DNA Replication

Question 1

Craig Venter and Gibson

Answer 1

Created life from a synthetic genome.
synthesised an M. mycoides yeast genome (circular genome), input into a membrane with cytoplasmic proteins to make it into this specific species.

Question 2

Who put lyrics into DNA

Answer 2

Venter and Gibson, why? just because they could.

Question 3

Taxol

Answer 3

Yew tree derived anticancer drug, at risk populations so they have figured out how to make it through E coli.

Clone bacteria with the genes for taxol synthesis, and now make it from recombinant DNA

Question 4

S-reticuline

Answer 4

Morphine, naturally in opium but takes a lot of work to harvest the seeds, but it can be made in recombinant yeast far easier.

Enabled by an enzyme-coupled biosensor.

Question 5

Where did insulin initially come from?

Answer 5

Live animals, pig mostly as it was close to the human synthesis.
It can now be made in bacteria cloned with the genes for insulin synthesis, mostly in yeast.

Question 6

Bacterial genome and replication characteristics

Answer 6

Circular, semiconservative in a rolling circle
Two replication forks - do the work in half the time

Question 7

Meselson and Stahl

Answer 7

Radiolabelled the parent DNA with N15 and N14 (heavy and light nitrogen) and proved that one strand was in each daughter cell and the second generation had half cells with completely new DNA.

amount of parent dna in each - 100 - 50/50 - 25/75 - 12/82.

Question 8

Fidelity in DNA replication

Answer 8

High quality to ensure fitness.
- base selection
- 3’5’ exonuclease
- mismatch repair
only 1 in every ten billion replication events is there a mutation

Question 9

Where are nucleotides added

Answer 9

The 3’ end

Question 10

Key points of DNA replication

Answer 10

DNA polymerases can only extend exist nucleic acid chains (such as primers
DNA synthesis occurs bidirectionally
- continuous on the leading strand
- in okazaki fragment on the lagging strand

Question 11

Primase (DnaG)

Answer 11

Binds and makes 12 nt piece of RNA

Question 12

Polymerase

Answer 12

Initiates DNA synthesis by adding deoxyribonucleic acids one at a time to the 3’ end of the chain

Question 13

Dna A

Answer 13

Bonds specific sequences in the riC with help from the HU proteins causing DNA to unwind in an ATP dependent manner

Question 14

Polymerase I

Answer 14

5’-3’ exonuclease activity that removes the RNA primers

Question 15

DNA ligase

Answer 15

Seals the breaks in DNA left after the RNA primer has been removed

Question 16

Helicase (DnaB)

Answer 16

Binds to both ends of the open complex to give two replication forks in opposite directions
Unwinds the replication fork

Question 17

Tus/ter

Answer 17

Tus binds to ter and causes disassembly of one replisome, and is subsequently removed by the second replisome as bacterial DNA replication is terminated

Question 18

HU

Answer 18

Histone-like proteins composed of compact DNA that are small and abundant. May be site specific or more general, and serve as accessory factors to help initiate replication and recombination.
Bind to the oriC and help Helicase bind and stabilise the replication fork

Question 19

SSB proteins

Answer 19

Single-strand binding proteins
Bind DNA to keep it the open complex open

Question 20

Prepriming complex

Answer 20

DnaA, HU, SSBs, Helicase (DnaB)
The initial open complex

Question 21

What does the oriC consist of, how does it change shape and why

Answer 21

13 mers and 9 mers to which the HU, ATP and dnaA bind
Winds around these proteins to initiate the open complex

Question 22

Formation of the prepriming complex is energy ____________

Answer 22

dependent!

Question 23

How big are okazaki fragments

Answer 23

1000 bp

Question 24

Pol I, Pol III, polA

Answer 24

A family of polymerases discovered by the same family.

Question 25

Arthur Kornberg

Answer 25

Discovered Pol I Nobel prize

Question 26

Thomas Kornberg

Answer 26

polIII and Pol III No nobel prize

Question 27

Roger Kornberg

Answer 27

Studies RNA polymerases Nobel prize

Question 28

Paula De Lucia

Answer 28

Isolated the gene for Pol I, known as polA

Question 29

Replisome

Answer 29

Model proposed by Arthur Kornberg Looping out of the parent DNA at the replication fork for synthesis of the lagging strand Still in fragments but much closer to the replication fork and thus more energetically favourable.

Question 30

Termination of replication from the nonpermissive side

Answer 30

Replication fork approaches ter (a DNA sequence to which Tus is bound) and Tus binds even tighter to initiate disassembly of the replisome by stopping helicase (DnaB)

Question 31

Termination of replication from the permissive side

Answer 31

Second replisome causes Tus to dislodge from ter and the finally segment is replicated - replication is finished.

Question 32

Final step of bacterial DNA replication

Answer 32

“Tangled” chromosomes are resolved through either recombination or decantenation

Question 33

Replicated DNA dimer resolution

Answer 33

Site specific recombination at dif which is then localised to the septum and cleaved at a productive synapse and cell.

Question 34

FtsK in DNA replication

Answer 34

lines up the dif sites on two different DNA copies

Question 35

XerC/D

Answer 35

Recombinase that acts on dif sites during septation and chromosome dimer recombination.

Question 36

DNA supercoiling

Answer 36

Energy stored in DNA molecule as a twist or writhe. Superhelical tension that may be used to aid processes such as packaging, open complex formation, etc.

Question 37

Positive supercoiling

Answer 37

Overwinding of the DNA

Question 38

Underwinding

Answer 38

Negative supercoiling

Question 39

DNA gyrase

Answer 39

Introduces negative supercoils

Question 40

Topoisomerases

Answer 40

Removes/introduces DNA supercoils by breaking the phosphate bond of DNA Includes topoisomerase I, gyrase, topoisomerase IV

Question 41

Topoisomerase I

Answer 41

removes supercoils

Question 42

Topoisomerase IV

Answer 42

removes cantenations, important during replication to resolve the “tangled” replicated chromosomes.

Question 43

Histone like proteins can also act as __________

Answer 43

General repressors