DNA replication

Question 1

Semi-conservative

Answer 1

One conserved parental strand, one newly-synthesized daughter

Question 2

DNA replication is

Answer 2

semi-conservative

Bidirectional

Question 3

How many sites of origin do prokaryotes and eukaryotes have?

Answer 3

Prokaryotes have one

Eukaryotes have many

Question 4

Process of replication

Answer 4

Replication forks are the sites at which DNA synthesis is occurring.

• First, origin binding proteins recognize and bind to origins of replication, which are ATrich sequences. The bacterial genome has one origin of replication, humans have 100s
• Next, the parental strands of DNA separate and the helix unwinds ahead of the replicationfork by helicases.
• While helicases unwind the double helix, single-strand binding proteins bind to eachsingle strand of DNA and hold it in a single-stranded conformation.

Question 5

Topoisomerase

Answer 5

Topoisomerases act to prevent the extreme supercoiling of the parental helix that would result as
a consequence of unwinding at a replication fork.
- Topoisomerases break and rejoin DNA chains.
- DNA gyrase, a topoisomerase inhibited by quinolones, is found mostly in prokaryotes

Question 6

Origin binding protein

Answer 6

DNA A prokaryotes
ORC euk
Recognizes and binds to origins of replication. Recruits DNA pol III to origin of replication.

Question 7

Helicase

Answer 7

Dna B prok
MCM eukaryotes
Unwinds parental DNA strands ahead of the replication fork by breaking H bonds.

Question 8

What proteins are involved in DNA replication?

Answer 8

origin binding proteins, helicase, single-strand binding proteins, DNA ligase, primase, DNA pol I

Question 9

Single-strand binding proteins

Answer 9

RPA in humans
Bind to each single strand of DNA and hold them apart. Protect strands from helicases. More important for okazaki fragments.

Question 10

DNA ligase

Answer 10

It binds Okazaki fragments by catalyzing the phosphodiester bond b/w 3’ OH and 5’P. ATP is a cofactor in eukaroytes (NAD+ for prok).

Question 11

Primase

Answer 11

Enzyme that catalyzes addition of RNA primer to being replication. DNA dependent, RNA polymerase.
Pol-alpha primase in eukaryotes.

Question 12

DNA Polymerase I

Answer 12

in Prokaryotes. NO CLAMP, distributive polymerase. Does “clean up” during replication/repair. Mediates replacement of RNA primers with DNA by 5’ to 3’ EXONUCLEASE activity (removes RNA primer).
5’ to 3’ DNA polymerase activity (fills gap) and 3’ to 5’ proofreading exonuclease.

Question 13

DNA polymerase

Answer 13

Catalyze the synthesis of DNA by adding deoxyribonucleotides to the 3’- hydroxyls of the RNA primers and subsequently to the ends of the growing DNA strands…

Question 14

Prokaryotic

Answer 14

DNA Pol I and Pol III. DNA Pol III is the major replicative enzyme because it has a sliding clamp that keeps it attached to the DNA template over a long distance. Thus, DNA Pol III has much higher processivity than DNA Pol I. DNA Pol I performs clean-up function during DNA replication and repair. DNA Pol I mediates replacement of RNA primers with DNA through its 5’-to-3’ exonuclease activity and 5’-to-3’ DNA polymerase activity

Question 15

Eukaryotic DNA replication

Answer 15

Requires at least three DNA polymerases, Pol α, Pol δ, andPol ε.
The DNA Pol α holoenzyme is a multi-protein complex that has both primase activity and DNA polymerase activity, although no proof-reading activity. DNA Pol
α synthesizes the first ~20 deoxyribonucleotides after the RNA primer, and is then swapped for Pol δ and Pol ε for lagging and leading strand synthesis, respectively.

Question 16

Which have a sliding clamp?

Answer 16

Polymerase III and DNA polymerase delta to attach to DNA template for a long distance.
Processive polymerases have a clamp. And do not dissociate easily.
PCNA is the sliding clamp for DNA pol delta.
Beta-subunit is sliding clamp for Pol III

Question 17

Difference between pol delta and pol epsilon?

Answer 17

Pol delta is for lagging strand

Pol epsilon for leading strand

Question 18

What is required for DNA polymerase?

Answer 18

• DNA polymerase cannot initiate de novo synthesis of new strands.
• RNA serves as the primer for DNA polymerase in cells.
  You need to copy parental DNA strand in a rxn catalyzed by primase… it’s complementary (you can’t synhtesize DNA de novo)
• The RNA primer (~10 nucleotides) is formed by copying of the parental DNA strand in a reaction catalyzed by primase, a DNA-dependent RNA polymerase

Question 19

Telomerase

Answer 19

enzyme ensures telomeres in immortal structures (like germ cells) do not shorten.
Act as reverse transcriptases. Bind to ends of DNA sequences and add extra dNTP. Carries its own RNA template. Works because telomeres are essentially uniform repetitiutous seq.
RNA-dependent DNA polymerase.
Repressed in somatic cells.

Telomeres get shorter with each cell division, get too short, apoptosis. Normal aging.
Great cancer target

Question 20

Reverse transcriptase?

Answer 20

Enzyme responsible for synthesis of DNA from RNA template. Retroviruses use these (HIV).

Question 21

DNA pol I vs DNA pol III?

Answer 21

Pol I has NO clamp, it’s distributive. And can remove RNA primer, has 5’ –> 3’ exonuclease activity.
Pol III has a clamp and is processive (NO 5’–>3’ activity, cannot remove primer).

BOTH have 3’–>5’ exonuclease activity that mediates proofreading.

Question 22

Tell me a little about directions

Answer 22

DNA pol reads in 3’ to 5’ direction

Synthesizes in 5’ to 3’ direction

Question 23

How does DNA pol do it?

Answer 23

It creates a phosphodiester bond. Adds dNTP onto the hydroxylated group of a previous nucleotide on a chain.

Question 24

How do you fix shit?

Answer 24

Exonucleases moving 3’ to 5’ direction

Question 25

Replication in leading vs. lagging?

Answer 25

Leading:

1. ) Origin binding protein binds origin
2. ) DNA melted by helicase
3. ) Topoisomerase relieves tension/supercoiling
4. ) Single stranded binding proteins keep replication fork open
5. ) Primase lays out RNA primer for DNA pol
6. ) DNA pol III (or pol epsilon) elongates DNA in one long strand 5’ to 3’ direction
7. ) 2 strands anneal (semi-conservative)

Lagging:
1-4 same.
5.) Primase attaches RNA primer to lagging strand. primase moves with machinery cuz needed multiple times.
6.) DNA pol III (pol delta) synthesizes okazaki frag in 5’ to 3’ direction
7.) RNA primer removed and replaced with DNA by polymerase I (5’ to 3’ exonuclease activity)
8.) Fragments sealed with DNA ligase
9.) Annealing of strands

Question 26

End-replication problem

Answer 26

With leading strand replicate to end, but with lagging strand you need to attach RNA primer and this can’t happen at end (nowhere to attach), so END OF LAGGING strand is not syntehsized and chromo decreases in length

Question 27

How is fidelity of replication so high?

Answer 27

Polymerases discriminate between the correct and incorrect nucleotides to incorporate based on the ability to form hydrogen bonds between the complimentary bases A-T and G-C AND the common geometry of the A-T and G-C base pairs that allow them to fit into the active site of the polymerase.
Only 1 wrong per 10,000-100,000.
- Errors (insertion of an inappropriate nucleotide) during replication can be proofread during replication by a 3’-to-5’
exonuclease activity associated with the polymerase complex.
Proof reading increases the accuracy of replication by 100-1,000 fold so 1 wrong nucleotide remains per million
to 100 million correct nucleotides.
- Post-replicational repair processes (e.g. mismatch repair, see DNA Repair Lecture notes) also increase the fidelity of repair.