Week 4

Question 1

ingredients for DNA synthesis

Answer 1

• origin of replication
• primers
• dNTPs
• ATP as an energy source
• DNA polymerase
• accessory proteins

Question 2

DNA synthesis procedure

Answer 2

1. separate DNA strands
2. synthesise DNA
3. proofread newly synthesised DNA

Question 3

bacterial DNA replication

Answer 3

1. origin of replication attracts initiator proteins, which bind to the origin of replication sequence.
2. unwinding by a helicase
3. binding of single-strand DNA binding proteins (ssps) which keeps replication fork open
4. RNA primers made by primase
5. DNA polymerase makes the new DNA strand
6. sliding clamp holds polymerase onto DNA
7. nick sealing by DNA ligase (getting rid of RNA primers, filling the gap, and sealing gaps between Okazaki fragments)

Question 4

initiator proteins for replication in e.coli

Answer 4

1. binds to origin - very specific and regulate when DNA replication happens.
2. destabilise the helix
3. helps helicase bind, acting as a signpost for DNA helicase to bind to helicase-loading proteins
4. requires ATP. don’t hydrolyse it until DNA synthesis has begun because of regulation. until new ATP is achieved a new cycle of DNA replication cannot be started

Question 5

describe how helicase works

Answer 5

• two types of helices exist
• predominant one moves 5’ to 3’ along the lagging strand template
• helicases require ATP
• quaternary structure

Question 6

single-stranded binding proteins

Answer 6

• following the action of helicase, single strand binding proteins keep DNA strands separated by binding ssDNA, which has short regions of base-paired ‘hairpins’
• single-strand binding protein monomers undergo cooperative protein binding, which straightens the region of the chain

Question 7

RNA primers made by primase

Answer 7

• primase (a type of RNA polymerase) used to make RNA primers
• in order to begin, DNA polymerase requires a bound primer
• primase proceeds in 3’ to 5’ along template strand, primase synthesis occurs in the 5’ to 3’
• primase joins together two ribonucleotides and continues
• associated with Helicase

Question 8

dna polymerase

Answer 8

• DNA is made in a complementary and antiparallel way using base pairing
• nucleotides are added onto the 3’ OH
• incoming nucleoside triphosphate pairs with a base in the template strand
• DNA polymerase catalyses covalent linkage of nucleoside triphosphate onto growing new strand

Question 9

sliding clamp

Answer 9

circular protein
- does not impede progress of DNA polymerase

Question 10

how are Okazaki fragments on lagging strand linked together?

Answer 10

• DNA polymerase adds nucleotides to 3’ end of new RNA primer to synthesise Okazaki fragment
• DNA polymerase finishes Okazaki fragment
• previous RNA primer removed by nucleases and replaced with DNA by repair polymerase
• tiny gap in the phosphodiester bond, nick, (no bases missing) sealed by DNA ligase

Question 11

how are nicks healed?

Answer 11

ATP hydrolysed, AMP released

Question 12

bacterial replisome

Answer 12

example of a molecular machine responsible for DNA replication in bacteria

Question 13

bacterial primosome

Answer 13

DNA helicase and primase

Question 14

what is the unwinding problem and how is it solved?

Answer 14

• as helicase unwinds DNA, supercoiling and tosional strain increase in the absence of topoisomerase, as DNA cannot rapidly rotate
• problem in circular chromosomes and large linear eukaryotic chromosomes
• some torsional stress is released by DNA supercoiling
• DNA topoisomerase creates a transient single-stranded break
• torsional stress ahead of the helicase is relieved by free rotation of DNA around the phosphodiester bond opposite the single-strand break; the same DNA topoisomerase that produced the break reseals it

Question 15

what happens at the ends of linear chromosomes? (eukaryotic problem only)

Answer 15

• no 3’ OH group available at the end of the chromosome on the lagging strand
• lagging strand is incompletely replicated
• the potential shortening of the 5’ end of the daughter DNA is a problem as it may lead to a loss of sequence information

Question 16

telomerase

Answer 16

• contains an RNA template which determines the repetitive sequence that is added to the 3’ end of the parental strand
• able to add DNA nucleotides to the 3’ end of parental DNA in the absence of a primer or DNA template
• RNA template -> DNA comp. copy

Question 17

what enzyme does telomerase resemble?

Answer 17

reverse transcriptase but has an RNA template

Question 18

what is generated by telomere replication?

Answer 18

G-rich end

Question 19

telomeres and cancer

Answer 19

• telomerase is abundant in stem and germ-line cells, but not in somatic cells
• loss of telomeres, which occurs normally during DNA replication limits the number of rounds of cell division
• most cancer cells produce high levels of telomerase

Question 20

3-20 kb of (TTAGGG)
reduced kb of (TTAGGG)
ends with no kb of (TTAGGG)
up to 55kb of kb of (TTAGGG)

Answer 20

embryonic or stem cell - indefinite replication
somatic cell - limited replication
senescent cell - breakage-fusion-bridge-cycle, chromosome instability, apoptotic cell death
cancer cell - persistent growth, but also chromosome instability - breakage and deletions

Question 21

does solving the end replication problem require an RNA primer?

Answer 21

yes

Question 22

RNA/DNA polymerases have an error rate of

Answer 22

1 in 10^4; 1 in 10^9

Question 23

the human genome (3x10^9) in a haploid cell is only changed about — nucleotides every time a cell divides

Answer 23

3

Question 24

2 separate mechanisms of DNA proofreading and repair

Answer 24

1. 3’ to 5’ exonuclease - removes the disincorporated nucleotide, but only from the ends. DNA polymerase has proofreading exonuclease activity. flips the newly synthesised strand from the polymerising domain into the editing domain (the 2 domains of DNA polymerase), removing the base from 3’ to 5’.
2. strand-directed mismatch repair - this is a DNA replication error repair process (if proofreading fails). initiated by detection of distortion in the geometry of the double helix generated by mismatched base pairs.

Question 25

Describe strand-directed mismatch repair in detail

Answer 25

1. MutS protein recognises and locks onto DNA mismatch. 2. MutS recruits MutL and scans DNA. 3. sliding clamp is encountered. MutL nuclease is activated and initiates strand removal 4. strand removal completed 5. repair DNA synthesis by polymerase

Question 26

methylation in prokaryotes

Answer 26

MutL detects which strand is methylated to determine which is the original (correct) strand and which is the newly synthesized (possibly incorrect) strand.

Question 27

defects in repair mechanisms

Answer 27

- linked to a variety of human diseases - eg breast, colon, skin cancers

Question 28

DNA can be damaged by:

Answer 28

1. oxidation 2. radiation 3. heat 4. chemicals

Question 29

thymine dimer

Answer 29

UV radiation may cause: one base is covalently bonded to the other; this causes a problem because when DNA polymerase is trying to replicate DNA, because these bases are stuck to each other, the polymerase will skip over the dimer and keep on going. C-C and C-T dimers may also occur

Question 30

depurination

Answer 30

the base can be chopped off of the DNA strand for guanine and adenine

Question 31

deamination

Answer 31

cytosine becomes uracil as NH2 is replaced by O

Question 32

two general mechanisms of DNA repair long after DNA replication is over

Answer 32

- base excision repair - nucleotide excision repair

Question 33

base excision repair

Answer 33

repairs one nucleotide - Uracil DNA glycosylase looks for U's (deaminated C's) that don't belong in the DNA helix - once it finds it, it will remove the base - AP endonuclease and phosphodiesterase remove the remaining sugar phosphate - DNA polymerase adds new nucleotide using bottom strand as a template - DNA ligase seals the break

Question 34

nucleotide excision repair

Answer 34

larger types of repairs ('bulky' lesions) - eg pyrimidine dimers or remove benzylpyrine in smokers - an excision nuclease will excise a big chunk of DNA from where the problem is - DNA helicase is also needed to open things up to remove the fragment - DNA polymerase adds new nucleotides using bottom strand as a template - DNA ligase seals break

Question 35

non-homologous end joining

Answer 35

- there is an accidental double-strand break - processing of DNA end by nuclease - end joining by DNA ligase - the break is thus repaired with some loss of nucleotides at the repair site

Question 36

homologous recombination

Answer 36

- there is an accidental double-stranded break - there is processing of the broken ends by recombination-specific nuclease - the double-strand break is accurately repaired using undamaged DNA as the template - the break is thus repaired with no loss of nucleotides at the repair site

Question 37

when does DNA polymerase undergo a small structural rearrangement that allows it to catalyse the nucleotide-addition reaction?

Answer 37

only when the base-pairing between each incoming nucleoside triphosphate and the template strand is correct

Question 38

clamp loader

Answer 38

uses the energy of ATP hydrolysis to lock the sliding clamp onto DNA