Curs 4

DNA replication

DNA and RNA are polymers of nucleotides

Phosphate group Nitrogenous base Sugar Phosphate group Nucleotide Nitrogenous base (A, G, C, or T)

Thymine (T)

Sugar (deoxyribose)

DNA nucleotide Polynucleotide Sugar-phosphate backbone

 Each strand of the double helix is oriented in the opposite direction

5 end

3 end

3 end

5 end

P P

Mitoza este procesul de diviziune celular, specific celulelor eucariote, prin care dintr-o celul-mam rezult doua celule-fiice, identice din punct de vedere genetic

CICLUL CELULAR

decondensarea cromozomilor

dublarea cantitii de ADN, ARN i proteine i condensarea cromozomilor

sinteza unor proteine necesare formrii fusului de diviziune i sinteza de ATP

How can entire chromosomes be replicated during S phase?

DNA replication begins at many specific sites

Origin of replication Parental strand Daughter strand

Bubble

Two daughter DNA molecules

Replicarea ADN este semiconservativa

Replication of DNA:

base pairing allows each strand to serve as a template for a new strand new strand is 1/2 parent template and 1/2 new DNA

Replication: 1st step

Unwind DNA
helicase enzyme
unwinds part of DNA helix stabilized by single-stranded binding proteins
helicase

single-stranded binding proteins

replication fork

DNA Polymerase
Bidirectional synthesis of the DNA double helix Corrects mistaken base pairings Requires an established polymer (small RNA primer) before addition of more nucleotides Other proteins and enzymes necessary

Replication: 2nd step

strand

Build daughter DNA

add new complementary bases DNA polymerase III

DNA Polymerase III

Energy of Replication
energy energy

ATP CTP TTP GTP

modified nucleotide

CMP TMP GMP AMP ADP

 The nucleotides arrive as nucleosides

DNA bases with PPP
P-P-P = energy for bonding

Energy of Replication

DNA bases arrive with their own energy source for bonding bonded by enzyme: DNA polymerase III

ATP

GTP

TTP

CTP

5
energy Replication DNA

Adding bases

Polymerase III
energy

can only add nucleotides to 3 end of a growing DNA strand

need a starter nucleotide to bond to

DNA Polymerase III DNA Polymerase III

energy

strand only grows 53

energy DNA

Polymerase III
3 5

Leading & Lagging strands

Limits of DNA polymerase III

can only build onto 3 end of an existing strand

3 3 growing replication fork 3 5

ligase

Lagging strand

5 3

Leading strand
3

Lagging strand

5 3

Okazaki fragments joined by ligase

DNA polymerase III

Leading strand

continuous synthesis

DNA Replication
Priming:
1. RNA primers: before new DNA strands can form, there must be small pre-existing primers (RNA) present to start the addition of new nucleotides (DNA Polymerase). 2. Primase: enzyme that polymerizes (synthesizes) the RNA Primer.

DNA Replication
Synthesis of the new DNA Strands:
1. DNA Polymerase: with a RNA primer in place, DNA Polymerase (enzyme) catalyze the synthesis of a new DNA strand in the 5 to 3 direction.
5
RNA Primer

3
5

Nucleotide

DNA Polymerase

DNA Replication

2. Leading Strand: synthesized as a single polymer in the 5 to 3 direction.

5
RNA Primer

5
Nucleotides

DNA Polymerase

DNA Replication
3. Lagging Strand: also synthesized in the 5 to 3 direction, but discontinuously against overall direction of replication (replication fork)
5 3
5
Leading Strand

3
5 3

DNA Polymerase

RNA Primer

5
Lagging Strand

DNA Replication
4. Okazaki Fragments: series of short segments on the lagging strand.

Okazaki Fragment RNA Primer

DNA Polymerase

5
3
Lagging Strand

3
5

DNA Replication
5. DNA ligase: a linking enzyme that catalyzes the formation of a covalent bond joining fragments
Example: joining two Okazaki fragments together.
DNA ligase

5 3

Okazaki Fragment 1

Okazaki Fragment 2

3 5

Lagging Strand

 DNA polymerase works in only one direction: 5 to 3

Telomere sequences are lost with each replication. Cancer, aging

DNA polymerase molecule 5 end Parental DNA 5 3

3 5

Daughter strand synthesized continuously

Daughter strand synthesized in pieces

3 5

5 3

telomeres

DNA ligase

Overall direction of replication

Repeating, non-coding sequences at the end of chromosomes = protective cap

Telomeres

limit to ~50 cell divisions

3 growing replication fork

5 3

5 3

telomerase
5

Telomerase

TTAAGGGTTAAGGG 3

enzyme extends telomeres can add DNA bases at 5 end different level of activity in different cells

Mutations can change the meaning of genes

Mutations are changes in the DNA base sequence
caused by errors in DNA replication or by mutagens change of a single DNA nucleotide causes sicklecell disease

 The information constituting an organisms genotype is carried in its sequence of bases

The DNA is transcribed into RNA, which is translated into the polypeptide
DNA

TRANSCRIPTION

RNA

TRANSLATION
Protein

The Nobel Prize in Physiology or Medicine 2012 John B. Gurdon eliminated the nucleus of a frog egg cell and replaced it with the nucleus from a specialised cell taken from a tadpole. The modified egg developed into a normal tadpole. Subsequent nuclear transfer experiments have generated cloned mammals

Shinya Yamanaka
Shinya Yamanaka studied genes that are important for stem cell function. When he transferred four such genes into cells taken from the skin, they were reprogrammed into pluripotent stem cells that could develop into all cell types of an adult mouse. He named these cells induced pluripotent stem (iPS) cells.

 iPS cells can now be generated from humans, including patients with disease. Mature cells including nerve, heart and liver cells can be derived from these iPS cells, thereby allowing scientists to study disease mechanisms in new ways.