replication

What is dna replication ?

Biological process by which a cell duplicates its DNA molecule, producing 2 identical copies from 1 original DNA strand

What ensurs replication ?

It ensurs the transmission of genetic information during cell division

What is the difference between an OGM and a chimera ?

OGM : mutation done by scientist

Chimera : Organism with mutations that happened in real life

How dna is organised in cells ?

It’s packed and condensed in chromosome

What is a nucleosome ?

Structural unit of chromatin, consisting of a segment of DNA wound around a core of 8 histone proteins

What are the purines ?

Adenine (A) and guanine (G) (double-ring nitrogenous bases)

What are the pyrimidines ?

Cytosine (C) and thymine (T) (single-ring nitrogenous bases)

What is the structural difference between purines and pyrimidines ?

Purines have a double-ring structure (a six-membered ring fused to a five-membered ring), while pyrimidines have a single six-membered ring

What is this ?

Cytosine

What is this ?

Thymine

What is this ?

Adénine

What is this ?

Guanine

Name those

What links 2 bases ?

Général rules of DNA organisation

• 5’ : phosphate group

• 3’ : hydroxyl group

• AT and CG complementarity

• 2 strands antiparallel

• Chargaff’s rules : %A=%T and %G=%C and C+G = A + T except for ssDNA like virus

Which migrates faster in electrophoresis ?

The supercoiled DNA bc it’s twisted so hydrodinamic so slip easily through agarose

What happened in 1953 ?

Watson Crick and Franklin disco discovered the double helical structure of DNA

3 models of DNA replication

• conservative model : parental DNA strands remained associated in one DNA molécule while new daughter strands remained associated in newly formed dna molecule

• Dispersive model : all resulting DNA strands have régions of db stranded parental dna and régions of db stranded daughter dna

• Semi conservative model : right one

Which enzyme unwinds the dna hélix ?

Helicase : break the h bonds between base pairs : generates 2 ss dna template ready for replication

Which protein stabilizes the unwind dna hélix ?

Single strand binding protein : prevent it from reannealing and to protect it from nuclease

Topoisomerase vs helicase

Topoisomerase relaxes supercoilling

Helicase unwinds dna

=> Topoisomerase works in front of helicase and allows it to unwind

Rules of replication

• semi conservative

• Primer is required

• Template is required

• Élongation occurs 5’→3’

• Template is read 3’→5’

• Proof reading is done by 3’→5’ exonuclease

• Semi discontinous

• Bidirectional

What means semi conservative ?

Each strand of DNA molecule serves as template for the next génération : after replication, each dna molecule contains one strand from the parent molecule and one newly synthetised strand

Step of DNA réplication

1. Replication fork formation

2. Primer binding

3. Elongation

4. Termination

Where replication begins ?

At an origin of replication

• oriC in prokaryotes : dnaA bind to oriC causing DNA to unwind (atp dependant)

• ORC in eukaryotes : recognized by a group of protein : pre RC complex

Replication speed

• E : between 1 and 2 kb / min

• P : ~ 1kb / sec

=> bc mutation are more dangerous in E so proofreading takes more Time

2 types of Topoisomerase

• Type 1 : makes a ss break in the dna

• Type 2 : makes a ds break in the dna

Both reseals the break afterwards

Which prokaryotic enzyme synthetise RNA primers ?

Primase : provide a starting point for DNA synthesis bc DNA polymerase can only add nucleotides to an existing strand

Leading strand vs lagging strand

Leading strand : 5’→3’ and Single rna primer

Lagging strand : 3’→5’ and multiples rna primers are needed, discontinous synthesis generating okazaki fragments

which enzyme does élongation in prokaryotes ?

Dna pol III : add nucleotides to the 3’end of the RNA primer

Which enzyme replace RNA primers in prokaryotes ?

DNA Pol I : removes RNA primers and replace them with DNA nucleotides.

Which enzyme ligate fragments in prokaryotes ?

Ligase : seals the Nick between okazaki fragment in the lagging strand, forming a continous dna strand (générâtes phosphodiester bonds)

6 Major différences between eucaryotic and prokaryotic replication

• single site vs multiple site for the origin of réplication

• Initiation by dnaA and dnaB vs by multi subunit protein (origin recognition complex)

• Two réplication forks vs multiple réplication forks

• Large vs short okazaki fragments

• Fast vs slow process

• Circular vs linear ds dna

What is the replisome ?

Multiprotein molecular machinery which replicate dna

Which protein recognizes the eucariotic origin of réplication

ORC and Cdc6 binds to it

Which enzyme on which strand in eucaryotes ?

On the lagging strand : Pol alpha

On the leading strand : Pol epsilon

Function of exonuclease

• cleave nucleotides from the free ends

• DNA damage repair

• Apoptosis

• Inflammation

Function of exonuclease

• cleave the phosphofiester bond at a specific position with in the dna strand

Pol 1 structure (procaryotes)

• monomeric enzyme with 3 domains

  • fingers domain : interact with the incoming dntp

  • Palm domain : catalyses phosphoryl transfer réaction

  • Thumb domain : interacts with duplex dna

Which co factor is essentiel to dna polymerase ?

Mg2+

What is the klenow fragment ?

It’s the larger fragment of the cleaved dna Pol I after proteolytic cleavage.

=> loss of 5’3’ exonuclease activity which corresponds to the RNA primer dégradation

How does polymerase activity work ?

Pol III structure (procariotes)

Multi subunit complex with 17 polypeptides organised in 4 sub assemblies :

• the core polymerase

  • alpha : polymerase

  • Epsilon : 3’5’ exonuclease

  • Thêta : stimulator of exonuclease

• The t subunit : responsible for dimerization of the core

• The slidind clamp

• Clamp loader function

=> asymmetric enzyme

Holoenzyme

Complète, functional enzyme which is catalytically active

Holoenzyme = apoenzyme + cofactor

Eukaryotic dna polymerase

• alpha : synthesize primers (primase activity)

• Epsilon : synthesize the leading strand

• Delta : synthesize the lagging strand

• Gamma : main replicative enzyme for mitochondrial dna

What is PCNA ?

Proliferating cell nuclear antigen in Pol delta eucaryote : principal conductor at the réplication fork

Post translational modification of pcna

Must go through ubiquitination, deubiquitination, sumoylation, phosphorylation

chemotherapy drugs target dna réplication

• topoisomerase inhibitor

• Nucleotides precursor synthesis inhibitor

Common uses of PCR (polymerase chain reaction)

• dna cloning

• Génétic testing

• Pathogen détection

…

What is included in a PCR mix ?

• taq dna polymerase

• Primers

• d NTPs

• Buffer with Mg2+

• DNA

Steps of PCR

• Initial dénaturation : 3/5 min à 95 C

• dénaturation : 95 C 10/30 sec

• Annealing : 55/68 C 10/30 sec

• Elongation : 72 C 10/30 sec par kb

• Final extension : 72 C 10 min

Types of PCR polymerase

• pfu dna polymerase : higher fidelity when copying dna

• Taq dna polymerase : most commonly used

4 key characteristics of dna polymerase

• specificity

• Thermostability

• Fidelity

• Processivity

Rules for a good primer

• length of 18-24 pb

• 40/60% GC content and 1-2 at start and end

• Melting température of 50/60 C

• No complementary régions between F and R

• Melting température of the 2 within 5 C from each other

What are ddNTPs ?

Dideoxtnucleotides : chain elongating inhibitors of DNA polymerase, used to terminate the synthesis of a growing dna strand, resulting in partially replicated dna fragments

Steps of sanger sequencing

• dénature ds dna using heat

• Make multiple copies of a segment

• Attach a primer

• Added to four polymerase solutions

• Grow complementary chain until termination dye

• Dénature the grown chains

• Electrophorese the four solutions

What is sanger sequencing ?

Also known as « the chain termination method » : laboratory technique used to détermine the exact nucleotide séquence of a dna segment