Week 2: DNA REPLICATION - DNA Replication Machinery

Question 1

What does semi-conservative mean ?

Answer 1

Each molecule has an original and a new strand

Question 2

What synthesises DNA?

Answer 2

DNA polymerase

Question 3

What phase of the cell cycle does DNA rep occur?

Answer 3

S-PHASE

8hrs

Question 4

What does DNAp require to synthesis new DNA strand?

Answer 4

all the (A,T,G,C) deoxyribonucleoside triphosphates,
Mg2+,
3’ OH paired to DNA template

Question 5

What direction is continuous DNA rep?

Answer 5

5->3

Question 6

What side are DNA bases continually added?

Answer 6

3’

Question 7

What is the byproduct of DNA polymerisation?

Answer 7

di-phosphate group

Question 8

What is the site where DNA rep starts?

Answer 8

origin

Question 9

How is the 3’->5’ direction replicated?

Answer 9

in fragments, discontinuous.

called Okazaki fragments

Question 10

Draw the structure of the DNA backbone…

Answer 10

ANSWER

Question 11

What is chromatin?

Answer 11

Highly conserved DNA

Many nucleosomes that create a chromatin fibre and eventually a chromosome

Question 12

What makes a histone?

Answer 12

octamer

4 x dimers

Question 13

How much DNA can an octamer of HIS wrap?

Answer 13

~1.7 turns or ~147 bases

Question 14

What are the HIS presenting a DNA octamer?

Answer 14

H2A, H2B, H3, H4

Question 15

What makes nucleosome?

Answer 15

An octamer of Positive HIS proteins (core)
negative backbone of DNA wraps around positive His.
“bead”

Question 16

What alters the structure and number of nucleosomes?

Answer 16

Chromatin remodelling Enzymes

Question 17

What can recruit chromatin remodelling enzymes?

Answer 17

Transcription activators

bind to enhancer sequences

Question 18

What is an issue with nucleosomes and DNA rep?

Answer 18

hinderance at the origin.

machinery can’t access it

Question 19

What is dormant origin, when are the activated?

Answer 19

activated when 2 chain are about to meet and an origin lies in the middle.

It allows no bases to be missed and chains can meet

ligase enzyme binds the 2 chains together

Question 20

How many replication origins does EUK vs PRO have?

Answer 20

PRO = 1 
EUK = clusters (many)

Question 21

What 3 essential elements are needed at the replication origin?

Answer 21

1. BS for initiator/ O rep P
2. Start site for rep
3. Elements for unwinding/distortion of DNA (helices) at fork

Question 22

What are the non-essential elements for the rep O?

Answer 22

BS for activators/ chromatin remodellers

Question 23

What is the role of the origin Rep Complex? and what does it form?

Answer 23

recruit other replication p to the origin & helicase

form pre-replactitive complex

Question 24

what assists helicases in opening the strand?

Answer 24

• Start site binding P
• Cdc45 and GINS bind to MCM complex and make it active.
• only occurs when S phase goes ahead.
• Protect & stabilise exposed start site DNA templates.

Question 25

What are the first things that bind to the origin of rep in EUK and PRO?

Answer 25

PRO -> initiator P EUK -> origin recognition complex ORC Recruit the DNA helicases to the origin region.

Question 26

What binds to the origin of rep first?

Answer 26

ORC | origin rep complex

Question 27

What proteins make up the helicases molecules? | active vs inactive

Answer 27

inactive =MCM hexamer- Cdt1 complex Active= CMG helices complex, Cdc 45, MCM + GINS 2 bind and when S phase is initiated they separate to the each strand

Question 28

How many proteins make up ORC?

Answer 28

6

Question 29

What is the shape of the helicase?

Answer 29

ring-shape and hexameric | similar in PRO and EUK

Question 30

What is the section that separates the DNA strands?

Answer 30

NTP hydrolysis

Question 31

How does helicase move and unwrap DNA? | reaction?

Answer 31

NTP hydrolysis | change shape of protein, move rapidly along DNA strand and unwrap as it goes.

Question 32

What McM subunits bind and create a gate between with the ORC-Cdc6 complex?

Answer 32

2-5

Question 33

What element does DNAp need to bind to DNA?

Answer 33

priming group

Question 34

What is the function of the priming group?

Answer 34

provides the 3'OH group paired to DNA template required by DNA polymerases to initiate Replication

Question 35

How can priming be achieved?

Answer 35

1. nick the DNA at origin to pose the 3'OH group 2. pre-formed RNA acts as a primer 3. primase enzymes synthesise RNA primers in PRO and EU

Question 36

What enzyme synthesis RNA primers?

Answer 36

primases

Question 37

What are primases?

Answer 37

small RNA polymerases binds to NTP Creates a short stretch or RNA with functions as the primer (chain of ribonuclease)

Question 38

Is the RNA polymerase removed?

Answer 38

YES, it is replaces with DMAp with high fidelity | transiently associated with DNA

Question 39

PRO primase?

Answer 39

DnaG | has a Zn2+ binding region for protein-protein/ protein-DNA binding

Question 40

Where does the palm on the DNAp bind to on the DNA?

Answer 40

palm binds the primer template junction and the newly synthesised DNA through interactions with the phosphate backbone of the minor groove.

Question 41

What mediates DNTP binding?

Answer 41

by base pairing through H bonding and binding to a divalent metal cation (Mg2+)

Question 42

What is the role of the finger region of DNAp?

Answer 42

closes around the bound dNTP, prevents hydrolysis and allows nuceophillic attack of oxygen of the 3' hydroxyl on the alpha phosphate to form a phospodiesteir bond.

Question 43

What can cause spontaneous mutation?

Answer 43

misfiring to tautness during DNA rep | occur very infrequently

Question 44

Do DNAp have proofreading activity?

Answer 44

YES!

Question 45

What is exonuclease?

Answer 45

enzymes that work by cleaving nucleotides one at a time from the end of the polynucleotide chain

Question 46

What reaction does a exonuclease undergo?

Answer 46

hydrolysis | breaks the phosphate bonds

Question 47

What are the DNAp in E.coli? PRO

Answer 47

DNA i - doesn't replicate most of the DNA | DNAp(iii)

Question 48

how many pol3 are involved in e.coli rep?

Answer 48

3 | 1 for leading and 2 for Okazaki segments

Question 49

How are the RNA primers removed in PRO?

Answer 49

POL1 last step exonucleases and uses RNAp to synthesise gaps

Question 50

What DNAp are involved in DNA synthesis?

Answer 50

alpha delta epsilon

Question 51

What sub of the alpha DNAp synthesises the initiator complex? euk

Answer 51

alpha sub | 3' OH of initiator DNA is used to prime DNA synthesis of the DNAp epsilon and delta.

Question 52

What POL synthesis leading nd lagging stands?

Answer 52

leading epsilon lagging delta 3'->5' exonuclease proof reading activity

Question 53

What elements are RNA primers removed at the end of DNA rep? EUK

Answer 53

combined activity of DNAp and Fen1 (flap endonuclease 1)

Question 54

How does DNAp displace RNA primers? EUK

Answer 54

It extends the fragments until it displaces the RNA primers, forming a flap Fen1 removes this flap (its an endonuclease)

Question 55

How does DNA ligase work? (general)

Answer 55

catalyses formation of a phosphodiester bond between the adjacent 3'OH and 5'P ends of the polynucleotides H-bonded to a complementary strand

Question 56

How does DNA ligase work in EUK?

Answer 56

enzyme nucleotide intermediate

Question 57

What is adenylyl

Answer 57

.

Question 58

How to topoisomerases reduce positive supercoils?

Answer 58

formation of covalent protein-DNA intermediate allowing the DNA to rotate and re-ligate one or both strands to prevent torsional strain

Question 59

POL can only hold on to the DNA for a small amount of time (low processivity), what factors aid the prolonged binding? PRO/EUK

Answer 59

A clamp is required to increase the processivity of P can synthesise long DNA strands

Question 60

What is the processivity of POLiii like? PRO

Answer 60

low, catalyses ~11phosdiester bonds | unbinds and binds repeatedly

Question 61

What enzyme is associated with POLiii to increase processivity?

Answer 61

HOLO enzyme | addition of many subunits

Question 62

What makes up the HOLO enzyme processive? PRO/EUK

Answer 62

many subunits but the last to attach is the clamp and makes the enzyme highly processive catalyses thousands of phosopdiester bonds before dissociating

Question 63

What subunit is Clamp on HOLO enzyme? PRO

Answer 63

beta

Question 64

What is more processive POL1 OR POL3? E.coli PRO

Answer 64

3 as it has holo enzyme so does not dissociate from DNA as frequently no loss of accuracy

Question 65

What is the processivity factor in EUK?

Answer 65

PCNA

Question 66

How do clamps associate with DNA?

Answer 66

the aid of clamp loader

Question 67

What are clamp loaders?

Answer 67

ATPases

Question 68

What are telomeres?

Answer 68

tandem repeats of a short conserved seq with a block of G nucleotides

Question 69

What is telomerase ?

Answer 69

essentially a reverse transcriptase | a DNA polymerase, synthesises DNA using an RNA template

Question 70

When are tolomerases present in the cell cycle?

Answer 70

on the ends of chromosome at the end of G1 phase, active during S phase