The flow of Genetic information

DNA ReplicationDNA ReplicationDNA is a double-helical molecule Watson and Crick Predicted Semi-conservative Replication of DNA The mechanism: Strand separation, followed by copying of each strand. Each separated strand acts as a template for the synthesis of a new complementary strand.Each strand of the helix must be copied in complementary fashion by DNA polymerase DNA replication takes place in a semiconservative manner. That is, a parent double helix forms two daughter double helices, each composed of one parent DNA stand and on newly synthesized stand.

Semiconservative Semiconservative

DNA ReplicationDNA ReplicationTwo identical new copies of

the DNA double helix are

produced during replication Each new strand is

complementary to its old

template strand In each new helix, one

strand is the old template

and the other is newly

synthesized.

SemiconservatiSemiconservative DNA ve DNA ReplicationReplication

• E. coli genome size = 4.6 X 106 bp

• Bacteria have circular chromosome with single origin of replication.

• Replication rate is ~1000 base pairs per second.

• Duplicate chromosome in 38 minutes.

• Eukaryotes have larger genomes 3 X 109 bps

• Rate of Eukaryote chromosome replication is slower

• But because eukaryote chromosomes have multiple origins of replication,

it takes about the same amount of time to replicate complete genome.

Eukaryote

prokaryote

DNA ReplicationDNA Replication

Stages of DNA ReplicationStages of DNA Replication Initiation: perpriming complex Elongation: RNA primer and DNA polymerase Termination

InitiationInitiation

DNA should unfold before the beginning of replication. This

process is performed by a group of proteins called the

prepriming complex:

• The first important protein is a small enzyme called DNAa

protein.

• 20-50 monomers of this enzyme will recognize and bind to a

consensus sequence called the origin of replication (a region

on DNA rich in the nucleotides Adenosine ad Thymidine).

• DNAa then causes a local opening or local melting to appear.

• This process is energy consuming; the energy is obtained by

DNAa form of ATP.

• Once the strands are separated, a

protein called single strand binding

protein (SSB protein) will bind to

each strand preventing their refolding

into a double helical form.

• The binding of SSB proteins does not

require energy.

•Another function of SSB proteins is to

protect the single DNA strands from

the action of endonucleases. Since

the single DNA strand is a substrate

for these enzymes.

InitiationInitiation

Helicase Is an enzyme that uses energy (ATP) to separate the two DNA strands apart beginning at the local opening formed by DNAa protein. Its action is focused at the replication fork.

Endonucleases and ExonucleasesEndonucleases and Exonucleases

Priming and Replication

DNA polymerase III is unable to form a nucleotides sequences

from scratch it requires a primer or an already existing

nucleotide which is RNA primer

•The starting point for DNA polymerase is a short segment of

RNA known as an RNA primer.

•The primer is RNA strand complementary to the DNA template

synthesized by an enzyme known as RNA polymerase or

Primase.

• The RNA primer consists of about 10 ribonucleotides

complementary to the sequence on the DNA strand.

•The DNA polymerase (once it has reached its starting point as

indicated by the primer) then adds nucleotides one by one in

an exactly complementary manner, A to T and G to C.

Elongation and replication Elongation and replication • Primase Complex Synthesizes short RNA primers.• The class of enzymes which perform DNA synthesis are called the polymerase enzymes. The polymerases can only read DNA in a 3` →5` direction they synthesize DNA only in 5` to 3` direction. • The DNA double helix has the following polarity:

Leading strand 3` → 5` lagging strand is 5` → 3` DNA polymerase uses the parent strands as template and synthesizes a new 5` → 3` strand

U

Synthesis along the leading strand is continuous and is carried

out by polymerase III. On the other hand, synthesis along the

lagging strand is discontinuous and is produced in fragments

called Okazaki fragments. and takes place via two enzymes:

polymerase III and polymerase I.

DNA Replication is DNA Replication is SemidiscontinuousSemidiscontinuous

DNA ReplicationDNA Replication

Replication of lagging strand by Okazaki fragments.Replication of lagging strand by Okazaki fragments. -The enzyme responsible for synthesis of these fragments is polymerase III - It requires an RNA primer -the synthesis takes place from 5` → 3`, in an opposite direction to that in which the replication fork progresses. -As the replication fork spreads and more of the double helix is unfolded, an RNA primer is added to the lagging strand at the replication fork, the polymerase III then proceeds in adding deoxynudeotides to the OH end of the RNA primer, until it reaches another primer.- A gap or a nick between the Okazaki fragments and the RNA primer. The nick is recognized by polymerase I, that removes the ribonucleotides of the primer and replace them by deoxynucleotides complementary to the parent DNA strand-This forms a discontinuous strand with stretches of DNA separated by gaps or nicks.- Another enzyme, ligase, seals these nicks by forming phosphodiester bonds. The polymerase I also replaces the RNA primer on the leading strand with deoxynucleotides.

Replication of lagging strand by Okazaki fragments.Replication of lagging strand by Okazaki fragments.

The Enzymology of DNA Replication The Enzymology of DNA Replication

• If Watson and Crick were right, then there should be an enzyme that makes DNA copies from a DNA template

• In 1957, Arthur Kornberg and colleagues demonstrated the existence of a DNA polymerase -

• Three DNA polymerases in E. coli- DNA polymerase I – DNA repair and participates in

synthesis of lagging strand- DNA polymerase II – DNA repair- DNA polymerase III – major polymerase involved in DNA

replication.- Elongation involves DnaB helicase unwinding, SSB

binding to keep strands separated.- Primase Complex synthesizes short RNA primers.- DNA polymerase grinding away on both strands - Topoisomerase II (DNA gyrase) relieves supercoiling that

remains

DNA Polymerase also has proof reading functionDNA Polymerase also has proof reading function

• The polymerization reactions have an error rate of 1 mistake for every 100,000 base pairs incorporated (1 X 10-5 errors per base)

• Polymerases have proof reading functions. As they synthesize DNA, they double check for any incorrectly paired bases.

• If any mismatches are found, they are removed and replaced by the correct base This means that polymerase III has 3` → 5` exonuclease activity or it proofreads DNA from 3` → 5`.

• Therefore proof reading function helps eliminate errors which could lead to detrimental mutations.

• However proof reading exonuclease has error rate of 1 mistake for every 100 base pairs (1 X 10-2 errors per base)

• Overall error rate is 1 X 10-7 errors per base.

• Polymerase I has a 5` → 3` exonuclease activity in order to remove the RNA primer. In addition to that, it also possesses 3` → 5` exonuclease activity which aims at proofreading the DNA sequence that has replaced the RNA primer.

Polymerase actionPolymerase action

DNA polymerase I has 5’ to 3’ exonuclease activity that removes RNA primer. Also has 5’ to 3’ DNA polymerase activity to fill in the gap. (proofreading 3’-5’ exonuclease activity)Ligase connects loose ends. Used NAD+ in phosphoryltransfer reaction,

DNA Polymerase I/ Ligase Required to Join Okazaki Fragments

•Polymerase I has a 5`→ 3` exonuclease activity in order to remove the RNA primer. In addition to that, it also possesses 3`→ 5` exonuclease activity which aims at proofreading the DNA sequence that has replaced the RNA primer.

DNA ReplicationDNA Replication

Enzymes that relief supercoiling (Super-twisting )Enzymes that relief supercoiling (Super-twisting ) During the replication the two strand of the DNA should be away from each other Supercoiling should be removed so the DNA have to rotate opposite of the direction of the coiling but this method costs energy and lead to another supercoiling Topoisomerase enzymes solve this problem. There are two types:Topoisomerase I works only on one strand of the DNA and cut that strand, then allows the other strand to pass through to solve the supercoil, then it connects the strand again [nuclease activity and ligase activity] Topoisomerase II works on the two strands of the DNA at the same time, and it is more efficient than topoisomerase I. Topoisomerase II cuts both strands of the DNA, allows the other one to move through it, and basically it will relieve supercoil.

Supercoiling During DNA strand separationSupercoiling During DNA strand separation

Enzymes that Relief SupercoilingEnzymes that Relief Supercoiling

DNA Replication in EukaryotesDNA Replication in Eukaryotes

• Occurs similarly to what occurs in prokaryotes.

• Multiple origins of replication

• Replication is slower than in prokaryotes.

• 5 different DNA polymerases in Eukaryotes.

Eukaryotic DNA PolymerasesEukaryotic DNA PolymerasesAlpha (Pol )– Primer synthesis and DNA repair Beta – DNA repair Gamma – Mitochondrial DNA replicationDelta (Pol )– Leading and lagging strand synthesis, and

DNA repair Epsilon (Pol ) – Repair and gap filling on lagging

strand.

Termination of Termination of ReplicationReplication

• Termination occurs at ter (terminus )region of E. coli chromosome.

• ter region rich in Gs and Ts, signals the end of replication.

• Terminator utilization substance (Tus) is a protein which binds to ter region.

• Tus prevents replication fork from passing by inhibiting helicase activity.

The EndThe End

DNA Repair • A fundamental difference from RNA,

protein, lipid, etc. • All these others can be replaced, but

DNA must be preserved • Cells require a means for repair of

missing, altered or incorrect bases, bulges due to insertion or deletion, UV-induced pyrimidine dimers, strand breaks or cross-links

• Two principal mechanisms: methods for reversing chemical damage and excision repair.

Repair of UV Induced Thymine

Dimers

General excision-repair

pathway

•Excision-repair systems scan DNA duplexes for mismatched bases, excise the mispaired region and replace it

Repair of damage resulting from the

deamination of cytosine• Deamination of cytosine to

uracil is one of most common forms of DNA damage

• DNA glycosylases cleave bases at N-glycosidic linkages. Leaving sugar-phosphate backbone.

• Endonuclease identifies abscent base and sugar phosphate.

• Gap then filled in by DNA polymerase and ligase.