DNA Function, Structure And Replication

Question 1

Prokaryotes

Answer 1

No nuclear membrane
DNA arranged often in a single chromosome

In E.coli it is circular (4x106np)

Question 2

Eukaryotes

Answer 2

DNA in the nucleus

DNA is bound to proteins to form the chromatin complex

Different appearance according to the functional moment- e.g looks large and dark in mitosis since chromatin condense into visual aggregates

Some DNA in mitochondria (purely maternal DNA)

Question 3

Functions of DNA

Answer 3

DNA acts as a template and regulator for transcription and protein synthesis

DNA is the genetic material, structural basis of heredity and genetic diseases

Question 4

Chromosomes (DNA) in E.coli (prokaryotic)

Answer 4

Supercoiled

Circular chromosome

2.5 x 106 kDa

4 x 106 bases

2mm linear

Question 5

Chromosomes (DNA) in humans (eukaryotic)

Answer 5

Complex packaging

(22 x 2) + X,Y or X,X

4 x 109kDa

3 x 109bp

2m linear

Question 6

DNA is tightly packaged
Histones can be modified by methylation

Answer 6

Chromatin can exist in different densities: heteronormative and eurochromatin

Question 7

DNA replication process

Answer 7

1. Prior to cell division, topoisomerase unwinds DNA and DNA helicase separates DNA apart to expose two single DNA strands and create two replication forks. DNA replication takes place simultaneously at each fork.
2. SSB’s (single-strand binding protein) coat the single DNA strands to prevent re- annealing or ‘snapping back together’.
3. The primase enzyme then uses the original DNA sequence on the parent strand to synthesise a short RNA primer. Primers are necessary since DNA polymerase can only extend a nucleotide chain, not start one
4. DNA polymerase begins to synthesise a new DNA (via complementary base pairing using free floating nucleotides) strand by extending an RNA primer in the 5’ to 3’ direction. Each parental strand is copied by one DNA polymerase.
5. As replication proceeds, RNAse H recognises RNA primers bound to the DNA template and removes the primers by hydrolysing the RNA
6. DNA polymerase can then fill the gap left by RNAse H
7. DNA replication process completed when the ligase enzyme joins the short DNA pieces (Okazaki fragments) together into one continuous strand.

Question 8

How fast is DNA replication

Answer 8

700-1000 bp per second

Question 9

Human DNA origins of replication

Answer 9

Multiple points of origin

Question 10

Enzymes involved…

Answer 10

DNA Polymerases (5’ to 3’): Reads 3’ to 5’ and synthesises DNA on daughter strand 5’ to 3’ - creates DNA by working in paris to make 2 new strands of DNA. Starts at a primer

DNA Helicase: Separates the DNA apart, by breaking hydrogen bonds between bases, exposing nucleotides

Ligase

Nuclease

Primase: RNA polymerase that synthesises the short RNA primers needed to start the strand replication process

Topoisomerase: Unwinds the double helix by relieving the supercoils

RNAse H: removes the RNA primers that previously began the DNA strand synthesis

Question 11

DNAPolymerase 5’ 3’

Answer 11

• DNA polymerase reads 3’ to 5’, prints 5’ to 3’

• Substrates are deoxyribonucleotides triphosphates

• Enzyme stays on the strand, at the same times extends and proof-reads

Question 12

Opening the strands

Answer 12

• Helicase opens it

• Single stranded binding proteins (SSB) proteins keep it open

• Topoisomerase unwinds it (relieves supercoiling)

-slide 30

Question 13

DNA Polymerase

Answer 13

Has editing function
Detects incorrect insertion of base and will excise and repeat
(Slide 31)

Question 14

Polymerase Chain Reaction

Answer 14

• Aim: to synthesise fragments of DNA

• Its the basis for forensic testing

• Primers: short synthetic pieces of DNA that have complementary bases to DNA you are trying to synthesise/amplify

• One cycle takes 5-20mins

Question 15

DNA Damage from sources like…

Answer 15

Chemical

Radiation

Spontaneous insertion of incorrect bases during replication

Question 16

Smoking

Answer 16

It is a DNA-adduct (directly adds to it)

It reacts with bases to form a bulky group that disrupts replication

Question 17

Ionising Radiation

Answer 17

Can damage bases

Cause breaks in phosphate backbone

Question 18

UV

Answer 18

Damages bases

In particular the formation of thymine dimers

Question 19

Mutations

Answer 19

DNA damage:
chemicals, UV, radiation, chance

DNA repair :
- Base or nucleotide excision (removal)
-Mismatch repair

Question 20

Tumour suppressor gene p53

Answer 20

Linked in cell cycle control

Detects DNA damage and initiates repair

Question 21

p53

Answer 21

1) DNA damage detected, initiate repair mechanism

2) Pauses cell cycle until repair is carried out
Halt cell cycle if DNA not repaired

3) Apoptosis- command cell to commit suicide if DNA damage not repaired

Question 22

Inhibitors of nucleotide synthesis

Answer 22

Methotrexate

MTX

5-Fluorouracil (FU)

Question 23

Methotrexate

Answer 23

folic acid analogue

inhibits childhood leukemia

some lymphom

Question 24

MTX

Answer 24

synthesis of purines

rheumatoid arthritis

psoriasis

Question 25

5-Fluorouracil (FU)

Answer 25

inhibits thymidilate synthase GI

head and nech

breast carcinomas

Question 26

DNA polymerase inhibitors

Answer 26

Cytosine Arabinoside

(AraC)

Acyclovir (Acy)

Question 27

DNA template damaging agents

Answer 27

Cyclophosphamide

Cysplatin (CPPD)

Question 28

Inhibitors of DNA topoisomerase

Answer 28

Doxorubicin

DOX

Question 29

The 5’ end of DNA strand…

Answer 29

Forms diester bond with the 3’ end so that complementary strand runs in opposite direction

(the enzyme polymerase is complementary to the 3’ end therefore the direction of DNA replication goes from 5’ to 3’)

Strands are copied simultaneously but at different rates
(Slide 15)

Question 30

Chromosomes

Answer 30

Each chromosome is made of 2 identical stands of (chromatids) joined in the centre (centromere).

Question 31

What do free phosphate groups in DNA provide?

Answer 31

energy for the reaction to go Through

Question 32

DNA structure relating to replication

Answer 32

DNA polymerase reads the template strand from 3’ to 5’ thus DNA is synthesised on the daughter strand from 5’ to 3’ since DNA runs antiparallel, the daughter strand is synthesised from 5’ to 3’ since phosphate at the 5’ is used by enzyme as a source of energy for reaction to occur

(ACTIVATION ENERGY) - reason why DNA CAN ONLY BE SYNTHESISED FROM 5’ TO 3’

Question 33

DNA structure relating to replication

Answer 33

• From diagram , at one end of the molecule is an unreacted oxygen (3’) whereas at 5’ end there is a phosphate group thus DNA can only be synthesised from 5’ to 3’ since if it was 3’ to 5’ there would be no phosphate group available to provide the energy for the reaction to occur
Unreacted oxygen at 3’ thus DNA polymerase must start at 5’ with phosphate as energy source
• DNA strands run antiparallel to each other i.e one runs 5’ to 3’ whereas the other runs 3’ to 5’

Question 34

Why is DNA replication semi conservative?

Answer 34

Because each resulting DNA double helix retains one strand of the original DNA

Question 35

Primer

Answer 35

short strand of DNA that is the start point for DNA synthesis as DNA polymerases can only add nucleotides on to an existing strand of DNA

Question 36

Single strand binding protein (SSB)

Answer 36

keeps two strands of DNA apart whilst synthesis of new DNA occurs - prevents annealing to form double stranded DNA