N & 14N/15N

6- Third generation: 2- 14N/14N, & 15N/14N

Question 8

What did Cairns experiments show?

Answer 8

• E. Coli chromosome is circular
• There is a single origin of replication in E. Coli

Question 9

What are DNA polymerases?

Answer 9

• Template-directed phosphoryl transfer machines
• DNA Pol I catalyzes the addition of complementary dNTP to the 3’OH end of a chain
• Mechanism is a nucleophilic displacement

Question 10

What are the 4 essential requirements for the activity of DNA Pol I?

Answer 10

1) Template 2) Primer 3) Free 3’OH end 4) dNTPs (as the Mg++ salt)

Question 11

What are the 3 different enzymatic activities of DNA Pol I?

Answer 11

1) 5’ to 3’ DNA polymerizing activity 2) 3’ to 5’ exonuclease activity 3) 5’ to 3’ exonuclease activity

Question 12

What is DNA polymerase structure like?

Answer 12

DNA Pol resembles a right hand:

• Palm: active site (polymerase & exonuclease), binds metal ions, primer terminus, a-phosphate of incoming dNTP
• Fingers: contact the ss template & incoming dNTP
• Thumb: contact duplex DNA

Question 13

What is the 3’ to 5’ exonuclease activity of DNA Pol?

Answer 13

It excises the mismatched nucleotides

Question 14

What gives ride to the high fidelity of DNA synthesis?

Answer 14

• Polymerization error 1/ 10^5 -3’ to 5’
• exonuclease 1/ 10^2
• Mismatch repair error 1/ 10^2
• In humans, there are 3.2x10^9 bp, so 3 nucleotides mutate each time a cell divides

Question 15

How is the DNA replication fork asymmetrical?

Answer 15

–> Both strands are polymerized 5’ to 3’ directions

1) Leading strand: continuous synthesis [5’ to 3’]
2) Lagging strand: discontinuous; synthesis of short poly-nt segments (Okazaki fragments) that are then ligated [5’ to 3’]

*Length of Okazaki fragments is due to processivity (ability to catalyze consecutive reactions w/o releasing its substrate

Question 16

What is the bacterial DNA replication machinery?

Answer 16

The replisome is composed of:

• Ori binding protein (DnaA)
• DNA hexameric helicase (DnaB)
• Primase (DnaG)
• ss-DNAbp (SSB)
• DNA topoisomerases / gyrase
• DNA ligase
• DNA polymerase III
• Sliding clamp & clamp loader

Question 17

What occurs in bacterial DNA replication initiation?

Answer 17

1. DnaA binds 9bp-oriC using ATP
2. Local unwinding of AT-rich sequence serves as loading zone for helicase
3. DnaB helicase is activated and moves in 5’ to 3’ direction
4. Dna B interacts with DnaG (primase), which synthesizes short RNA primer for DNA Pol III holoenzyme

Question 18

How does DNA helicase work?

Answer 18

• Disrupts the H-bonds between the strands
• Hexameric ring protein that encircles DNA to catalyze ATP-dependent unwinding of dsDNA to generate the replication fork
• ATP binding and hydrolysis change protein conformation in a cyclical manner
• Nearly unlimited processivity

Question 19

What is the role of SSB (ss DNA binding protein)?

Answer 19

• Binds DNA cooperatively (8nt) w/o covering the bases to remove the “hairpin” structure of ssDNA
• Aid helicases by stabilizing the unwound ssDNA
• Also involved in repair and recombination

Question 20

What is the role of DNA primase?

Answer 20

• RNA Polymerasethat makes RNA primers
• Limited processivity, chains shorter than 12 nt’s

Question 21

What is the role of DNA topoisomerases?

Answer 21

Resolve DNA winding & prevent tangling during replication by cutting DNA backbone:

• Type I: reversibly cut one strand. Have nuclease & ligase activities. Reuse energy from phosphodiester bond cleavage.
• Type II: bind double helix & make transient breaks in both strands. Enzyme then causes 2nd stretch of DNA double helix to pass through the break, reseals break. Required for daughter chromosome separation.

Question 22

What is the effect of camptothecin & doxorubicin?

Answer 22

• Camtothecin: it’s a cytotoxic quinoline alkaloid which i_nhibits the DNA enzyme topo I_
• Doxorubicin (adriamycin): used to treat breast cancer because it _inhibits topo II a_ctivity.

Question 23

What is the structure of DNA Pol III?

Answer 23

1) Core subunit: α-subunit (polymerase activity) & ε-subunit (3’->5’ exonuclease activity). Sufficient to replicate, but not processive (10-15nt)

2) γ/δ-subunit: clamp loader; multiple protein complexes that bind primer-template junctions. DNA-dependent ATPase activity

3) β-subunit: sliding clamp

Question 24

What are all the DNA Pols found in E. Coli?

Answer 24

Pol I: RNA primer removal, DNA repair

Pol II: DNA repair

Pol III: DNA replication enzyme

Pol IV and V: DNA repair

Question 25

How do nucleoside analog drugs work?

Answer 25

* Block DNA replication by becoming incorporated to chain or modifying sugar moieties * Ex. Cytarabine (anti-cancer therapy), adenine arabinoside (anti-viral), AZT (anti-HIV agent)

Question 26

What occurs in bacterial DNA replication elongation?

Answer 26

**1) Leading strand:** 1st RNA primer 4-12 nt is synthesized by primase, & Pol III synthesizes **2) Lagging strand**: * Pol III complex must be partially disassembled * new RNA primer is then synthesized * Pol III reassembled to elongate * RNA primer must be removed * Okazaki fragments, ~1-2K nt, are ligated (DNA ligase joins 5'PO₄ on chain made by DNA Pol III and 3'OH on chain made by DNA Pol I)

Question 27

What is the lagging strand synthesis in eukaryotes?

Answer 27

* Okazaki fragments are about 100 nt * The primase/polymerase alpha synthesizes about _10 nt of RNA plus 10-20 nt of DNA_ * Primer extended by a complex: _DNA Pol δ, PCNA, and RFC_

Question 28

What is the eukaryotic DNA replication machinery?

Answer 28

* MCM proteins (helicase) * RPA (like SSB, protects ssDNA) * Primase-Pol α * Pol δ/ε /γ (3' to 5' nuclease, proofreading) * DNA topo I and II * Lig 1 (ligates DNA) * DNA polymerase δ/ε (elongates DNA chain) * PCNA (processivity factor, like sliding clamp) * RFC (load PCNA, like clamp loader)

Question 29

What are the human DNA-dependent DNA polymerases?

Answer 29

**Pol α**: primer (has primase), initiates synthesis **Pol β**: base excision repair **Pol γ**: mitochondrial DNA synthesis, proofreading activity **Pol δ:** for lagging strand synthesis, proofreading **Pol ε:** elongates the leading strand

Question 30

What disorders are associated with mutation of human DNA polymerases?

Answer 30

1) _Opthalmoplegia, Alper syndrome_, other neurodegenerative disorders (Pol γ) 2) In _tumors_ (Pol β)

Question 31

What is the eukaryotic regulation of DNA replication?

Answer 31

* It is cell cylce-regulated, governed by cyclins & cell division cycle (cdc) gene products that encode kinases * different combinations of cyclins and kinasesregulate different phases of the cycle * Kinases activate Cdk (G1/S-cdk, S-cdk, and M-Cdk)

Question 32

What are the 3 checkpoint in the cell cycle?

Answer 32

1) **Start G1** checkpoint: if environment is favorable, cell enters S phase 2) **G2/M checkpoint:** if all DNA is replicated and environment is favorable, enter mitosis 3) **Metaphase checkpoint:** if all chromosomes are attached to spindle, trigger anaphase and proceed to cytokinesis

Question 33

How are origins of replication different in prokaryotes & eukaryotes?

Answer 33

- Prokaryotic have only 1 origin of replication - In yeast, they are known as Autonomously Replicating Sequences (ARS), 400 - In mammalian cells: origins are 30-300kb apart, & rate of synthesis is 50bp/s - Active chromatin (containing actively transcribed genes) is replicated early.

Question 34

What is the role of nucleosomes?

Answer 34

* Histones and Mg2+ neutralize - charged DNA * Nucleosome structure: 2 of each: H2A, H2B, H3, and H4 (all are +charged bc of Lys and Arg) * DNA double helix wraps around nucleosomes twice forming ionic bonds * H1 binds to linker DNA chain between nucleosome beads -

Question 35

Why does trinucleotide repeat expansion occur?

Answer 35

* From strand slippage during replication * From unequal strand exchange between chromatids * Oxidative damage * Ex. Kennedy's disease, Huntington's, & SCA

Question 36

How is the "end-replication problem" solved for the linear eukaryotic DNA?

Answer 36

* Problem at 5' end of lagging strand bc when last RNA primer is removed, a shortened 5' end remains that cannot be filled in bc there is no addition of OH groups * RNase H and Fen 1 remove primers * Fixed by telomerase

Question 37

What are telomerases?

Answer 37

* Ribonucleoprotein enzyme that replicates telomeres * RNA subunit (TR) of telomerase is complementary to the telomere sequence (CCCCAA) * TERT component is homologous to HIV RT motif * Newly synthesized DNA is used as a template as the enzyme moves

Question 38

What is the Hayflick limit?

Answer 38

* Number of times a normal human cell population will divide until cell division stops * Telomeres' DNA gets a little shorter w/ each division until they reach a critical short length

Question 39

What are some factors that affect telomeres other than cell division?

Answer 39

* _Accelerate loss_: stress, smoking, obesity * TA-65 from astragalus plant is thought to "_turn on_" telomerase * Cancer _increase telomere length_ because telomerase is activated

Question 40

What are some premature aging syndromes?

Answer 40

- Ataxia telanglectasia (ATM) - Aplastic anemia (TERC/TERT) - Bloom syndrome (BLM) - Dyskeratosis congenita (DKC1, TERC) - Fanconi anemia (Fanc genes) - Nijmegen breakage syndrome (NBN) - Werner syndrome (WRN)

Question 41

What are telomeres?

Answer 41

* Long repeats of (GGGGTT)n that cap and protect chromosomes * Telomere is a nucleoprotein complex (proteins specifically associated with telomeres): Rap1, TRF1/2 (bind dsRNA), TPP1, POT1 (binds ssDNA), TIN2 * It has a high order structure (T-loop)