chapter 11: DNA replication

Question 1

conservative

Answer 1

two newly synthesized DNA strands are joined together
original strand remains together

Question 2

semi conservative

Answer 2

each replicated DNA molecule consists of one old and one new strand

Question 3

dispersive

Answer 3

parental strands are dispersed into two new double helices

Question 4

Messelson-Stahl experiment

Answer 4

N15 has higher density than N14

• in this experiment:
  ecoli is grown in a N15 heavy medium (high density on centrifugal force)
  then it is added to an N14 medium
  the cells replicate once in N14 ( N15/N14 ; band is in the middle)
  then replicated a second time in N14 ( two bands ; N14/N14 (on the low density side) and N15/N14 (in the middle) )
  replicated for a third time (more N14/N14 and less N15/N14)

conservative –> semi conservative –> mix of conservative and semi conservative

Question 5

origin of DNA replication (Ori)

Answer 5

where DNA replication begins

Question 6

replication fork

Answer 6

At ori the helix is gonna unwound to create replication forks
there are 2 replication forks because replication is bidirectional

Question 7

Replicon

Answer 7

length of DNA replicated
e coli replicon consists of entire genomes (4.6 million base pairs)

Question 8

Direction of DNA synthesis is 5’ to 3’

Answer 8

3’ end attaches to 5’ end

Question 9

biochemical properties of DNA polymerases 1

Answer 9

initiation of chain synthesis: (negative; it can not do it on its own because a primer is required)

5’-3’ polymerization: (positive)

3’-5’ exonuclease activity: (positive)

5’-3’ exonuclease activity: (positive)

molecules of polyemrase/cell: 400

Question 10

biochemical properties of DNA polymerases 2

Answer 10

initiation of chain synthesis: (negative; it can not do it on its own because a primer is required)

5’-3’ polymerization: (positive)

3’-5’ exonuclease activity: (positive)

5’-3’ exonuclease activity: (negative)

molecules of polyemrase/cell: ?

Question 11

biochemical properties of DNA polymerases 3

Answer 11

initiation of chain synthesis: (negative; it can not do it on its own because a primer is required)

5’-3’ polymerization: (positive)

3’-5’ exonuclease activity: (positive)

5’-3’ exonuclease activity: (negative)

molecules of polyemrase/cell: 15

Question 12

exonuclease activity

Answer 12

DNA proofreading

Question 13

DNA polymerase 3

Answer 13

an enzyme involved in DNA replication

Question 14

the components of the DNA polymerase III holoenzyme

Answer 14

holoenzyme: active form of DNA pol 3, it contains poly 3 core enzyme complex made up of 3 subunits
- α - 5’-3’ polymerization
- ɛ - 3’-5’ exonuclease
- θ - core assembly
core enzymes: elongate polynucleotide chain and proofreads

t (tau): dimerizes core complex

sliding DNA clamp loader ( Y): loads enzyme on the template (serves as a clamp loader)

sliding DNA clamp: binds the core enzymes and double-stranded DNA, keeps the core enzymes bound to DNA for a longer time allowing it to synthesize long stretches of DNA before it detaches from DNA

Question 15

OriC DNA composition

Answer 15

has three AT rich 13bp repeasts and five bp repeasts

Question 16

DnaA

Answer 16

DNA replication initiation in E coli requires dnaA
it is an initiator protein
it binds to the 9bp repeat of the oriC DNA

Question 17

Helicase

Answer 17

hexamer of dnaB subunits
subsequently recruits DNA pol 3 holoenzyme to bind replication fork and initiate repliaction
requires energy supplied by hydrolysis of ATP ti denature hydrogen bonds between base pairs that stabilize the double helix
assembles around exposed ssDNA

Question 18

DNA gyrase

Answer 18

cuts DNA to relax positive supercoils; or introduce negative supercoils
driven by energy released during ATP hydrolysis

Question 19

Single stranded DNA binding proteins (ssbp)

Answer 19

stablize the open conformation of helix
bind specifically to single strands of DNA

Question 20

Primase

Answer 20

RNA polymerase
recruited to replication fork by helicase
synthesize RNA primer
provide 3’-OH required by DNA pol 3 for elongation

Question 21

DNA polymerase I

Answer 21

removes primer and replaces it with DNA

Question 22

DNA ligase

Answer 22

catalyzes formation of phosphodiester bonds
seals nicks and joins DNA fragments

Question 23

leading strand

Answer 23

continuous DNA synthesis

Question 24

lagging strand

Answer 24

discontinuous DNA synthesis

Question 25

steps during DNA replication

Answer 25

1. strand separation at OriC (role of DnaA)
2. involvement of heicase (main role to separate DNA strands ahead of the replication fork)
3. binding to SSBP (help keep the strands separated at the fork)
4. relaxation of positive supercoils (DNA gyrase)
5. RNA primer synthesis (primase: RNA polymerase)
6. DNA synthesis (by DNA pol 3)
7. removal of RNA primer and filling in the gaps with DNA (DNA pol 1, RNAse H auxiliary role in removing RNA)

Question 26

concurrent DNA synthesis

Answer 26

achieved on both strands at single replication fork
lagging strand is looped and spooled out
inverts physical but not biochemical direction (still 5’-3’)
DNA clamp binds to the core enzyme and prevents core enzyme dissociation from template

find picture on presentation and explain what is happening

Question 27

temp sensitive mutations

Answer 27

produce function protein at 30 degrees celsius but a non-functional protein at 42 degrees celsius

provides a way to study the effect of lethal mutations affecting DNA replication

mutant cells grow at permissive temp and mutant phenotype expressed at restrictive temp to identify the defective function

Question 28

telomere replication

Answer 28

Telomerase: Ribonucleoprotein having reverse transcriptase activity plus RNA
- add repeats of six nucleotide sequence to 3’ end of DNA: GGGTTG

Question 29

telomeres

Answer 29

inert chromosomal ends that protect intact eukaryotic chromosomes from improper fusion or degradation

long stretches of short repeating sequences preserve the integrity/stability of chromosomes

Question 30

Shelterin

Answer 30

protein complex that protects the telomere
extends 3’ loop back on itself
T loop stabilized by protein complexes

Question 31

loss of telomere effect

Answer 31

loss of telmoeres –> loss of chromosome end protection –> chromosome instability –> aging, cancer, cell death, disease