Mary K. Campbell
Shawn O. Farrell
http://academic.cengage.com/chemistry/campbell
Chapter Ten
Biosynthesis of Nucleic Acids: Replication
Paul D. Adams • University of Arkansas
Replication of DNA
• Naturally occurring DNA exists in single-stranded
and double-stranded forms, both of which can exist
in ________ and ________ forms
• Difficult to generalize about all cases of DNA
replication
• We will study the replication of ________ ________
________ DNA and then of ________ ________
________ DNA
• most of the details we discuss were first investigated
in ________, particularly ________ ________
Flow of Genetic Information in the Cell
• Mechanisms by which information is transferred in
the cell is based on the “________ ________”
Prokaryotic Replication
• Challenges in duplication of ________________
double-stranded DNA
• achievement of continuous ________ and
separation of the two DNA strands
• ________ of ________ portions from attack by
________ that attack single-stranded DNA
• synthesis of the DNA ________ from one 5’ -> 3’
strand and one 3’ -> 5’ strand
• efficient protection from ________ in replication
Prokaryotic Replication (Cont’d)
• Replication involves ________ of
the two original strands and
________ of two new daughter
strands using the ________
strands as ________
• _________________ replication:
each daughter strand contains one
template strand and one newly
synthesized strand
• Incorporation of isotopic label as
sole _________ source (15NH4Cl)
• Observed that 15N-DNA has a
higher density than 14N-DNA, and
the two can be separated by
density-gradient ______________
Which Direction does Replication go?
• DNA double helix unwinds at a specific point called an
______________________________
• Polynucleotide chains are synthesized in both
directions from the origin of replication; DNA
replication is _______________ in most organisms
• At each origin of replication, there are two _________
_____________, points at which new polynucleotide
chains are formed
• There is ______ origin of replication and ________
replication forks in the circular DNA of prokaryotes
• In replication of a _______________ chromosome,
there are several origins of replication and two
replication forks at each origin
DNA Polymerase
• DNA is synthesized from its 5’ -> 3’ end (from the 3’ -> 5’
direction of the template)
• the ________ strand is synthesized continuously
in the 5’ -> 3’ direction toward the replication fork
• the ________ strand is synthesized __________
(________ fragments) also in the 5’ -> 3’ direction,
but away from the replication fork
• lagging strand fragments are joined by the
enzyme DNA ___________
DNA Polymerase
DNA Polymerase Reaction
• The 3’-OH group at
the end of the growing
DNA chain acts as a
_______________.
• The ______________
adjacent to the sugar
is attacked, and then
added to the growing
chain.
Properties of DNA Polymerases
• There are at least _______ types of DNA polymerase (Pol)
in E coli, _______ of which have been studied extensively
Function of DNA Polymerase
• DNA polymerase function has these requirements:
• all four deoxyribonucleoside triphosphates: dTTP,
dATP, dGTP, and dCTP
• Mg2+
• an _________ - 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 & repair enzymes
Supercoiling and Replication
• DNA _________ (class II
topoisomerase)
catalyzes rxn involving
relaxed circular DNA:
• creates a _______ in
relaxed circular DNA
• a slight unwinding at
the point of the nick
introduces _______
• the nick is _______
• The energy required for
this process is supplied
by
____________________
Replication with Supercoiled DNA
• Replication of supercoiled circular DNA
• DNA gyrase has different role here. It introduces a
nick in supercoiled DNA
• a _______ point is created at the site of the _______
• the gyrase opens and _______ the swivel point in
advance of the replication fork
• the newly synthesized DNA automatically assumes
the ___________ form because it does not have the
nick at the swivel point
• _______, a helix-destabilizing protein, promotes
unwinding by binding at the replication fork
• single-stranded binding (SSB) protein _______
single-stranded regions by _______ tightly to them
Primase Reaction
• The primase reaction
• RNA serves as a _______ in DNA replication
• _______ activity first observed in-vivo.
• Primase - catalyzes the copying of a short stretch of
the DNA template strand to produce RNA primer
sequence
• __________ and _________ 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
DNA Replication in Prokaryotes
• DNA synthesis is bidirectional
• DNA synthesis is in the 5’ -> 3’ direction
• the leading strand is formed continuously
• the lagging strand is formed as a series of Okazaki
fragments which are later joined
• Five DNA polymerases have been found to exist in E. coli
• Pol I is involved in synthesis and repair
• Pol II, IV, and V are for repair under unique conditions
• Pol III is primarily responsible for new synthesis
DNA Replication in Prokaryotes
• Unwinding
• DNA gyrase introduces a swivel point in advance of the
replication fork
• a helicase binds at the replication fork and promotes
unwinding
• single-stranded binding (SSB) protein protects exposed
regions of single-stranded DNA
• Primase catalyzes the synthesis of RNA primer
• Synthesis
• catalyzed by Pol III
• primer removed by Pol I
• DNA ligase seals remaining nicks
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
• ____________________: catalyzed by Pol I: cutting
is removal of the RNA primer and patching is
incorporation of the required deoxynucleotides
• ______________________: Pol I removes RNA primer
or DNA mistakes as it moves along the DNA and then
fills in behind it with its polymerase activity
• ______________________: enzymes recognize
that two bases are incorrectly paired, the area of
mismatch is removed, and the area replicated again
• ______________________: 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
Mismatch Repair in Prokaryotes
• Mechanisms of mismatch repair encompass:
Eukaryotic Replication
• Not as understood as
prokaryotic. Due in
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.
Telomerase and Cancer (Biochemical Connections)
• Replication of linear DNA molecules poses particular
problems at __________________ of the molecules
• Ends of eukaryotic chromosomes called
______________________:
__________________________________________
• See figures on p. 282-283