DNA replication, enzymes at replication fork & DNA replication accuracy

Question 1

What is a chromosome?

Answer 1

nuclear genetic material distributed along chromsomes

Question 2

What are telomeres?

Answer 2

areas of highly repetitive DNA
protect chromosome ends from degradation, recombination & end fusion

Question 3

what is a centromere?

Answer 3

repetitive DNA
forms spindle attachment in mitosis

Question 4

What is the origin of replication?

Answer 4

sequence where duplication of DNA begins
has many

Question 5

what is semi-conservative replication?

Answer 5

discovered by Mehselton & Stahl
centrifugation with different isotopes of N and bacteria
each generation had half light and half heavy N
settle at different points in the test tube

Question 6

What is involved in DNA synthesis?

Answer 6

DNA polymerase
Mg2+
Single stranded template of DNA
Primer 3’-OH
Synthesis in 3’ to 5’ direction

Question 7

differences between eukaryotic and bacterial genomes

Answer 7

euk are large & arranged as linear chromosome
bact are small, compact and circular

Question 8

what is the replication fork?

Answer 8

the site here both strands of DNA are copied at
in 5’ to 3’ direction

leading strand is continuous
lagging strand is discontinuous

Question 9

what are the discontinuous fragments called?

Answer 9

Okazaki fragments

Question 10

What are the steps in lagging strand synthesis?

Answer 10

1. Primase synthesises RNA primer
2. DNA polymerases elongate primer with new DNA
3. Nucleases removed RNA at 5’ end of neighbouring fragment
4. DNA polymerase fills the gap
5. DNA ligase connects adjacent Okazaki fragments

Question 11

What are the different enzymes present at the replication fork?

Answer 11

helicase - unwinds DNA
ss-binding protein - stabilises DNA
primase (RNA polymerase) - synthesises RNA primer
DNA polymerase- creates new DNA strand & proof read
Sliding clamp - keeps DNA polymerase on DNA
clamp loader - loads clamp onto DNA
nuclease - trim Okz fragments
DNA ligase - joints Okz fragments

Question 12

What is primase?

Answer 12

specialised RNA polymerase that creates RNA primers

Question 13

what is the speed of DNA replication?

Answer 13

50 base pairs s-1 at each fork
high as DNA polymerase attaches to sliding clamp

Question 14

What is a sliding clamp?

Answer 14

keeps DNA polymerase on the DNA
loaded on by DNA clamp loader

Question 15

What are the steps of clamp loading?

Answer 15

1. Loader binds to clamp
2. Opens the clamp
3. DNA then engages in clamp
4. ATP hydrolysis locks the clamp round the DNA by closing it
5. Release of clamp loader
6. DNA polymerase then binds to sliding clamp
7. Clamp loader can then be used again

Question 16

What do single stranded binding proteins do on the lagging strand?

Answer 16

prevent an y base pairing within the single strand until DNA polymerase has arrived

Question 17

what is a histone chaperone?

Answer 17

load histones onto newly synthesised DNA

Question 18

What is a topoisomerase?

Answer 18

untwist DNA by breaking and reforming phosphodiester bonds
preventing supercoiling by DNA helicase
unswivels the DNA before replication fork

Question 19

DNA polymerase accuracy

Answer 19

more accurate than RNA polymerase
5’ to 3’ polymerisation 1 in 10^5 errors per nucleotide

MutS detects incorrect base pairing in newly synthesised DNA