DNA Function, Structure And Replication Flashcards
Prokaryotes
No nuclear membrane
DNA arranged often in a single chromosome
In E.coli it is circular (4x106np)
Eukaryotes
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)
Functions of DNA
DNA acts as a template and regulator for transcription and protein synthesis
DNA is the genetic material, structural basis of heredity and genetic diseases
Chromosomes (DNA) in E.coli (prokaryotic)
Supercoiled
Circular chromosome
2.5 x 106 kDa
4 x 106 bases
2mm linear
Chromosomes (DNA) in humans (eukaryotic)
Complex packaging
(22 x 2) + X,Y or X,X
4 x 109kDa
3 x 109bp
2m linear
DNA is tightly packaged
Histones can be modified by methylation
Chromatin can exist in different densities: heteronormative and eurochromatin
DNA replication process
- 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.
- SSB’s (single-strand binding protein) coat the single DNA strands to prevent re- annealing or ‘snapping back together’.
- 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
- 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.
- As replication proceeds, RNAse H recognises RNA primers bound to the DNA template and removes the primers by hydrolysing the RNA
- DNA polymerase can then fill the gap left by RNAse H
- DNA replication process completed when the ligase enzyme joins the short DNA pieces (Okazaki fragments) together into one continuous strand.
How fast is DNA replication
700-1000 bp per second
Human DNA origins of replication
Multiple points of origin
Enzymes involved…
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
DNAPolymerase 5’ 3’
• 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
Opening the strands
• Helicase opens it
• Single stranded binding proteins (SSB) proteins keep it open
• Topoisomerase unwinds it (relieves supercoiling)
-slide 30
DNA Polymerase
Has editing function
Detects incorrect insertion of base and will excise and repeat
(Slide 31)
Polymerase Chain Reaction
• 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
DNA Damage from sources like…
Chemical
Radiation
Spontaneous insertion of incorrect bases during replication
Smoking
It is a DNA-adduct (directly adds to it)
It reacts with bases to form a bulky group that disrupts replication
Ionising Radiation
Can damage bases
Cause breaks in phosphate backbone
UV
Damages bases
In particular the formation of thymine dimers
Mutations
DNA damage:
chemicals, UV, radiation, chance
DNA repair :
- Base or nucleotide excision (removal)
-Mismatch repair
Tumour suppressor gene p53
Linked in cell cycle control
Detects DNA damage and initiates repair
p53
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
Inhibitors of nucleotide synthesis
Methotrexate
MTX
5-Fluorouracil (FU)
Methotrexate
folic acid analogue
inhibits childhood leukemia
some lymphom
MTX
synthesis of purines
rheumatoid arthritis
psoriasis
5-Fluorouracil (FU)
inhibits thymidilate synthase GI
head and nech
breast carcinomas
DNA polymerase inhibitors
Cytosine Arabinoside
(AraC)
Acyclovir (Acy)
DNA template damaging agents
Cyclophosphamide
Cysplatin (CPPD)
Inhibitors of DNA topoisomerase
Doxorubicin
DOX
The 5’ end of DNA strand…
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)
Chromosomes
Each chromosome is made of 2 identical stands of (chromatids) joined in the centre (centromere).
What do free phosphate groups in DNA provide?
energy for the reaction to go Through
DNA structure relating to replication
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’
DNA structure relating to replication
• 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’
Why is DNA replication semi conservative?
Because each resulting DNA double helix retains one strand of the original DNA
Primer
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
Single strand binding protein (SSB)
keeps two strands of DNA apart whilst synthesis of new DNA occurs - prevents annealing to form double stranded DNA
