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BIOCHEMISTRY 2 > Qiao1-Replication > Flashcards

Qiao1-Replication Flashcards

1
Q

What can the cell do to preserve genome integrity?

A

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

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2
Q

Why is cancer said to be a genomic disorder?

A
  • 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.
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3
Q

What are caretaker and gatekeeper genes?

A

*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.

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4
Q

What are some characteristics of DNA?

A
  • 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
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5
Q

How does Dactinomycin work?

A
  • Cytotoxic effect by intercalating into the narrow groove of the DNA helix interfering with DNA/RNA synthesis
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6
Q

What are some general characteristics of DNA replication?

A
  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
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7
Q

What is the Meselson-Stahl experiment?

A

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

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8
Q

What did Cairns experiments show?

A
  • E. Coli chromosome is circular
  • There is a single origin of replication in E. Coli
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9
Q

What are DNA polymerases?

A
  • 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
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10
Q

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

A

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

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11
Q

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

A

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

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12
Q

What is DNA polymerase structure like?

A

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
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13
Q

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

A

It excises the mismatched nucleotides

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14
Q

What gives ride to the high fidelity of DNA synthesis?

A
  • 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
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15
Q

How is the DNA replication fork asymmetrical?

A

–> 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

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16
Q

What is the bacterial DNA replication machinery?

A

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
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17
Q

What occurs in bacterial DNA replication initiation?

A
  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
18
Q

How does DNA helicase work?

A
  • 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
19
Q

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

A
  • 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
20
Q

What is the role of DNA primase?

A
  • RNA Polymerasethat makes RNA primers
  • Limited processivity, chains shorter than 12 nt’s
21
Q

What is the role of DNA topoisomerases?

A

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.
22
Q

What is the effect of camptothecin & doxorubicin?

A
  • 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.
23
Q

What is the structure of DNA Pol III?

A

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

24
Q

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

A

Pol I: RNA primer removal, DNA repair

Pol II: DNA repair

Pol III: DNA replication enzyme

Pol IV and V: DNA repair

25
Q

How do nucleoside analog drugs work?

A
  • 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)
26
Q

What occurs in bacterial DNA replication elongation?

A

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)
27
Q

What is the lagging strand synthesis in eukaryotes?

A
  • 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
28
Q

What is the eukaryotic DNA replication machinery?

A
  • 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)
29
Q

What are the human DNA-dependent DNA polymerases?

A

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

30
Q

What disorders are associated with mutation of human DNA polymerases?

A

1) Opthalmoplegia, Alper syndrome, other neurodegenerative disorders (Pol γ)
2) In tumors (Pol β)

31
Q

What is the eukaryotic regulation of DNA replication?

A
  • 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)
32
Q

What are the 3 checkpoint in the cell cycle?

A

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

33
Q

How are origins of replication different in prokaryotes & eukaryotes?

A
  • 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.
34
Q

What is the role of nucleosomes?

A
  • 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 -
35
Q

Why does trinucleotide repeat expansion occur?

A
  • From strand slippage during replication
  • From unequal strand exchange between chromatids
  • Oxidative damage
  • Ex. Kennedy’s disease, Huntington’s, & SCA
36
Q

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

A
  • 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
37
Q

What are telomerases?

A
  • 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
38
Q

What is the Hayflick limit?

A
  • 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
39
Q

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

A
  • Accelerate loss: stress, smoking, obesity
  • TA-65 from astragalus plant is thought to “turn on” telomerase
  • Cancer increase telomere length because telomerase is activated
40
Q

What are some premature aging syndromes?

A
  • 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)
41
Q

What are telomeres?

A
  • 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)

BIOCHEMISTRY 2 (12 decks)

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