DNA Replicatio n Vipin Shankar

In the early 1930s, biologists began
speculating as to what sort of molecule could have the kind of stability that the gene demanded, yet be capable of permanent, sudden change to the mutant forms that must provide

What is the genetic material?
• Is it
– The proteins, that make up the enzymes? – The complex proteins of the chromosomes? – The amino acids that make up the proteins? – Or, the seemingly simple nucleic acids that make up the chromosomes?

Avery’s Bombshell
• Oswald T Avery, Colin M MacLeod and Maclyn McCarty (Rockefeller Institute, New York), based on original observations by Griffith. • DNA can carry genetic specificity.

The Double Helix

The Cell Cycle

Replication
• A template directed nucleic acid synthesis reaction. • Replication leads to doubling of the DNA, preserving the genetic information, for transmission to the next generation. • Occurs in the S phase of the Cell Cycle. • Replication requires a template to provide sequence information.

DNA Replication – The possible mechanisms.
“… It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material….” - Watson and Crick (in the paper describing DNA structure)

The possible mechanisms…
• The Conservative model
– Both parental strands remain together and the two new strands of DNA would form an entirely new DNA molecule.

• The Dispersive model
– The strands get broken as frequently as ten base pairs and are used to prime the synthesis of similarly short regions of DNA, which get subsequently joined to form the complete DNA strand.

The possible mechanisms…
• The Semi-conservative model
– The two strands separate during replication and each strand act as the template for a new strand. – Thus the new DNA molecule is made up of a newly synthesized strand and a strand from the original molecule.

Experimental evidence for strand separation during replication
• Mathew Meselson and Frank Sthal (1958), at the California Institute of Technology.

The Meselson - Sthal experiment
• They grew E. coli in a medium containing 15NH4Cl as the only source of nitrogen. • After growing for several generations, on the 15N-media, the DNA was found to be denser. • The density of the strands were determined by CsCl-density gradient centrifugation. • Meselson and Sthal, transferred the E. coli, with the heavy (15N) DNA, to a media containing 14NH4Cl as the only

More proof for semi-conservative replication
• Taylor et al, labeled Vicia fava (broad bean) root tip cells with 3 H-thymidine and allowed them to grow in unlabelled Dr. J Herbert Taylor medium. • The metaphase chromosomes were analyzed by autoradiography. • Observations:
– Both chromatids were labeled after one generation.

• • • •

Chromosome from parent cell labeled with 3Hthymidine. Cells grown in medium without 3H-thymidine. Chromatids separate during cell cycle. And each chromatid produces its sister chromatid The newly produced chromatids are not labeled.

More proof…
• Based on the use of 5bromodeoxyuridine (BrdU), an analogue of thymidine. • DNA with BrdU in place of thymidine, does not stain with fluorescent dye (33258 Hoechst). • When cells labeled with BrdU are subsequently grown in a medium without the analogue
– Only one chromatid takes up the stain while its sister does not.

Dr. Cairns’ Experiment
• Dr. J Cairns (1963) used autoradiography to demonstrate semi-conservative model of replication. • He grew E. coli on a medium containing 3H-thymine. • The DNA was then extracted and carefully subjected to autoradiography.

Dr. Cairns experiment: inferences
• The E. coli DNA is a circle. • The DNA is replicated while maintaining the integrity of the circle. • An intermediate theta structure forms (topologically similar in shape to the Greek letter ‘θ’.) • Replication of the DNA seems to be occurring at one or two moving Yjunctions in the circle. • The DNA is unwound at a given point, and replication proceeds at a Yjunction, in a semi-conservative

The rolling circle replication
• This form of replication is initiated by a break in one of the nucleotide strands that creates a 3’-OH group and a 5’-phosphate group. • New nucleotides are added to the 3’end of the broken strand, with the inner (unbroken) strand used as a template. • As new nucleotides are added to the 3’end, the 5’end of the broken strand is displaced from the template, rolling out like thread being pulled off a spool. • The 3’end grows around the circle, giving rise to the name rolling-circle model.

The rolling circle mode…
• The replication fork may continue around the circle a number of times, producing several linked copies of the same sequence. • With each revolution around the circle, the growing 3’ end displaces the nucleotide strand synthesized in the preceding revolution. • Eventually, the linear DNA molecule is cleaved from the circle, resulting in a double stranded circular DNA molecule and a single-stranded linear DNA molecule. • The linear molecule circularizes either before or after serving as a template for the

The replicon
• Stretches of DNA with a single origin of replication (Francois Jacob, Sydney Brenner & Jacques Cuzin (1963)). • The entire DNA replicated from a single origin. • 2 components:
– The initiator – The replicator

The replicon…
• The initiator
– A protein that specifically recognize the replicator. – Recruits the replication machinery to the origin of replication.

• The replicator
– The entire set of DNA sequences sufficient to direct the initiation of replication. – Composed of two parts • A recognition site for the initiator

The origin of replication
• A stretch of AT rich DNA, that unwinds readily but not spontaneously. • Called oriC in E. coli.
– Contains 2 repeated motifs. – A 9-mer motif repeated 5 times & – A 13-mer motif repeated 3 times.

The mechanism of replication
• Tightly controlled process,
– occurs at specific times during the cell cycle.

• Requires:
– A set of proteins and enzymes, – Energy.

• Two basic steps:
– Initiation – Elongation.

• Two basic components:
– Template – Primer.

Process of semi-conservative replication
• Identification of the origins of replication. • Unwinding (denaturation) of ds-DNA to provide an ss-DNA template. • Formation of the replication fork. • Initiation of DNA synthesis and elongation. • Formation of replication bubbles with ligation of the newly synthesized DNA segments.

Enzymology of DNA replication
• DNA Helicase: Unwinds DNA ahead of replication fork • DNA Polymerase: DNA synthesis and repair of gaps • DNA Ligase: Joins fragments of DNA • DNA Primase: Syntheses primers • DNA Topoisomerases: Releases torsional strain caused by helicase activity • SSBs: Stabilizes single stranded

DNA Polymerases
• First identified in lysates of E. coli by Arthur Kornberg (1963). • The first polymerases to be isolated was named DNA Pol – I • Isolation of polymerases represent a landmark discovery in molecular biology, since the ability of these enzymes to accurately copy a DNA template provided biochemical basis of the mode of replication provided by Watson & Crick.

DNA Polymerases: more studies
• Cultures of E. coli were treated with chemical mutagens. • Mutants deficit in Pol-I activity were isolated and sub-cultured. • These strains grew normally in normal media. • But, these strains were extremely sensitive to agents that cause DNA damage.

DNA Polymerases: more studies…
• Inferences:
– Pol-I may not be required for DNA replication. – Pol-I may be involved in the repair of DNA damage, rather than in DNA replication.

DNA Polymerases: further studies
• DNA polymerases II & III were isolated from E. coli. • The potential role of these enzymes were studied by the isolation of appropriate mutants. • Its been confirmed that
– Pol-III & Pol-I are the major enzymes responsible for replication. – Pol-II is responsible for ‘error-prone’ DNA repair.

• Eukaryotic cells contain 5 different

DNA Polymerases: Types

DNA Polymerases: Functions

DNA Polymerase: Mechanism
• Requirements:
– All 4 dNTPs; viz, dATP, dCTP, dGTP & dTTP. – A primer – template junction.

• The new chain is synthesized by adding appropriate dNTPs at the 3’ end of the primer at the primer – template junction. • A phosphodiester bond is formed between the 3’OH of the primer and the α-phosphate group of the

DNA Polymerases: Mechanism…
• Hydrolysis of the pyrophosphate drives the reaction. XTP + (XMP)n → (XMP)n+1 + P ~ P P ~ P → 2Pi XTP + (XMP)n → (XMP)n+1 + 2Pi

DNA Polymerases: Mechanism…
• Have a single active site to catalyze the addition of all four dNTPS. • This is done by exploring the nearly identical geometry of the A:T & G:C base pairs. • The DNA polymerase monitors the ability of the incoming nucleotide to form a correct base pair. • Only when a correct base pair is formed a phosphodiester bond is formed between the 3’OH of the

DNA Polymerase: Mechanism…
• Incorrect base-pairing leads to lower rates of nucleotide addition, due to catalytically unfavorable alignment of the substrates : Kinetic selection. • The rate of incorporation of an incorrect nucleotide is 10,000 fold slower. • DNA polymerase can distinguish between rNTPs and dNTPs, by steric exclusion.

DNA Polymerase: Mechanism…
• DNA Polymerase resembles a Hand that Grips the Primer : Template junction • 3 domains –
– The palm: • contains the primary elements of the catalytic site. • Has 2 divalent metal ions – Mg2+ or Zn2+. • Catalysis of the phosphodiester bond. • Monitors the accuracy of base

DNA Polymerase: Mechanism…
• Domains
– The Fingers: • Bind to the incoming nucleotide. • Once the correct base pair is formed, the finger domain moves and enclose the dNTP, thus enhancing catalysis. – The Thumb: • Not intimately involved in catalysis. • Maintains the correct position of the primer in the active site. • Maintains a strong association between the polymerase and the

Processivity of DNA Polymerase
• Processivity is a characteristic of enzymes that operate on polymeric substrates. • DNA polymerases are capable of adding as many as 1,000 nts per second. • The degree of processivity is defined as the average number of nucleotides added each time the enzyme binds a primer : template junction. • Each DNA polymerase has a

Processivity….
• The initial binding of the polymerase to the primer : template junction is the rate limiting step. • The processivity of DNA Pol is increased by a Sliding Clamp. • The Sliding clamp is an association of proteins that assemble in the shape of a doughnut. • The clamp encircles the newly synthesized ds DNA and keeps DNA Pol associated with the primer :

Proof Reading by Polymerase
• Mediated by nuclease activity that remove the incorrectly base paired nucleotides. • The 3’ to 5’ exonuclease activity of the palm domain, checks for the incorrect base pairing. • The proof reading gives a second chance to correct mistakes and to add the correct nucleotide.

DNA Replication: The process
• The initiator protein recognizes and binds to the replicator sequence. • The initiator protein recruits DNA Helicase to the Origin of replication. • DNA Helicase unwinds the ds DNA at the origin of replication – formation of the Replication Fork. • Other proteins of replication machinery assemble at the replication fork.

DNA Replication: The process…
• Single Strand Binding Proteins (SSBs) stabilize the replication fork by binding to the newly opened ss DNA, preventing the recoil. • Topoisomerases prevent the supercoiling formed during helicase activity.
– 2 classes of topoisomerases are present. – Class II topoisomerases also called Gyrases are the important ones in DNA

The need for topoisomerases

DNA Replication: The process…
• Primase synthesizes RNA primer for the action of DNA polymerase. • PROBLEM.
– DNA Polymerase can synthesize DNA only in the 5’ to 3’ direction. – So synthesis on one strand is continuous. – What happens on the other strand?

• The strand on which continuous DNA synthesis proceeds is called the Leading Strand.

DNA Replication: The process…
• The second strand is called the Lagging Strand. • On the lagging strand DNA synthesis happens in short fragments (1000 – 2000 nts in bacteria and 100 – 400 in Ek.cells) called Okazaki Fragments. • Each Okazaki fragment requires a new primer. • The RNA primers are removed by RNAase H. • The single stranded nicks produced

DNA Replication: Trombone Model
• At the replication fork, the leading strand and the lagging strand are synthesized simultaneously. • This limits the amount of ss DNA present in the cell during replication. • To co-ordinate the replication of both the strands, multiple DNA Polymerases act together. • A large multi-protein complex called the DNA Pol –III holoenzyme in which the core enzyme is associated with

Finishing replication
• Completion of replication is different in circular and linear chromosomes. • In a circular chromosome (like in E. coli), the 2 replication forks meet at a region called the termination region or ter region. • Termination utilization substance (tus protein), forms a complex with the ter region and stops the progress of the replication fork. • This results in two daughter molecules linked to one another (Catenane). • These are seperated by the action of class II

Replication of circular chromosomes

Finishing Replication…
• The requirement of a RNA primer for the synthesis of all Okazaki fragments on the lagging strand, creates a dilemma for the replication of the end of the linear chromosome – the end replication problem. • Once RNA molecule is removed from the last Okazaki fragment, a short region of un-replicated DNA will remain on the lagging strand. • This means that each round of replication would result in the

Telomerase
• Telomerase is a DNA polymerase that does not require a exogenous template. • Telomerase is an enzyme which includes both protein and RNA components (ribonucleoprotein). • The RNA component acts as a template for adding the telomeric sequence to the 3’ terminus at the end of the chromosome and thus solves the end replication problem.

The End Replication Problem

Assembling newly replicated DNA into nucleosomes
• When eukaryotic DNA is replicated, it complexes with histones. • This requires synthesis of histone proteins and assembly of new nucleosomes. • Transcription of histone genes is initiated near the end of G1 phase, and translation of histone proteins occurs throughout S phase.

Assembly of nucleosomes