GENET 270: Lec 2 - Bacterial chromosomes pt. 1

Which scientists discovers that DNA = genetic material

Griffith, Hershey + Chase

Which scientists discovered that DNA = double helix

Franklin + Wilkins

Which scientists discovered/modeled DNA structure?

Watson + Crick

*****What were the 3 classic experiments that determined DNA = genetic material

1. Griffith: S type Cells + mice (transforming principle)

2. Avery, Macleod + McCarthy: What was transforming principle

3. Hershey + chase: T2 Phage (DNA = unit of heredity)

*****Explain Griffiths Experiment + what/how it gave the conclusion it did?

Streptococcus Pneumonia = Protected by glycocalyx (SMOOTH)= protect from mice immune system = kill mice

• Mice + S type cell = mice die

• Mice + Heat killed S = live

• Mice + R(rough no glycocalyx) = mice live

• Mice + Heat killed S + Live R = Mice Die

Hypothesis = S cells have a transforming principle that allows R type cells to be converted to S type

****In Griffiths experiment, what type of bacteria (S or R) was isolated from the dead mice of Treatment: Mice + Heat killed S + Live R = Mice Die

S type bacteria = isolated from dead cells

*****Explain Avery, Macleod + McCarthy Experiment What were the Methods?

1. They prepared cell extracts from heat-killed virulent (S strain) S. pneumoniae.

2. Treated the extracts with enzymes that selectively destroyed:

   • Proteins → using proteases

   • RNA → using RNases

   • DNA → using DNases

3. Mixed each treated extract with live, non-virulent (R strain) bacteria.

****What were the RESULTS of Avery, Macleod + McCarthy

• Protease-treated extract → still transformed → proteins NOT the genetic material.

• RNase-treated extract → still transformed → RNA NOT the genetic material.

• DNase-treated extract → no transformation → DNA must be the genetic material.

****Explain Hershey + Chase Experiment What were the Methods?

Infect Bacteria with T2 phage radioactively labelled:

• DNA = 32P

• Protein = 35S

After phage infected bacteria = wash phage coats off from outside of bacteria + isolate progeny from infected bacteria + look for S + P labels

****What were the RESULTS of Hershey + Chase Experiment?

Phage coat content: 80% S label 70% P label = same as 1st gen (infecting phage)

Progeny = 4% S label + 30% P label

• DNA not protein of parent phage = enters bacteria THERFORE = HEREDITARY MATERIAL

****Other than DNA = Heredity material. What else did the 3 experiments suggest about heredity?

Heredity = similar in both bacteria + Higher level organisms as both contain DNA as gametic material

What E is used for the polymerization of DNA?

3 phosphates at end of dNTP

• lose last 2 of the 3 = E

****What are the Greek letters corresponding to each PO4- on the dNTP? from closest to sugar to farthest

Closest

• Alpha

• Beta

• Gamma

Farthest 

Fill in the Blank: Nucleotides are joined by ______ bond that connect the ___’___ of the sugar to the ___’____?

Joined by phosphodiester bond

• 5’ C to 3’ C

True or false: Bacterial DNA = Heavily condensed however unlike eukaryotic DNA has no specific organization

False

• Bacteria nucleoid IS very condensed

• BUT it is also very COORDINATED/ORGANIZED

(not sure how important) Isolated Bacterial chromosomes reveal ___# loops each ____ kb of DNA connected to central core

~400 loops of DNA, each ~10 kb long

********Is the bacterial Nucleoid Static or Dynamic?

Dynamic

• level of super coiling always varies + various proteins are always working on DNA

*****What influences the Dynamics of the nucleoid?

Replication, recombination + translation

• all need changes in supercoiling to occur

******What are the 2 phases of growth that the bacteria can be in that affect the DYNAMICS of the NUCLEOID?

1. Experimental growth phase

2. Stationary phase

******What are the Events occurring at Exponential vs. stationary phase that affects the DYNAMICS of the nucleoid

Exponential: Nucleoid = very supercoiled, many proteins + enzymes working to replicate DNA

Stationary: Not supercoiled or active

***What are NAPS, what do they do? what does the acronym stand for?

Nucleoid Associated Proteins

• contribute to ORGANIZATION of nucleoid + GENE regulation replication, transcription, recombination, and repair.

*****What things Do NAPS DO carry out to organize + regulate gene expression

• They compact, bend, bridge (hold 2 pieces together), and organize DNA into the nucleoid.

• Different DNA binding modes affect regulation + nucleoid shape

Need to relax supercoil to read DNA = regulate gene expression

****What are the 2 types of supercoiling

1. Positive (over wound)

2. Negative (underwound)

*****What are the 3 proteins affecting supercoiling?

1. NAPs

2. Enzymes (RNA + DNA poly)

3. Topoisomerases

***What are NAPS how do they affect supercoiling?

CONSTRAIN supercoils = prevent twisting

• changes in nap binding = lead to unconstrained super coils = help with strand separation during central dogma

*****How do ENZYMES affect super coiling?

RNA + DNA poly = unwind DNA = INTRODUCE ve+ Supercoiling downstream

******What are the 2 categories of TOPOISOMERASE? How many supercoils do they each remove with one cut?

Type 1 = cut 1 strand = remove 1 super coil

Type 2 = Cute both strands = Removes 2 super coils

*****What is an EXAMPLE of Type 1 + What are 2 examples of TYPE 2? What do they each do?

Type 1:

• TopA = removes NEGATIVE supercoils

Type 2:

• Topo 4 = Decatenates chromosomes after replication

• Gyrase (special class) = INTRODUCE ve- supercoils

True or false Bacterial genome = less repetitive than eukaryotic?

True

• no non-coding DNA

What issue does having less repetitive DNA + Few/no introns cause?

Mutations = more likely to cause issues since each gene codes for something

****Bacterial genome = exhibit high degree of synteny Define the word

Conservation in genetic linkage

• a lot of similar genes ordered in same way

• Dna organization is very similar within bacteria

*****What Disrupts areas Synteny? How are these disruptions acquired?

Disrupted by INSERTIONS

= Acquired through HORIZONTAL GENE TRANSFER (dna from other sources not from parents (vertical)

*****What enzymes/proteins SYNTHESIZE DNA in replication?

Polymerases

****What are the 2 types of POLYMERASES in replication + what do they do?

• Poly 3 = main job of DNA rep

• Poly 1 = Replace primers with DNA + Ligates (5’ exonuclease)

*****What enzymes/proteins SYNTHESIZE PRIMERS in replication?

Primases

****What is THE PRIMASE in replication? (Dna ?)

Dna G

*****What enzymes/proteins DEGRADE DNA in replication?

Nucleases

****What are the 2 types of NUCLEASES in replication + what do they do?

• Endonuclease

• Exonuclease

  • 5’ = poly 1

  • 3’ = editing

*****What enzymes/proteins LINK/GLUE DNA in replication?

Ligases

****What bond do LIGASES form?

Phosphodiester bonds

• 5’ PO4-- to 3’ OH

*****What are the 4 ACCESORY PROTEINS involved in DNA replication? + What do they each do?

1. Helicase = unwinds dsDNA

2. Topoisomerase = Relieves supercoiling down stream

3. Clap = Clamps polymerase to DNA

4. Clamp loader = Loads clamp, Binds poly on both strands + helicase

*****What is Dna B?

Helicas

*****What does Helicase do?

Form ring that travels down ONE strand of DNA prying strand apart

****What is a down side of Helicase?

E EXPENSIVE = uses a lot of ATP

*****What is Dna C?

Needed to load Helicase onto DN

*****What is Dna N

Sliding clamp

***Do leading + lagging strand travel in same or opposite direction?

SAME

*******What is done to COORDINATE the replication of leading + lagging strand?

LARGE PROTEINS COMPLEXES

• lagging + leading move together at same speed in same direction at same time

*****What are the 5 proteins associated with the LARGE PROTEINS complex that coordinated replication?

1. DNA poly on both strand interact with…

2. DnaN (sliding clamp)

3. DNA on both strands also connected by… TAU proteins

4. Tau proteins also binds Dna B (helicase)

5. Helicase interacts with Primase

*****What is the TROMBONE MODEL for coordinating replication of 2 RNA templates?

Laggin strand = DNA poly jumps ahead + slides back/synth DNA until it run into previous Okazaki fragment then jumps forward again

****Each time DNA poly 3 finishes a Okazaki fragment what is left behind?

Sliding clamp (Dna N)

****What are the 3 frequent blocks DNA poly encounters during replication?

1. Broken DNA

2. Damaged DNA

3. Physical blocks

*****What are the 2 types of Physical blocks?

1. Supercoiling

2. Proteins bound/acting on DNA eg. transcription(RNA poly)

*****How does DNA poly deal with DAMMAGED DNA on lagging strand?

• At lesion = Poly 3 released —> LEAVE CLAMP BEHIND

• JUMPS to next primer + restarts synthesis

leaves gap that is repaired by another mechanism

*****How does DNA poly deal with DAMMAGED DNA on Leading strand?

DNA poly stall = Signal DnaG (primase) to synth new primer 

• Poly 3 = release/Jump + restart synth at new primer

*****How does DNA poly deal with TRANSLESIONS?

Use OTHER POLYMERASE that can synthesize over lesion

******What is the OTHER POLYMERASE often used for lesions?

Poly 2

*****Why can OTHER polymerases synthesize over lesions but poly 3 can’t?

DNA poly 3 = small groove for base pair specificity

Others = large groove = can slide over lesions

*****What is a concern with these OTHER polymerases?

Accuracy + Processivity

Bigger groove = lower accuracy + processivity

****What is one of the biggest problems for replication when it come to PHYSICAL BLOCKS?

Transcription

******What are the 2 types of conflicts replication has with transcription. Which is more detrimental + why?

1. Head on (DNA vs RNA poly) = more detrimental

• causes dsDNA break = kill cell

2. Co-directional conflicts = doesn’t usually break DNA

***Why do Co-directional conflicts occur?

REPLICATION = 10-20x’s FASTER than transcription- 

• poly 3 = 1000 ntd/SECOND

******What are the 5 mechanisms for dealing with physical blocks to DNA replication. What type of Physical block do they prevent?

1. Genome Organization = prevent HEAD ON

2. RNA poly terminator proteins = stalled RNA collisions

3. RNA poly modulators = Stalled RNA collisions

4. Extra helicases = prevent HEAD ON

5. Poly 3 removal of RNAP + restart replication = CO-DIRECTIONAL

****How does Genome organization prevent head on collisions that could be lethal?

Organize genes with high frequencies of translation to be oriented in SAME DIRECTION of replication

• Co-directional collision = not as bad

****How does RNA poly terminator proteins prevent collisions?

Bind to stalled RNA poly at LESION + KICK it OFF

*****What is an example of a RNA poly terminator protein?

Mfd (mutation freq. decline)

*****Other than kick stalled RNA poly off at lesions, what else does RNA Terminator proteins do?

Recruits UvrAB proteins that fix Lesions

****How does RNA poly Modulators prevent head on collisions that could be lethal?

Dislodge RNA poly stalled at DNA lesions + just in normal growth without lesions

*****What are 3 examples of RNA poly modulators + how do they work?

DksA, GreA + GreB

• Insert coiled-coil domain into 2econdary channel of RNA poly

*****Under only WHAT CONDITIONS does RNAP Modulators work?

High stress

• modulators increase during high stress

• indicated by polyphosphate guanosine

****How do HELICASES prevent head on collisions that could be lethal?

Knock off RNA poly from DNA

*****What are the 3 helicases that can do this action?

Rep, UvrD + DinG

****What areas of gene does helicase help the replication fork get through? (2)

• Where DNA Binding proteins are

• Rec A coated ssDNA

****How does Poly 3 removal of RNA pol t+ restart of replication prevent the type of collision that it does? What type of collision is it?

Co-directional

• Replisome can knock RNAP off DNA when it smack into the back

******On which strand does Poly 3 removal of RNA pol t+ restart of replication occur

LEADING

*****What does Poly 3 removal of RNA pol t+ restart of replication use as the PRIMER to restart replication?

mRNA transcript left behind by RNA poly from transcription

******What are the 3 things that have recently changed regarding replication

1. DNA G (primase) can synth primers on LEADING STRAND + leading strand synthesis can be DISCOUNTINUOS

2. MAYBE DNA poly can use mRNA transcript left behind by RNA poly to use a primer to restart replication after co-directional transport

3. DNA replisome can switch out poly 3 when needed for another poly 3 or a different poly (2,4,5)