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9 views

replication

Lec

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ain't your saintess
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© © All Rights Reserved
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Biosynthesis of Nucleic Acids:

Replication

Feb. 25, 2016

CHEM 281
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
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
 protection 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
Prokaryotic Replication (Cont’d)
 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
Evidence for Semiconservative Replication
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
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.
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
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
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
Replication Fork General Features
Summary of DNA Replication in Prokaryotes
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

 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
DNA Polymerase Repair
DNA Polymerase Repair
DNA Polymerase Repair
DNA Polymerase Repair
Mismatch Repair in Prokaryotes
Why T and not U?
DNA Recombination
 Genetic Recombination- When genetic information is
rearranged to form new associations

 Homologous - Reactions between homologous


sequences

 Nonhomologous- Different nucleotide sequences


recombine
Recombination
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
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
().
The Eukaryotic Replication Fork
 The general features of DNA replication in eukaryotes are similar to
those in prokaryotes. Differences summarized in Table 10.5.
The Eukaryotic Replication Fork
Telomerase and Cancer
 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


Telomerase and Cancer
Telomerase and Cancer

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