Lecture 3-4 - DNA Replication, Repair, and Recombination

Question 1

What conditions are required for DNA polymerase to begin replication?

Answer 1

RNA primer with an available 3’ hydroxyl, sufficient dNTPs, and a template DNA strand.

Question 2

Which direction is DNA synthesized by DNA polymerase what does this result in?

Answer 2

5’ to 3’

Because strands are antiparallel one strand is synthesized continuously following the replication fork (leading strand) while the other must be synthesized in small segment (Okazaki fragments) away from the replication fork (lagging strand).

Question 3

Describe DNA proofreading performed by DNA polymerase.

Answer 3

After the addition of each nucleotide, DNA polymerase undergoes a conformational change to “squeeze” the DNA to ensure that the correct base pair was made (proper base pairing results in DNA having a constant diameter). When an incorrect base pair is detected DNA polymerase exhibits a 3’ to 5’ endonuclease activity which removes the improper base pair.

Question 4

Describe lagging strand synthesis.

Answer 4

Primase occasionally creates an RNA primer on the DNA strand which DNA polymerase uses as a template. This results in the production of Okazaki fragments. The primer is removed by RNAseH which recognizes RNA-DNA hybrids. Primer is replaces with DNA by DNA polymerase. Nicks at the union of Okazaki fragments and replacement of primers are sealed by DNA ligase.

Question 5

What is DNA helicase?

Answer 5

Protein that separates double stranded DNA strands. It is propelled down DNA by conformation change caused by hydrolyzing ATP.
It consists of 6 identical subunits.

Question 6

What is single-stranded binding protein?

Answer 6

Proteins which bind exposed SS DNA. This helps to stabilize the DNA, and prevent it from forming hairpin loops.
Found primarily on the lagging strand just after the replication fork.
Bases remain exposed.

Question 7

What is the sliding clamp and the sliding clamp loader?

Answer 7

The sliding clamp loops around the DNA and holds proteins associated with the replication on the DNA strand while sliding.
The clamp loader hydrolyzes ATP to load the sliding clamp on the DNA template at the end of the primer.

Question 8

What is mismatch repair?

Answer 8

Removes most of the mismatched base pairs that were missed by proofreading.
MutS binds newly synthesized DNA at sites of mismatch.
MutL scans for nicks further along on the DNA (nicks indicate newly synthesized strand vs. original strand)
The newly synthesized strand is removed and resynthesized.

Question 9

What are topoisomerases? What is the difference between type I and type II topoisomerases?

Answer 9

Proteins which resolve structural issues in DNA.

Type I release tension from supercoiling which is caused by helicase and DNA replication. It creates a single stranded break which allows the DNA to twist around the intact strand. Energetically favorable, no ATP used.

Type II separates interlocked DNA which causes tangling. It forms a double stranded break in one strand which it uses to pass the other strand through. Process requires ATP at several steps.

Question 10

What factors are required for an origin of replication?

Answer 10

• binding site for ORC (origin recognition complex)
• A-T rich sequence
• binding site for proteins which attract ORC

Question 11

How is DNA replication limited to only occurring once?

Answer 11

In eukaryotes several proteins are phosphorylated by Cdks which activates them. ORC is one of these proteins. It stays associated with the origin and is nonfunctional after its phosphorylated.

In prokaryotes replication cannot occur until the new strand is methylated.

Question 12

How are chromatin structure and histone modifications maintained following replication?

Answer 12

Histones disassemble at the replication fork. H3-H4 is added behind the replication fork by chaperones and H2A/H2B associate later.

Because the H3-H4 dimers are randomly passed on to either strand, reader-writer complex’s can spread histone modifications to histones that were not directly inherited.

Question 13

What are telomeres and what is their function?

Answer 13

Long repetitive sequences at the end of chromosomes made by telomerase so lagging strand synthesis has a template at the ends.
The 3’ end is longer and folds back to form a loop which protects the chromosome end from being targeted from repair.

Question 14

What is base excision repair?

Answer 14

• DNA glycosolases (multiple types) look for altered bases
• base is “flipped out” and glycosyl bond is cleaved
• AP (apurininc/apyrimidinic) endonuclease and phosphodiesterase remove backbone
• DNA polymerase and ligase fill and seal gap

Question 15

What is nucleotide excision repair?

Answer 15

• enzyme complex looks for lesions/distortions in the double helix
• backbone is cleaved on either side of he damage
• helicase “peels” the damaged DNA off
• DNA polymerase fill and seal the gap

Question 16

What is transcription-coupled repair?

Answer 16

• RNA polymerase stalls during transcription when it comes to damaged DNA
• recruits DNA repair mechanisms to the site
• ONLY transcribed strand is repaired

Question 17

What is Cockanye’s syndrome?

Answer 17

-defect in transcription-coupled repair causing RNA polymerase to permanently stall at damaged DNA

Question 19

How does the structure of DNA make it ideal for damage detection?

Answer 19

Deaminated bases result in bases that are not used in DNA and therefore can be easily identified.

Question 20

Why are methylated cytosines problematic?

Answer 20

• when deaminated they become thymine
• special glycosylase is capable of identifying and removing the thymine
• 3% of cytosines are methylated, however, this is responsible for 1/3 of point mutations

Question 21

What are translesion polymerases and what is the disadvantage of using them?

Answer 21

• DNA polymerase is unable to replicate DNA at sites of DNA damage; translesion polymerases are used instead
• only add a few nucleotides before switching back to DNA polymerase
• translesion polymerases lack exonuclease capability and are more prone to producing uncorrected mismatching

Question 22

What are cell cycle checkpoints and what is their relation to DNA damage?

Answer 22

• checkpoint are certain conditions that must be cleared before a cell can move into a different stage in the cell cycle
• ATM is the protein responsible for generating signals DNA damage
• transition from G1 to S phase is blocked by DNA damage
• transition from S phase to G2 is slowed by DNA damage
• transition from G2 to M phase is blocked by DNA damage

Question 23

What is homologous recombination?

Answer 23

-exchange of DNA sequences from homologous chromosomes resulting in heteroduplex

Question 24

How can homologous recombination be used to repair double stranded DNA?

Answer 24

• 5’ ends are degraded by nuclease
• 3’ end invades homologous chromosome which is facilitated by RecA
• DNA synthesis occurs using homologous chromosome as a template
• strands dissociate after damaged DNA has been moved past

Question 25

What does the term loss of heterozygosity refer to?

Answer 25

• non-functional gene is used to repair its functional homolog homologous recombination
• can change phenotype of a a cell; possibly creating cancer

Question 26

What is non-homologous end joining?

Answer 26

• double stranded break repair
• both ends are processed resulting in loss of some nucleotides and then ends are ligated together
• primary mechanism of repair; deletion typically non-problematic due to small percentage of protein coding DNA