Molecular Biology Lecture 7 - DNA Replication Flashcards Preview

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Flashcards in Molecular Biology Lecture 7 - DNA Replication Deck (23):
1

DNA synthesized in the ________ direction by ________

5’ → 3’ , DNA polymerase

2

Proofreading is done by ______ in the _______ direction

exonuclease, 3’ → 5’

3

What are nucleoside triphosphates (dNTPs)?

Nucleoside triphosphate (NTP) is a molecule containing a nucleoside bound to three phosphates. These include the four bases needed for DNA replication.

4

Primer

A particular arrangement of single-stranded DNA (ssDNA)

5

Correct base pairing provides specificity

3’ OH and α Phosphate correctly positioned.

6

Right hand glove model for DNA pol

Draw and label

7

DNA polymerase form processive reaction. This means?

Processivity is a characteristic of enzymes that operate on polymeric substrates. In the case of DNA processivity is defined as the average number of nucleotides added each time the enzyme binds a primer:template juction

1. Binding of pol to DNA is the rate-limiting step
2. Polymerization is very fast
3. Processive – multiple nucleotide additions per polymerase binding

8

Proofreading of DNA....

....is done by the exonuclease in the 3' to 5' direction. Mispaired DNA alters the geometry of the 3'-OH. This will decrease the rate of addition and increase the rat of exonucleases

1. Unpaired (SS) DNA slows down the polymerase
2. Exonuclease removes mismatched base
3. 10-5 vs. 10-7 errors
4. Post-replication repair reduces errors

9

Replication fork

DNA synthesis on two strands
Two DNA polymerase molecules
Leading and lagging strands - Okazaki fragments

10

RNA primer

Primase are short RNA primers (5-10 nucleotides long) on an ssDNA template.

DNA polymerase requires a primer to copy ssDNA, RNA primer can be created de novo (the method of creating a transcriptome without a reference genome)

11

RNA primer Con't.....

1. RNA not DNA primer
- 5-10 bases

2. Primase
- RNA polymerase
- Does not require a primer

3. Recognizes simple sequence (GTA in E. coli)

4. Stimulated when bound by helicase
- restricts activity to replication fork

12

Removal of primer

To RNA primers with DNA, an enzyme RNaseH recognizes and removes most of each RNA primer. RNaseH recognizes and degrades RNA base paired with DNA.

RNaseH can only cleave bonds between RNA:DNA

The removal of RNA leaves a gap in the dsDNA.

DNA polymerase I fills this up from the 5'-3' end

DNA ligase – covalent linkage, requires ATP

13

DNA helicase

1. DNA polymerase does not unwind DNA efficiently

2. Helicase moves in front of polymerase
Moves along SS and denatures DS

3. Requires ATP

4. Hexameric protein - circles DNA

5. Polarity of movement
5’ → 3’ (lagging strand)
7. Requires DNA to be SS first (initiation).

14

ssDNA-binding proteins

After DNA helicase passes, the newly separated ssDNA must remain free for DNA polymerase to use a template for DNA synthesis.

ssDNA proteins (SSB's) rapidly bind to the separated strands

15

SSBs characteristics

1. Cooperative binding

2. Cannot denature DNA

3. Stabilize ssDNA

4.Non-specific binding: electrostatic interactions

5. Easily displaced

16

Topoisomerase II

As the strands of DNA are uncoiled by DNA Helicase on the replication fork it increasingly becomes super-coiled.

1. Topoisomerase II relieves supercoiling

2. DNA gyrase (+1 to -1 in figure)

3. Remember that the E. coli chromosome starts out negatively supercoiled (DNA gyrase).

17

Prokaryotes and eukaryotes have multiple DNA polymerases. In E.coli ......

1. E. coli
- DNA pol I – primer removal, gap filling
- accurate, not processive
- DNA pol III – main replication pol
- accurate, highly processive
- Other DNA pols involved in DNA repair

18

DNA polymerases in Eukaryotes

Eukaryotes
- DNA pol α/primase – primer synthesis, initial DNA synthesis
- DNA pol δ – lagging strand synthesis
- DNA pol ε – leading strand
- Other DNA pols involved in DNA repair or mit DNA replication

19

DNA polymerase switching

??

20

Overview of DNA replication

1. DNA synthesis

2. Proofreading

3. Primer removal

4. Filling in gap and covalently sealing

5. Producing SS template

6. Relieve positive supercoils

7. Processivity - sliding clamp and clamp loader

8. Regulation
- initiation of replication
- cell-cycle regulation

21

Sliding clamps

These are proteins composed of multiple identical subunits that assemble in the shape of a donut.

in the absence of a replication fork, DNA Polymerase dissociates and diffuses away from the template DNA on average one every 20-100bp synthesized.

In the presence of the sliding clamp DNA polymerase may still disengage but will not diffuse away from the DNA

22

Sliding clamp mechanism

Draw & label

23

How do you put a sliding clamp on DNA?
Sliding clamp loaders

A sliding clamp is a closed ing in solution but most open to encircle DNA.

Sliding clamp loaders catalyze the opening and placement of sliding clamps onto DNA