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Molecular Biology > Lecture #3 > Flashcards

Lecture #3 Flashcards

1
Q

Base Review

A

Bases: Cytosine, Thymine, Adenine, Guanine
Nucleoside: Base + sugar
- Cytidine, Thymidine, Adenosine, Guanosine
Nucleotide: Base+ Sugar+ Phosphate
- Mono, Di, Tri Phosphate
- Nucleoside monophosphate
- CMP, TMP, AMP, GMP

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2
Q

Nucleotide Addition

A

Nucleotides are always added in a 5’ and 3’ direction. (always added to 3’ carbon)

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3
Q

How is DNA replicated?

A 
Semiconservative: 
 - Template each parent strand 
  - hybrids created  
Dispersive: 
  - Strands copied in short segments 
   -  Mixture of parent and daughter  
Conservative: 
   - Parent molecule remains intact 
   - original + daughter
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4
Q

“Most beautiful experiment in biology”

A
  • Matt Meselson
  • Frank Stahl (both grad students)
    Two sets of e.coli grown
    • 15N media (heavy)
    • 14 N media (light)
    • Incorporated into DNA
      Isolated from E. coli
    • mix w/ cesium chloride
      Centrifuge (way to separate molecules/ DNA based on density)
    • density gradient forms
    • equilibrium density centrifuge
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5
Q

The Experiment 1

A
  • Grow bacteria in 15N media
  • transfer to 14N media
  • Grow another 20 min.
  • Extract DNA
  • centrifuge
  • Heavy or light? (two bands: conservative. one band: semi-conservative).
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6
Q

The Experiment 2

A

Heat DNA before centrifuging to denature the helices.

- heavy or light? (one band: dispersive. two bands: semi: conservative)

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7
Q

Characteristics of replication

A

DNA is replicated VERY quickly
- 1000nt/sec for bacteria (circular chromosome) –> fully extended
- 100nt/sec for human (highly complex chromosomes)
Each strand acts as a template for the synthesis of a new strand.

The helix needs to be separated in order for replication to occur one of the first things needed to happen.

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8
Q

Opening of DNA Double Helix

A

The double Helix is VERY stable
- Numerous hydrogen bonds
G-C pairs more stable
100 degrees C is needed to denature the DNA
In vivo the helix is broken apart by “Initiator Proteins”
- Break the hydrogen bonds

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9
Q

Replication begins at Replication Origins

A

replication begins at replication origins
- One in bacteria, 10,000 in humans
- Why?
- A/T rich regions
Opening of the helix creates a “bubble”
- Proteins bind and act on DNA
-Replication fork formed, bi-directional replication
Replication occurs on the top and bottom strands

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10
Q

Replication Fork is Asymmetrical

A

Replication occurs in a 5’ –>3’ direction
- ALWAYS? Yes
Replication occurs in an asymmetrical manner
Leading strand: continuous replication
Lagging strand: discontinuous replication

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11
Q

Lagging strand synthesis

A 
Generates Okazaki fragments 
   - small sequences of DNA 
   - later joined together 
Depends on which "end" of the replication fork 
  - Right end-top strand 
  - left-end bottom strand

stud figure in slide

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12
Q

Replication Machinery

A
  • initiator protein
  • DNA helicases
  • single-strand DNA biding proteins
  • primase
  • sliding clamp
  • clamp loader
  • DNA polymerase
  • Endo (within)/ Exo (end of DNA) nucleases
  • Telomerase
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13
Q

Step 1: Recognition of the Replication Origin

A

Initiator protein used
- E. coli: DNA
- Eukaryotes: origin recognition complex: ORC
DNA sequence specific

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14
Q

Step 2: Loading the Pre-replication complex

A

Late “M” phase + Early G1 phase of the cell cycle (right before synthesis starts)
DNA helicase is loaded
- Release tension on the end

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15
Q

Step 3: Recruitment of the Replisome

A 
DNA helicase 
Clamp Loader 
  - RFC: Replication Factor C 
Sliding Clamp 
  - PCNA: Proliferating Cell Nuclear Antigen 
Topoisomerase 
Single Stranded BInding Protein (SSB) 
  - RPA: Replication Protein A 
Primase 
DNA Polymerase 
RNase H 
DNA Ligase
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16
Q

The Replisome

A 
DNA Helicase- *unwinds the DNA/splits double helix*
   - Recruited to the DNA by the ORC 
    - upstream of the polymerase 
    - Ring shaped 
   - 5' --> 3' or 3' --> 5'  
   - Located at the front of the replication fork 
Pries apart the helix and unwinds DNA 
  - Breaks H bonds 
ATP dependent 
Selects for ssDNA over dsDNA 
  - Narrowest point is only 13A
17
Q

The Replisome: Topoisomerase

A
  • Relieves positive supercoiling generated by the helicase

- can break one or both strands

18
Q

The Replisome: Single Stranded Biding Protein (SSb; RPA)

A
  • Sequence independent
  • INteracts with DNA through electrostatic interactions with the backbone
  • cooperative
19
Q

The Replisome: The Clamp Loader (RPA)/ The Clamp (PCNA)

A

The Clamp Loader (RPA)
- Loads the clamp onto the DNA; ATP dependent
The Clamp (PCNA)
- Ensures that the DNA polymerase remains attached to the template DNA –> sliding clamp

20
Q

The Replisome: Primase and RNase H

A

Primase:
- RNA polymerase, deposits an RNA primer complementary to the DNA templates. Does not have deoxyribose. Cannot start a DNA replication without a primer.
RNase H:
- Recognizes RNA/ DNA hybrids; removes RNA primer

21
Q

The Replisome: DNA Polymerases/DNA Ligase

A

DNA Polymerases

  • Pol (alfa): extends the RNA primer another 20 nt.
  • Pol (symbol) and Pol (symbol): extends primer on the leading strand and at Okazaki fragments.
  • check slides for symbols*

DnA Ligase
- Joins the Okazaki fragments by ligating the 3’ OH w/ the 5’ phosphate
ATP dependent

22
Q

Filling in the Okazaki Fragments

A

DNA polymerase adds to the RNA primer
- Ends at the next RNA primer
RNase H degrades the RNA primer

DNA polymerase replaces the sequence with DNA

DNA ligase joins the two ends together
know image on this slide

23
Q

The Job of DNA Polymerase

A

Some DNA polymerases are more processive than others
- The # of nucleotides added each time a polymerase sits down/binds
- sitting down is the rate limiting step
- varies for each polymerase
DNA Polymerase contains two domains
- Polymerization
- Editing/ Exonuclease

24
Q

Addition of nucleotides using DNA Polymerase

A

All Polymerases add nucleotides in a 5’ –> 3’ direction

Incoming nucleotide
- 5’ end contains a triphosphate

25
Q

Addition of nucleotides using DNA Polymerase

A

Formation of phosphodiester bond
- Tyrosine in polymerase interacts with base
- Arginine and lysine in polymerase interact with phosphates
Coordinated by two metal ions
- Usually Mg2+
- Neutralizes the (-) charges

26
Q

Addition of nucleotides using DNA Polymerase

A
  • Energy required for phosphodiester bond is supplied by…
    • Release of pyrophosphate
    • Hydrolysis of pyrophosphate by pyrphosphatase

Translocation

  • Hydrogen bonds between polymerase and template broken.
  • Electrostatic interactions between polymerase and template remain.
27
Q

What if a mistake is made?

A

DNA polymerase contains a 3’ –> 5’ exonuclease domain
- higher affinity for ssdNA for dsDNA

A problem is noticed due to a change in geometry
- Incoming nucleotide doesn’t fit well
(This property helps ensure that the nutation rate is 1 every 10^10 bp)

28
Q

What happens if the Polymerase was 3’ –> 5’

A

If nucleotides were added to the 5’ end

  • Phosphate is released from the 5’ end.
  • Proofreading results in a monophosphate end.
  • No energy is available to join the new nucleotide.
29
Q

But what happens at the end of the chromosome?

A

End Replication Problem

  • Cap is left at the end of the chromosome
  • Lagging strand

Telomerase (Its own RNA primer, own polymerase) recruited to the ends of the chromosomes

  • Contains an RNA template
  • Binds the 3’ end of the template DNA
  • Uses its RNA template to extend the 3/ end of the template DNA
  • Final Okazaki fragment is generated

Molecular Biology (8 decks)

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