DNA Structure

Question 1

What did Maurice Wilkins and Rosaline Franklin find?

Answer 1

Experimental evidence that DNA = a helix

They were taking DNA in solution - adding ethanol at various ratios - then pulling out fibres of DNA from the liquid

Then putting these fibre into an intense X-ray beam - and following diffraction if the X-ray beam by the DNA

the crosswires of the diffractions the middle = show its a helix

Question 2

They found different forms of helix

Answer 2

Base pairs on inside & sugars and phosphates on outside

Maurice Wilkins - using diff levels of ethanol- produce different form of DNA = A form

= right handed DNA helix - more tightly wound - size of minor & major grooves = close (not much different to B from (predominant form)

Z DNA = left handed coil DNA - found inside cells ( function not established well)

Question 3

What are the 2 polynucleotide strands that make up the 2 chains of DNA in the helix composed of?

Answer 3

Composed of Nucleotides ( repeating base, deoxyribose sugar, phosphate) linked by 3’-5- phosphodiester bonds

Question 4

What is the DNA helix held together by?

Answer 4

Held together by hydrogen bonds between AT & CG bonds

When you look at DNA from a distance - the amt of A = roughly amount of T and same w C & G

Question 5

CG base pairs

Answer 5

Cytosine - Pyrimidine (single six membered ring)
Guanine - Purine - 2 runts

Form 3 hydrogen bonds between them

Question 6

AT base pairs

Answer 6

Thymine - six membered & pyrimidine
Adenine - purine

Only 2 hydrogen bonds - not as strong of an interaction of CG base pair

When AT & CG packed together in DNA = form very smooth and uniform helix - as both similarly sized

Question 7

Feature of a DNA helix

Answer 7

2 anti parallel polynucleotide chains for a right handed helix
Bases on inside of helix-phosphate & sugars on outside
Polynucleotide chains held together by Hydrogen bonds between AT & CG

One strand = complementary to other strand

Question 8

What structure forms an A from helix?

Answer 8

Transfer RNA ( tRNA )

Single RNA chain - has a lot of self complementarity - so can fall back in itself to form areas or A form helix

Question 9

What are some unusual DNA structures?

Answer 9

Left handed / Z-DNA
Formed by 5’…GCGCGCGC or 5’GTGTGTGT
alternating purine - pyrimidine sequence

Four stranded Junction = Holliday Junction

Tetraplex DNA = 4 stranded DNA helix - formed at the end of chromosomes - the telomeres of chromosomes - involves G rich DNA sequences

Question 10

What are Holliday Junctions?

Answer 10

Arise in living cells through
DNA strand exchange between 2 homologous chromosomes - red & blue duplexes

Important role in DNA repair & exchange of genetic information

Question 11

What are the levels of DNA structure?

Answer 11

Primary = Sequence of bases ( determined by DNA sequencing methods)

Secondary = Helical structure ( can be probed by X-ray techniques & chemistry)

Tertiary = DNA supercoiling - DNA helix urself = coiled in space ( can use electron microscopy)

Quaternary = Individual chromosomes that are interlocked.
Produced naturally in bacteria - at end of a round of replication -
where bacterial DNA = circular - the 2 daughter chromosomes are interlocked like 2 locks in a chain

Question 12

What can you infer from gene sequence?

Answer 12

What the protein sequence must be

Question 13

What is the most widely used method of DNA sequencing?

Answer 13

Sanger Sequencing

DNA strand = copied w DNA polymerase - in presence of inhibitors that arrest DNA synthesis - specifically at A,C,G,T

The DNA strands = separated by length on a polyacrylamide gel

If the DNA or incorporated inhibitor = radioactive or fluorescent - the DNA bands can be visualised & the sequence read

700-1000 bases per read

Question 14

Why is having DNA sequence so important?

Answer 14

Important to understand gene function

Identify anti-microbial or anti-cancer targets or vaccine candidates

Question 15

Describe bacterial / eukaryotic DNA

Answer 15

In most species - bacterial DNA = Circular ( e.coli= 3x106bp roughly)

Supercoiled in the cell - the DNA helix = twisted in space

Supercoiling caused by enzyme DNA Gyrase

Question 16

What is DNA Gyrase?

Answer 16

An enzyme that uses ATP - as an energy source - and takes a relaxed DNA circle & converts it into a supercoiled space

Question 17

Why is supercoiling important?

Answer 17

It is very important for facilitating bacterial DNA replication

An essential enzyme in bacteria

Question 18

What do the enzymes Topo IV & Topo I ( related to Gyrase) do?

Answer 18

Reverse reaction to Gyrase

Take out the supercoiled & allows DNA to become relaxed

In the bacterial cell - there is competition between those activities - sets the level of supercoiling at some intermediate level

Question 19

The size of bacteria is small compared to the size of its DNA, what happens?

Answer 19

The DNA has to be further compacted than what the supercoiling gives

E.g the E.coli chromosome = organised into 50 supercoiled loops so the DNA can fit inside

Question 20

What is a karyotype analysis?

Answer 20

Human DNA = 23 pairs chromosomes. Can be visualised using this

Where you arrest cells at mitosis - then break the nucleus & spread out all the chromosomes & label them w particular pieces of DNA - which Dye is attached

So you can identify the individual chromosomes and sort then out

Far more DNA in a eukaryotic cell than in a bacterium

Question 21

In the chromosomes, the DNA is complexed w histones. What are histone?

Answer 21

Basic proteins which interact w the negative charge in the phosphates in DNA and allow the DNA to be packaged

Question 22

What is chromatin?

Answer 22

There are also other proteins (as well as histones) involved in binding DNA

This whole nucleoprotein complex = called Chromatin

Question 23

What is the foundation of chromatin?

Answer 23

Nucleosome ( the building block)

First seen in the electron microscope

The DNA - seemed to be present when it came out of the nucleus as a ‘ BEADS ON A STRING’ structure

Regularly spaced nucleosomes interacting w DNA

DNA = string 
Nucleosomes = beads

Question 24

Structure of nucleosomes

Answer 24

Determined by X-ray crystallography

Made up of several different proteins & DNA

In the middle = 4 different histone types = histones 2a, 2b, 3 and 4

Highly basic, small proteins - able to interact w the negative charges from the phosphates of the DNA

This core = sometimes called the Histone Aptamer = because there are 2 copies of each of the 4 histones

The DNA = coiled on the outside of the histone core
Roughly 2 super helical turned of the DNA = wrapped outside the nucleosome

Core DNA = DNA - physically associated w the histones
Linker DNA = DNA - runs between adjacent histones

Question 25

Why must there be other ways for the DNA to be compacted other than the nucleosomes?

Answer 25

The nucleosomes = present at the lowest level organisation of chromatin

But can’t account for all the compaction needed to get the eukaryotic DNA to sit inside the nucleus of a cell

Question 26

What other ways can the DNA be compacted?

Answer 26

Take the beads on a string structure & coil it again in 3 dimensions

There are proteins that sit in nucleosomes

E.g another histone = histone 1 - sits at entry & exit point of the DNA in the nucleosome

These histone H1s = present on adjacent or nearby nucleosomes can come together &

result in 3D coiling of the DNA and nucleosome structure

Question 27

How does DNA damage cause mutations in the DNA?

Answer 27

Spontaneous ( spontaneously hydrolysed)

E.g loss of bases (abasic site - problematic in DNA replication)
Or hydrolysis of C to U ( when C = not stable) - result in mutation
The phosphodiester bonds can also be hydrolysed & give breaks in the DNA

Chemicals & radicals generated by oxidative metabolism
Change base structure ( also cyclophosphamide)
Insert between bases ( intercalators e.g doxorubicin widely used as anti cancer drug)

When ATP = generated by oxidative metabolism - energy available from food stores = greatly enhanced.
Problem = can generate free radicals that can damage bases in DNA

Many important anti- cancer drugs = damaging to DNAs

DNA = not immune to effects of radiation
If you have 2 adjacent thymine in same polynucleotide chain = can cross link to make thymine dimer - problem

Ionising radiation = can cause breakage to chromosome

Question 28

What does DNA repair do? And what is involved?

Answer 28

Maintains genome stability
More than 100 enzymes/proteins involved
Many inherited diseases - have a defect in DNA repair system
Other cancer prone families have DNA repair defects
Understanding DNA damage and repair = very important

Question 29

DNA has conformational ability, what does this mean?

Answer 29

It exists in more than one form.

A, B and Z DNA