DNA Replication

DNA Is Reproduced by Semiconservative

Replication
• The complementarity of DNA strands allows
each strand to serve as a template for
synthesis of the other
• Three modes of DNA replication are possible:
– Conservative
• Original helix is conserved and two newly synthesized
strands come together
– Semiconservative
• Each replicated DNA molecule consists of one "old"
strand and one new strand
– Dispersive
• Parental strands are dispersed into two new double
helices
• DNA replication begins at the origin of
replication
• Where replication is occurring, the strands of
the helix are unwound, creating a replication
fork
• Bacteria have a single circular DNA, and DNA
synthesis originates at a single point, the
origin of replication, called OriC
• The entire bacterial chromosome

• Eukaryotes
– Many origin of replications
– Linear

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DNA Synthesis Involves Many Proteins
• DNA polymerase catalyzes DNA synthesis and
requires a DNA template and all four
deoxyribonucleoside triphosphates (dNTPs)
• Chain elongation occurs in the 5' to 3'
direction by addition of one nucleotide at a
time to the 3' end
• As the nucleotide is added, the two terminal
phosphates are cleaved off, providing a newly
exposed 3'-OH group that can participate in
the addition of another nucleotide as DNA
synthesis proceeds

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5’  3’
• DNA polymerase is a complex enzyme
(holoenzyme) made up of many subunits
• Its 3' to 5' exonuclease activity allows
proofreading
• There are seven key issues that must be
resolved during DNA replication:
1. Unwinding of the helix
2. Reducing increased coiling generated during
unwinding
3. Synthesis of a primer for initiation
4. Discontinuous synthesis of the second strand
5. Removal of the RNA primers
6. Joining of the gap-filling DNA to the
adjacent strand
7. Proofreading
 1. Unwinding of the helix
• Proteins, which require the energy normally
supplied by the hydrolysis of ATP to break
hydrogen bonds and denature the double
helix, are called helicases
• Single-stranded binding proteins (SSBPs)
stabilize the open conformation
 2. Reducing increased coiling generated
during unwinding

• Unwinding produces supercoiling that is

relieved by DNA topoisomerases
• DNA topoisomerases makes single- or double-
stranded cuts to undo the twists and knots
created during supercoiling, which are then
resealed
 3. Synthesis of a primer for initiation

• To elongate a polynucleotide chain, DNA

polymerase requires a primer with a free 3'-
hydroxyl group
• Primase synthesizes an RNA primer that
provides the free 3'-hydroxyl required by DNA
polymerase III

• Priming is a universal phenomenon during

initiation of DNA synthesis
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Figure 11.10
 4. Discontinuous synthesis of the second
strand

• As the replication fork moves, only one strand

can serve as a template for continuous DNA
synthesis—the leading strand
• The opposite lagging strand undergoes
discontinuous DNA synthesis

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The lagging strand is
synthesized as
Okazaki fragments,
each with an RNA
primer
 5. Removal of the RNA primers
6. Joining of the gap-filling DNA to the
adjacent strand

• An RNAase removes the primer and DNA

polymerase replaces it with DNA
• The fragments are joined by DNA ligase
 7. Proofreading
Proofreadingand
• Proofreading correction are an
errorcorrectionare
and error
integral part of DNA replication
• All of the DNA polymerases have 3' to 5'
exonuclease activity that allows proofreading

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• Eukaryotic DNA replication is more complex
due to several features of eukaryotic DNA:
– There is more DNA than prokaryotic cells
– The chromosomes are linear
– The DNA is complexed with proteins

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• Eukaryotic chromosomes contain multiple
origins of replication to allow the genome to
be replicated in a matter of minutes to a few
hours

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• Eukaryotic origins also control timing of DNA
replication
• The prereplication complex (pre-Rc) assembles
at replication origins
• In early G1 phase of the cell cycle, replication
origins are recognized by a six-protein complex,
the origin recognition complex (ORC), which
tags the origin as the site of initiation

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The Ends of Linear Chromosome Are
Problematic during Replication

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• Telomeres at the ends of linear chromosomes
consist of long stretches of short repeating
sequences and preserve the integrity and
stability of chromosomes

• TTAGGG (x2500)

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• Lagging strand synthesis at the end of the
chromosome is a problem
• Why?

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• Telomerase directs synthesis of the telomere
repeat sequence to fill the gap
• This enzyme is a ribonucleoprotein with an
RNA that serves as the template for the
synthesis of its DNA complement
– Reverse transcription

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• In most eukaryotic somatic cells, telomerase is
not active
• With each successive cell division, telomeres
shorten and erode, causing further cell
division to stop
• Malignant cells maintain telomerase activity
and are immortalized

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– DNA helicase
– Single-stranded binding proteins
– DNA topoisomerase
– RNA primase
– DNA polymerase
– Sliding clamp & clamp loader
– RNase
– DNA ligase
– Telomerase