DNA Replication

“A structure this pretty just had to exist.”

—James Watson, The Double Helix,1968

PART IThe complete story•How DNA was found to be the hereditary material??

DNA: The Genetic Material

Chromosomes= DNA + Proteins

Mendel’s experiments showed that genes were the basic unit of inheritance.Genes are located on chromosomes

So question arises??

Which Chemical component is the Genetic material?

Protein OR DNA??

Fredrick Griffith Experiment 1920sTransforming Principle

• The S strain DNA contains the genes that form the protective polysaccharide capsule. While the bacteria had been killed, the DNA had survived the heating process and was taken up by the R strain bacteria.

• Griffiths Conclusion: Some material in the heat-killed S strain that was responsible for transforming the R-strain into a lethal form

Transformation = passing the inheritance factor from one organism to another THIS TRANSFORMING PRINCIPLE WAS LATER FOUND TO BE DNA

• Oswald Avery: He had reported that DNA, not protein (which was believed at the time), was the hereditary substance (transforming material).

• he treated samples known to contain the pneumococcal transforming principle to destroy different types of molecules—proteins, nucleic acids, carbohydrates, and lipids—and tested the treated samples to see if they had retained transforming activity

• . The answer was always

the same: If the DNA in the sample was destroyed, transforming activity was lost, but there was no loss of activity when proteins, carbohydrates, or lipids were destroyed

TO COFIRM WHETHER DNA OR PROTEIN IS THE

HEREDITARY MATERIAL• The Hershey–Chase Experiment• Using T2 bacteriophage :virus that infects

bacteria, consists of little more than a DNA core packed inside a protein coat

• When a T2 bacteriophage attacks a bacterium, part of the virus enters the bacterial cell. About 20 minutes later, the cell bursts, releasing dozens of viruses

• The entry of a viral component changes the genetic program of the host bacterial cell: it is converted from a bacterium into a bacteriophage factory. Hershey and Chase set out to determine which part of the virus protein or DNA—enters the bacterial cell

P32

LABELLED

S35

LABELLED

The Hershey–Chase Experiment

One batch of T2

viruses grown in presence of S35 had S35 in their protein coat.

The other batch of T2 viruses grown in presence of P32 had P32 in their DNA.

PART II DNA STRUCTURE•How DNA structure was established??

X-ray crystallography provided clues to DNA structure

Chargaff’s rule• The amount of adenine equals the amount of

thymine (A =T), and the amount of guanine equals the amount of cytosine (G = C). As a result, the

total abundance of purines (A + G) equals the total abundance of pyrimidines (T + C).

Watson and Crick Model of DNA Structure

Key features of DNA

Double stranded

Uniform diameter

Right handed helix twists like a Screw

Anti parallel

Hydrogen bonding

Complementary base pairing

DNA Structure- a revision

• Nucleotide• Nucleoside• Purines• Pyrimidines

Anti parallel strandsThe 5'-end (pronounced "five prime end") designates the end of the DNA or RNA strand that has the fifth carbon in the sugar-ring of the deoxyribose or ribose at its terminus (theend with the free phosphate)The 3'-end of a strand is so named due to it terminating at the hydroxyl group of the third carbon in the sugar-ring, and is known as the tail end(the end with the free hydroxyl)

the phosphodiester bond

-diester refers to the two bonds formed by -OHgroups reacting with phosphate groups

phosphodiester

ester

structure• In both RNA and

DNA, the backbone of the macromolecule consists of alternating pentose sugars and phosphates (sugar—phosphate—sugar—phosphate). The bases are attached to the sugars and project from the chain.

• The nucleotides

are joined by phosphodiester linkages between the sugar of one nucleotide and the phosphate of the next .The phosphate groups link carbon 3 in one pentose sugar to carbon 5 in the adjacent sugar.

Concept of complementarity• The two anti parallel polynucleotide chains of

double-helical DNA are not identical in either base sequence or composition. Instead they are complementary to each other

• Every base pair consists of one purine (A or G) and one pyrimidine (T or C). This pattern is known as complementary base pairing. Because the AT and GC pairs are of equal length and fit identically into the double helix, the diameter of the helix is uniform

• information contained in the sequence of one strand is conserved in the sequence of the other

Why does A bond only with T and C only with G? What do you

notice?• Nitrogenous Bases T and C are single-ring Pyrimidines .

• A and G are double-ring Purines

• A single ring base bonds with a double

PART III DNA REPLICATION

•How identical copies of DNA are produced??

Determining the mechanism of DNA replication

Three models:• Conservative• Semi Conservative• Dispersive

DN

A R

epli

cati

on

The Meselson-Stahl Experiment

Can be explained only by the semi conservative model of DNA replication.

In the first round of DNA replication, the strands of the double helix—both heavy with 15N ,separated. Each strand then acted as the template for a second strand, which contained only 14N and hence was less dense. Each double helix then consisted of one 15 N strand and one 14 N strand, and was of intermediate density

In the second replication, the 14 N-containing strands directed the synthesis of partners with 14 N, creating low-density DNA, and the15 N strands formed new 14 N partners

• In conservative

replication, the first generation would have had both high-density DNA (15 N–15N) and low-density DNA (14N–14N), but no intermediate-density DNA.

• In dispersive replication, the density of the new DNA would have been half that of parental DNA

DNA replicationReplication: process of producing two

identical replicas from one original DNA molecule. Each strand of the original DNA molecule serves as template for the production of the complementary strand

• a huge protein complex called the replication complex

• the replication complex seems to be stationary, it is the DNA that moves

• DNA replicates in both

directions from the origin of replication (ori), forming two replication forks

• Small circular chromosomes, such as the 3-million-base pair DNA of bacteria, have a single origin of replication. Human chromosome with 80 million base pairs, there are hundreds of origins of replication.

DNA Replication Mechanism • Initiation

• DNA helicase uses energy from ATP hydrolysis to unwind the DNA

• special proteins called single strand binding proteins bind to the unwound strands to keep them from reassociating into a double helix

• DNA polymerases cannot start a strand from scratch. Therefore, a short single strand of RNA, called a primer, is required for replication. This RNA strand, is synthesized by an enzyme called primase (RNA Polymerase)

• Elongation:

• DNA polymerase then adds nucleotides to the 3′end of the primer and continues until the replication of that section of DNA has been completed.

• Then the RNA primer is degraded, DNA is added in its place, and the resulting DNA fragments are connected by the action of ligases

nucleotides are always added to the growing strand at the 3′end— the end at which the DNA strand has a free hydroxyl (—OH) group on the 3′ carbon of its terminal deoxyribose The three phosphate groups in a deoxyribonucleoside triphosphate areattached to the 5′position of the sugar. So when a new nucleotide is added to DNA, it can attach only to the 3′end

When DNA polymerase brings a deoxyribonucleoside

triphosphate with the appropriate base to the 3′ end of a growing chain, the free hydroxyl group on the chain reacts with one of the substrate’s phosphate groups.. The phosphate group still on the nucleotide becomes part of the sugar–phosphate

backbone

of the

growing

DNA

molecule.

• Termination:• Eukaryotes initiate DNA replication at multiple

points in the chromosome, so replication forks meet and terminate at many points in the chromosome.

• Termination requires that the progress of the DNA replication fork must stop or be blocked. Involves the interaction between two components: (1) a termination site sequence in the DNA, and (2) a protein which binds to this sequence to physically stop DNA replication,named the DNA replication terminus site-binding protein, or Ter protein

single strand binding proteins

Helicase

summary ENZYMES FOR DNA REPLICATION

• Helicase = separates 2 DNA strands (breaks H bonds)

• Primase =RNA primers at INITIATION• Topoisomerase = unwinding DNA• DNA Polymerase = Adding of DNA nucleotides• LIGASE = Binds the Okazaki fragments• single strand binding proteins = bind to the

unwound strands to keep them from reassociating into a double helix

• The DNA polymerases are

only able to “read” the parental nucleotide sequences in the 3'→5' direction, and they synthesize the new DNA strands only in the 5'→3' (antiparallel) direction. Therefore, the two newly synthesized stretches of nucleotide chains must grow in opposite directions—one toward the replication fork and one away from the replication fork .

• 1. Leading strand: The strand

that is being copied in the direction of the advancing replication fork is called the leading strand and is synthesized continuously.

• 2. Lagging strand :The strand that is being copied in the direction away from the replication fork is synthesized discontinuously, with small fragments of DNA being copied near the replication fork. These short stretches of discontinuous DNA, termed Okazaki fragments, are eventually joined (ligated) to become a single, continuous strand.

telomeres • Telomeres are

complexes of noncoding DNA plus proteins located at the ends of linear chromosomes. They maintain the structural integrity of the chromosome, preventing attack by nucleases

Following removal of the RNA primer from the

extreme 5'-end of the lagging strand, there is no way to fill in the remaining gap with DNA.

Consequently, in most normal human somatic cells, telomeres shorten with each successive cell division. Once telomeres are shortened beyond some critical length, the cell is no longer able to divide and is said to be senescent. In germ cells and other stem cells, as well as in cancer cells, telomeres do not shorten and the cells do not senesce. This is a result of the presence of telomerase, which maintains telomeric length.

Questions ???