Prokaryotic Replication (Cont’d)

• Replication involves the separation of

the two original strands and the
synthesis of two new daughter
strands using the original strands as
templates.
Chapter Ten • Semiconservative replication: each
daughter strand contains one
Biosynthesis of Nucleic Acids: template strand and one newly
Replication synthesized strand
• Incorporation of the 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 G0 indicates original strands.
Paul D. Adams • University of Arkansas ultracentrifugation G1 indicates new strands after the first generation
[Link]/chemistry/campbell G2 indicates new strands after the second generation.

Chapter Outline
10-1 The Flow of Genetic Information in the Cell
10-2 Replication of DNA
10-3 DNA Polymerase
10-4 Proteins Required for DNA Replication

Replication of DNA Evidence for Semiconservative Replication

• 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 Heavy DNA labeled with 15N forms a band at the
bottom of the tube.
• most of the details we discuss were first investigated in prokaryotes, particularly E. coli
Light DNA with 14N forms a band at the top.

DNA that forms a band at an intermediate position

has one heavy strand and one light strand.

Flow of Genetic Information in the Cell In Which Direction does Replication go?

• Mechanisms by which information is transferred in the cell are based on “Central Dogma” • DNA double helix unwinds at a specific point called the 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.

The yellow arrows represent general cases.

The blue arrows represent special cases (mostly in RNA viruses).

Prokaryotic Replication Bidirectional Replication

Challenges in duplication of circular double-stranded DNA

• achievement of continuous unwinding and separation of the two DNA strands
• protection of unwound portions from attack by nucleases that attack single-stranded DNA
• synthesis of the DNA template from one 5’ à 3’ strand and one 3’ à 5’ strand
• efficient protection from errors in replication
Bidirectional
The semidiscontinuous model for DNA replication (cont’d)
Replication

Remember that DNA is synthesized from its 5’à 3’ end (from the 3’à 5’ direction of the
template)
•the lagging strand is synthesized semidiscontinuously (Okazaki fragments) also in the 5’à 3’
direction but away from the replication fork
•lagging strand fragments are joined by the enzyme DNA ligase

Proteins Required for DNA Replication: Supercoiling and

Properties of DNA Polymerases
Replication
• There are at least five types of DNA polymerase (Pol) in E coli, three of which have been studied DNA gyrase (class II topoisomerase) catalyzes
extensively 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

Function of DNA Polymerase Replication with Supercoiled DNA

• Replication of supercoiled circular DNA
DNA polymerase function has the following requirements: • DNA gyrase has a different role here. It introduces a nick in supercoiled DNA
• all four deoxyribonucleoside triphosphates: dTTP, dATP, dGTP, and dCTP • a swivel point is created at the site of the nick
• Mg2+ • the gyrase opens and reseals the swivel point in advance of the replication fork
• an RNA primer - a short strand of RNA to which the growing polynucleotide chain • the newly synthesized DNA automatically assumes the supercoiled form because it does not have
is covalently bonded in the early stages of replication the nick at the swivel point
• helicase, a helix-destabilizing protein, promotes unwinding by binding at the replication fork
• DNA-Pol I: repair and patching of DNA • single-stranded binding (SSB) protein stabilizes single-stranded regions by binding tightly to them
• DNA-Pol III: responsible for the polymerization of the newly formed DNA strand
• DNA-Pol II, IV, and V: proofreading and repair enzymes

DNA Polymerase Primase Reaction

• The primase reaction
The addition of a nucleotide to a • RNA serves as a primer in DNA replication
growing DNA chain. • primer activity was first observed in vivo.
• Primase - catalyzes the copying of a short stretch of the DNA template strand to produce RNA
The 3'-hydroxyl group at the end of the primer sequence
growing DNA chain acts as a nucleophile. • Synthesis and linking of new DNA strands
It attacks the phosphorus adjacent to the • begun by DNA polymerase III
sugar in the nucleotide, which is added to
• the newly formed DNA is linked to the 3-OH of the RNA primer
the growing chain.
• as the replication fork moves away, the RNA primer is removed by DNA polymerase I
Pyrophosphate is eliminated, and a new
phosphodiester bond is formed.

The semidiscontinuous model for DNA replication How Does Replication Work With Supercoiled DNA?

General features of a
replication fork.

DNA is synthesized from its 5’à 3’ end (from the 3’à 5’ direction of the template)
• Newly synthesized DNA is shown in red. Because DNA polymerases only polymerize nucleotides
5’ à3', both strands must be synthesized in the 5’ à 3' direction. The DNA duplex is unwound by DNA gyrase and helicase, and the single strands are coated with
SSB (ssDNA-binding protein). Primase periodically primes synthesis on the lagging strand. Each
• Thus, the copy of the parental 3’ à 5' strand is synthesized continuously; this newly made strand
half of the dimeric replicative polymerase is a holoenzyme bound to its template strand by a b-
is designated the leading strand.
subunit sliding clamp. DNA polymerase I and DNA ligase act downstream on the lagging strand to
• The leading strand is synthesized continuously in the 5’ à 3’ direction toward the replication fork remove RNA primers, replace them with DNA, and ligate the Okazaki fragments.
Summary of DNA Replication in Prokaryotes

1) DNA synthesis is bidirectional.

2) The direction of DNA synthesis is from the 5’ end to 3’ end of the newly formed strand.
3) Five DNA polymerases have been found in E. coli.
4) DNA gyrase introduces a swivel point in advance of the movement of the replication fork.
5) Primase catalyzes the synthesis of an RNA primer. End of Chapter
6) The synthesis of new strands is catalyzed by Pol III.

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 the removal of the RNA primer, and patching is the
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 is 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

Eukaryotic DNA Polymerase

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

The Eukaryotic Replication Fork

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