DNA replication

Question 1

how are nucleotides joined to DNA sequences

Answer 1

nucleophilic attack (transfer of electron forming diester bond) on phosphate of new DNTP = binds new nucleotide and releases 2 phosphates

Question 2

how does DNA replication take place

Answer 2

1. DNA strands are unwound by DNA helicase
2. DNA polymerase binds at 3’ ligases strands back together

Question 3

what are Okazaki fragments

Answer 3

• RNA primers bind to DNA and act as template for DNA polymerase to bind to
• allow DNA polymerase to bind

Question 4

where are okazaki fragments found

Answer 4

• lagging strand = 5’→3’
  
  • needs okazaki fragments
• leading strand = 3’→5’
  
  • DNA synthesised continually as DNA P binds to 3’ OH

Question 5

why does DNA polymerase require primers

Answer 5

act as a hook to be able to bind to the lagging strand

Question 6

function of dna primase

Answer 6

produces primers
binds to short RNA sequence and inserts DNA hybrids for DNA polymerase to bind to

Question 7

what primers are produced on the leading strand

Answer 7

• short RNA primer is synthesised using template and NTPs by DNA Primase
• once RNA primer is in place, DNA polymerase extends it via 3’

Question 8

what primers are produced on the lagging strand

Answer 8

1. DNA primase makes RNA primer
2. primase end dissociates and joins further up strand
3. DNA polymerase extends RNA primer
4. ribonuclease H removes RNA primer = leaving gap
5. DNA polymerase extends across gap
6. DNA ligase seals nick

Question 9

diseases associated with replication errors

Answer 9

• mutation in genes encoding DNA helicase → werner syndrome (premmie aging)
• bloom gene mutation → bloom syndrome
  • LOF in RecQ DNA helicase

Question 10

what is speed of DNA polymerase dependent on

Answer 10

processivity (ability to catalyse consecutive reactions without losing the substrate)

Question 11

what is a high processivity for an enzyme?

Answer 11

• 1 nuc/sec = non-processive
• multiple nuc/sec = processive
  average no. nucleotides added each time enzyme binds to template

Question 12

function of sliding clamp

Answer 12

-> increases processivity of dna polymerase
- dependent on ATP
- binds to primer:template junction via clamp loader
- ATP breaks off → clamp loader releases → DNA polymerase binds on

Question 13

what are single stranded dna binding proteins (SSBs)

Answer 13

• hairpins when DNA coils up = stop polymerase
• SSBs bind to RNA and prevent hairpins

Question 14

DNA topoisomerases function

Answer 14

• prevent DNA from being tangled during replication
• enhance processivity of DNA polymerase
• DNA topoisomerases relax tension resealing the backbone of parental helix

Question 15

difference between type I and type II topoisomerases

Answer 15

• type I topoisomerases nick and reseal one of the DNA strands with no ATOP
• type II topoisomerases nick and reseal both DNA strands with ATP

Question 16

when do replicator selection and origin activation occur

Answer 16

1. replicator selection occurs in G1 = formation of pre-replicative complex
2. origin activation occurs in S phase = unwinding of DNA and recruitment of DNA polymerase

Question 17

what are replication origins controlled by

Answer 17

high levels of cyclin-dependent kinase (cdk) activity in S phase activates existing pre-RC but prevents formation of new pre-RCs
- G1 = low cdk = pre-c formation and no activation
- S1 = high cdk = no new pre-RC and activation

Question 18

cdk levels in G1

Answer 18

• G1 = low cdk = pre-c formation and no activation

Question 19

cdk levels in S1

Answer 19

• S1 = high cdk = no new pre-RC and activation

Question 20

what enzymes are involved in finishing replication

Answer 20

• DNA polymerase and ligase close all but one gap
• RNA primer at end of chromosome is removed by RNase H ⇒ gap = 3’ overhang

Question 21

what enzyme forms telomeres

Answer 21

telomerase

Question 22

what sequence is added to extend the 3’

Answer 22

TTAGGG

Question 23

structure of telomeres

Answer 23

ribonucleoprotein with intrinsic RNA component acting as template for telomere repeat sequences to be synthesised in step-wise process using reverse transcriptase = telomerase shuffle

Question 24

what is the telomerase shuffle

Answer 24

• telomerase rna pairs up with existing telomere repeat + 3’ overhang
• telomerase uses this as template to add 3’ nucleotides on
• shuffles forward to synth next 3 nucleotides and again to bind to next 3 nucleotides etc