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Biosynthesis of

Nucleic Acids:

Replication

Replication of DNA

Naturally occurring DNA exists in single-stranded and double-stranded forms, both of which can exist

in linear and circular forms

Difficult to generalize about all cases of DNA replication

We will study the replication of circular double-stranded DNA and then of linear double-stranded

DNA

most of the details we discuss were first investigated in prokaryotes, particularly E. coli

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Flow of Genetic Information in the Cell

Mechanisms by which information is transferred in the cell is based on Central Dogma

Prokaryotic Replication

Challenges in duplication of circular double-stranded DNA

achievement of continuous unwinding and separation of the two DNA strands

arotection of unwound portions from attack by nucleases that attack single-stranded DNA

synthesis of the DNA template from one 5 -> 3 strand and one 3 -> 5 strand

efficient protection from errors in replication

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Prokaryotic Replication (Contd)

Replication involves separation of the two original strands and synthesis of two new daughter strands using the original strands as templates

Semiconservative replication: each daughter strand contains one template strand and one newly synthesized strand

Incorporation of isotopic label as sole nitrogen source (15NH4Cl)

Observed that 15N-DNA has a higher density than 14N-DNA, and the two can be separated by density-gradient ultracentrifugation

DNA replication - process of copying a double-stranded

DNA molecule

Dogma of Genetic Information

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The replication process

1. DNA molecule unzips

the double helix separates into the individual DNA strands

the "unwound" DNAs are

exposed

to free nucleotides, and

base

pairs complementarily

2. both strands can serve as templates

3. the individual nucleotide

complements are

joined together by

phosphodiester bonds

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4. new strands -high fidelity (one for every 109 -1010

bases) are made as free nucleotides combine with

their complementary bases.

Parent strand

Daughter strand

Daughter strand

Parent strand

DNA replication is semiconservative

DNA synthesis is from 5 to 3

Which Direction does Replication go?

DNA double helix unwinds at a specific point called an origin of replication

Polynucleotide chains are synthesized in both directions from the origin of replication; DNA

replication is bidirectional in most organisms

At each origin of replication, there are two replication forks, points at which new polynucleotide chains are

formed

There is one origin of replication and two replication forks in the circular DNA of prokaryotes

In replication of a eukaryotic chromosome, there are several origins of replication and two replication forks

at each origin

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Bidirectional Replication

DNA Polymerase Reaction

The 3-OH group at the end of the growing DNA chain acts as a nucleophile.

The phosphorus adjacent to the sugar is attacked, and then added to the growing chain.

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DNA Polymerase

DNA is synthesized from its 5 -> 3 end (from the 3 -> 5 direction of the template)

the leading strand is synthesized continuously in the 5 -> 3 direction toward the replication fork

the lagging strand is synthesized semidiscontinuously (Okazaki fragments) also in the 5 -> 3 direction, but away from the replication fork

lagging strand fragments are joined by the enzyme DNA ligase

Properties of DNA Polymerases

There are at least five types of DNA polymerase (Pol) in E coli, three of which have been studied extensively

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Function of DNA Polymerase

DNA polymerase function has the following requirements:

all four deoxyribonucleoside triphosphates: dTTP, dATP, dGTP, and dCTP

Mg2+

an RNA primer - a short strand of RNA to which the growing polynucleotide chain is covalently bonded in the early stages of replication

DNA-Pol I: repair and patching of DNA

DNA-Pol III: responsible for the polymerization of the newly formed DNA strand

DNA-Pol II, IV, and V: proofreading and repair enzymes

Supercoiling and Replication

DNA gyrase (class II topoisomerase) catalyzes reaction involving relaxed circular DNA:

creates a nick in relaxed circular DNA

a slight unwinding at the point of the nick introduces supercoiling

the nick is resealed

The energy required for this process is supplied by the hydrolysis of ATP to ADP and Pi

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Replication with Supercoiled DNA

Replication of supercoiled circular DNA

DNA gyrase has different role here. It introduces a nick in supercoiled DNA

a swivel point is created at the site of the nick

the gyrase opens and reseals the swivel point in advance of the replication fork

the newly synthesized DNA automatically assumes the supercoiled form because it does not have the nick at the swivel point

helicase, a helix-destabilizing protein, promotes unwinding by binding at the replication fork

single-stranded binding (SSB) protein stabilizes single-stranded regions by binding tightly to them

Primase Reaction

The primase reaction

RNA serves as a primer in DNA replication

primer activity first observed in-vivo.

Primase - catalyzes the copying of a short stretch of the DNA template strand to produce RNA primer sequence

Synthesis and linking of new DNA strands

begun by DNA polymerase III

the newly formed DNA is linked to the 3-OH of the RNA primer

as the replication fork moves away, the RNA primer is removed by DNA polymerase I

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Replication Fork General Features

Summary of DNA Replication in

Prokaryotes

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Proofreading and Repair

DNA replication takes place only once each generation in each cell

Errors in replication (mutations) occur spontaneously only once in every 109 to 1010 base pairs

Can be lethal to organisms

Proofreading - the removal of incorrect nucleotides immediately after they are added to the growing DNA during replication (Figure 10.10)

Errors in hydrogen bonding lead to errors in a growing DNA chain once in every 104 to 105 base pairs

Proofreading Improves Replication Fidelity

Cut-and-patch catalyzed by Pol I: cutting is removal of the RNA primer and patching is incorporation of the required deoxynucleotides

Nick translation: Pol I removes RNA primer or DNA mistakes as it moves along the DNA and then fills in behind it with its polymerase activity

Mismatch repair: enzymes recognize that two bases are incorrectly paired, the area of mismatch is removed, and the area replicated again

Base excision repair: a damaged base is removed by DNA glycosylase leaving an AP site; the sugar and phosphate are removed along with several more bases, and then Pol I fills the gap

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DNA Polymerase Repair

Mismatch Repair in Prokaryotes

Mechanisms of mismatch repair encompass:

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DNA Recombination

Genetic Recombination- When genetic information is rearranged to form new associations

Homologous - Reactions between homologous sequences

Nonhomologous- Different nucleotide sequences recombine

Recombination

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Eukaryotic Replication

Not as understood as prokaryotic. Due in no

small part to higher level

of complexity.

Cell growth and division divided into phases: M,

G1, S, and G2

Eukaryotic Replication

Best understood model for control of

eukaryotic replication

is from yeast.

DNA replication initiated by

chromosomes that

have reached the G1

phase

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Eukaryotic DNA Polymerase

At least 15 different polymerases are present in eukaryotes (5 have been studied more extensively)

Structure of the PCNA Homotrimer

PCNA is the eukaryotic equivalent of the part of Pol III that functions as a sliding clamp ().

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The Eukaryotic Replication Fork

The general features of DNA replication in eukaryotes are similar to those in prokaryotes. Differences summarized in

Table 10.5.

Telomerase and Cancer (Biochemical Connections)

Replication of linear DNA molecules poses particular problems at the ends of the molecules

Ends of eukaryotic chromosomes called telomeres

Telomere- series of repeated DNA sequences