16374 DNA Replication

7/31/2019 16374 DNA Replication

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Kunjan Saxena

Asst. Prof.

Dept. of Biotechnology

L.P.U


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DNA replication Copying genetic information for transmission to the

next generation

Occurs in S phase of cell cycle Process of DNA duplicating itself

Begins with the unwinding of the double helix toexpose the bases in each strand of DNA


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Semi-Conservative DNA Replication

Watson and Crick proposed that the two strands in theparent DNA molecule separate, allowing each strandto act as a template for the daughter DNA molecules.Their model of DNA replication is the semi-

conservative model where each daughter DNAmolecule contains one intact strand of the parent DNAmolecule.

Watson and Crick did not have any experimentalevidence to support the semi-conservative model.Other scientists proposed two other possible modelsfor DNA replication the conservative model and thedispersive model.


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If the replication method were conservative, one

round of replication would yield a DNA molecule thatcontained two strands of parental DNA, and a secondDNA molecule that contained two new strands.

If the replication method were semi-conservative, one

round of replication would yield two DNA moleculesthat each contained one strand of parental DNA andone strand of new DNA.

If the replication method were dispersive, one round

of replication would yield two DNA molecules eachcontaining mixtures of fragments of the original DNAas well as new fragments of DNA.


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DNA replication is bidirectional The replication process does not begin at any random spot

along the DNA molecule. DNA replication begins at asingle origin of replicationknown asoriC.

Each bacterial chromosome contains a single origin ofreplication. The hydrogen bonds between the twocomplementary DNA strands break at this origin so thateach strand serves as a template for the synthesis of a newstrand.

The double helix unwinds at the replication forks,forming a replication bubble. Replication proceeds in bothdirections away from the origin of replication. Eventually,the two replication forks meet opposite the origin ofreplication, and the result is two separate and completecircular chromosomes.


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When the strands are separated, however, DNApolymerase cannot simply begin copying the DNA.DNA polymerase III can only extend a nucleic acidchain. To give the DNA polymerase III a place to start,an RNA polymerase called primase first copies a shortstretch of the DNA strand. This creates acomplementary RNA segment, up to 60 nucleotideslong that is called a primer.


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Now DNA polymerase III can copy the DNA strand. TheDNA polymerase III starts at the 3 end of the RNA primer,and, using the original DNA strand as a guide, begins tosynthesize a new complementary DNA strand. DNApolymerase then synthesizes a new strand of DNA byextending the 3' end of an existing nucleotide chain,

adding new nucleotides matched to the template strandone at a time via the creation of phosphodiester bonds. Theenergy for this process of DNA polymerization comes fromtwo of the three total phosphates attached to eachunincorporated base. (Free bases with their attached

phosphate groups are called nucleoside triphosphates.)When a nucleotide is being added to a growing DNAstrand, two of the phosphates are removed and the energyproduced creates a phosphodiester bond that attaches theremaining phosphate to the growing chain.


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Chemistry of DNA Polymerase


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DNA Synthesis Proceeds in a 53Direction and Is

Semidiscontinuous

A new strand of DNA is always synthesized in the53 direction, with the free3 OH as the point at which the DNA is elongated.

Because the two DNA strands are antiparallel, the strand serving as thetemplate is read from its 3 end toward its 5 end.

If synthesis always proceeds in the 53direction, how can both strands besynthesized simultaneously?

If both strands were synthesized continuously while the replication forkmoved, one strand would have to undergo 35 synthesis. This problem wasresolved by Reiji Okazaki and colleagues in the 1960s. Okazaki found that oneof the new DNA strands is synthesized in short pieces, now called Okazakifragments.

This work ultimately led to the conclusion that one strand is synthesizedcontinuously and the other discontinuously. The continuous strand, or leading strand, is the one in which53 synthesis

proceeds in the same direction as replication fork movement. The discontinuous strand, or lagging strand, is the one in which 53

synthesis proceeds in the direction opposite to the direction of fork movement.


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DNA replication is bidirectional and

semidiscontinuous


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Before the lagging-strand DNA exits the replication factory,

its RNA primers must be removed and the Okazakifragments must be joined together to create a continuousDNA strand.

The first step is the removal of the RNA primer. RNAse H,

which recognizes RNA-DNA hybrid helices, degrades theRNA by hydrolyzing its phosphodiester bonds.

Next, the sequence gap created by RNAse H is then filled inby DNA polymerase I which extends the 3 end of the

neighboring Okazaki fragment. Finally, the Okazaki fragments are joined together byDNA

ligase that hooks together the 3 end of one fragment tothe 5 phosphate group of the neighboring fragment


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REPLICATION IN ACTION

The process begins when the helicase enzyme unwinds thedouble helix to expose two single DNA strands and create tworeplication forks. DNA replication takes place simultaneously ateach fork. The mechanism of replication is identical at each fork

Single-strand binding proteins, or SSBs, coat the single DNAstrands to prevent them from snapping back together. SSBs areeasily displaced by DNA polymerase.

The primase enzyme uses the original DNA sequence as atemplate to synthesize a short RNA primer. Primers arenecessary because DNA polymerase can only extend a nucleotidechain, not start one.

DNA polymerase III begins to synthesize a new DNA strand byextending an RNA primer in the 5' to 3' direction. Each parentalDNA strand is copied by one DNA polymerase. Remember, bothtemplate strands move through the


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replication factory in the same direction, and DNA

polymerase can only synthesize DNA from the 5

endto the 3 end. Due to these two factors, one of the DNAstrands must be made discontinuously in short pieceswhich are later joined together.

As replication proceeds, RNAse H recognizes RNAprimers bound to the DNA template and removes theprimers by hydrolyzing the RNA.

DNA polymerase I can then fill in the gap left byRNase H.

The DNA replication process is completed when theligase enzyme joins the short DNA pieces together intoone continuous strand.


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DNA REPLICATION PROTEINS

DNA replication requires a variety of proteins. Each protein performs a specificfunction in the production of the new DNA strands.

Helicase, made of six proteins arranged in a ring shape, unwinds the DNAdouble helix into two individual strands.

Single-strand binding proteins, or SSBs, are tetramers that coat the single-stranded DNA. This prevents the DNA strands from reannealing to formdouble-stranded DNA.

Primase is an RNA polymerase that synthesizes the short RNA primers neededto start the strand replication process.

DNA polymerase is a hand-shaped enzyme that strings nucleotides togetherto form a DNA strand. The sliding clamp is an accessory protein that helps holdthe DNA polymerase onto the DNA strand during replication.

RNAse H removes the RNA primers that previously began the DNA strandsynthesis.

DNA ligase links short stretches of DNA together to create one longcontinuous DNA strand.

Topoisomerase relaxes the DNA from its supercoiled nature.


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Features of DNA Replication DNA replication is semiconservative

Each strand of template DNA is being copied.

DNA replication is semidiscontinuous

The leading strand copies continuously The lagging strand copies in segments (Okazaki

fragments) which must be joined

DNA replication is bidirectional

Bidirectional replication involves two replication forks,which move in opposite directions

16374 DNA Replication

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