DNA REPLICATION &
REPAIR
Student Edition
9/27/13
Dr. Brad Chazotte
213 Maddox Hall
chazotte@campbell.edu
Web Site: http://www.campbell.edu/faculty/chazotte
Original material only ©2000-14 B. Chazotte
Pharm. 304
Biochemistry
Fall 2014
GOALS
•Examine the mechanism of DNA replication.
•Learn what steps are involved in replicating DNA.
•Examine which enzymes/proteins are involved in DNA
replication.
•Understand how and why DNA is repaired
•Examine the similarities and differences between
prokaryotic and eukaryotic DNA replication.
•Examine repair mechanisms that insure replication
fidelity.
PROKARYOTE REPLICATION
DNA Replication
is
Semiconservative
The Meselson & Stahl
Experiment
Voet, Voet & Pratt 2013 Fig 25.1
DNA Replication by DNA Polymerase
•DNA is replicated using a
single strand as the template.
•The template strand is read in
the 3’ → 5’ direction
•The synthesis of the new
(replicated) complementary
strand by DNA polymerase 5’ PO4 group on
occurs only in the 5’ → 3’ sugar
3’ OH group of
Voet, Voet & Pratt 2013 Fig 25.2
sugar
Incoming dNTP
•The new strand forms a
double helix with the template
strand that is antiparallel.
Voet, Voet & Pratt 2013 Fig 3.3
Replication of the E. Coli
Chromosome
“θ structure”
DNA synthesis occurs at a replication fork
in the replication eye (θ structure).
There may be one fork (unidirectional
replication) or two forks (bidirectional
replication.
Found that θ replication in bacteria is
almost always bidirectional.
Voet, Voet & Pratt 2013 Fig 25.3; & 2008 Fig. 25.4
DNA Replication is Semidiscontinuous
DNA synthesis is ALWAYS 5’ →3’ direction.
Leading strand synthesis is ALWAYS 5’→3’ as well and is able
to be CONTINUOUS.
Lagging strand synthesis is STILL 5’→3’ direction but must be
DISCONTINUOUS. Lagging strand synthesis proceeds with 1K
to 2K size fragments called Okazaki fragments that are
subsequently linked together by another enzyme, DNA ligase.
Voet, Voet & Pratt 2013 Fig 25.4
DNA Replication Requires an RNA Primer
DNA polymerase requires a free 3’OH on the sugar moiety.
RNA primers synthesized complementary to template strand provide
a free 3’OH group for the DNA polymerase.
In E. Coli PRIMASE synthesizes the RNA primers 1-60 nucleotides
long (primer length is species dependent).
RNA primer is replaced by DNA later.
The leading strand needs one primer the lagging strand needs
multiple primers (for each Okazaki fragment).
Voet, Voet & Pratt 2013 Fig 24.5
Major Steps of Prokaryote Replication
Initiation
•
Find the designated replication initiation site
•
Separate the strands
•
Unwind the DNA
•
Prevent the single strands from reannealing
Synthesis (Elongation)
•
Replisome (has 2 polymerases) moves along the template strands and
synthesizes DNA. DNA Polymerase III is E. Coli’s replicase.
•
Check fidelity of replication (proofreading)
•
Joining of Okazaki fragments on the lagging strand (DNA ligase) after
removing RNA primer
Termination
Replication terminus has a number of terminator sites
Sequences in E. Coli Replication
Origin “oriC”
Enzymes need to “know” where to start replication process.
Want a start site that has familiar features “consensus” that a
protein and/or enzyme can recognize plus certain
chemical/physical properties.
Note that the sequence on the left is AT-rich.
Lehninger (Nelson & Cox) 2005 Fig 25.11
Initiation of Replication
•
•
•
•
•
•
In E. Coli replication begins at a region know as
oriC – highly conserved sequence in gram(-)
bacteria.
Multiple copies of protein DnaA bind to oriC. Cause
~45 bp AT-rich sequence (13 bp repeats) to separate
into single strands. (“melting”).
Hu histone-like protein binds and stimulates
initiation
Helicase enzyme DnaB recruited by DnaA to oriC.
One DnaB binds to each melted strand to further
separate the strands. (Helicases act to unwind DNA
by traveling along one strand of the double helix.)
Single Strand binding protein (SSB) electrostatically
binds to DNA behind the advancing helicase to
prevent reannealing of the strands.
DNA Gyrase (a topoisomerase II) relieves the stress
on DNA molecule due to helicase driven unwinding
Lehninger (Nelson & Cox) 2005 Fig 25.12
DNA Synthesis (Elongation)
Basic Concept
Involves two related, yet distinct, operations: Lagging
and Leading strand syntheses.
Accomplished via the replisome, a single multiprotein
particle, that includes TWO polymerase III enzymes
Problem? - DNA polymerase synthesis is 5’→3’ and
strands are antiparallel. How can you synthesize the
lagging strand?
Berg, Tymoczko & Stryer 2012 Fig 28.9
PRIMING for DNA Synthesis
Primosome – a protein assembly that creates a RNA primer
complementary to the template strand so DNA polymerase can
synthesize DNA.
In E. Coli the primosome includes the DnaB helicase and DnaG
an RNA-synthesizing primase.
Primosome moves in 5’→3’ direction along the LAGGING
strand, i.e. towards the replication fork, while displacing SSB.
The primosome must initiate EACH Okazaki fragment on the
lagging strand. (The leading strand needs only one primer.)
Okazaki Fragment Syntheses
the
Lagging
Strand
DNA Topoisomerase
relieves strain while
unwinding DNA. Type II
cuts both strands and then
reseals.
Helicase separates dsDNA.
Binds at initiator site and
uses energy to separate
strands
SSB keep single strands
separate
Primase synthesizes RNA
primer
DNA polymerase III synthesizes DNA
Has to add to a free 3’ OH group
Lehninger (Nelson & Cox) 2005 Fig 25.13
E. Coli DNA Polymerase I Klenow
Fragment with helical dsDNA
Polymerization
adds to
3’OH
Voet, Voet & Pratt 2013 Fig 25.9
Voet, Voet & Pratt 2006 Fig 24.10
Comparison of E. Coli DNA
Polymerases
Replaces RNA primers
Main DNA
Synthesis
Voet, Voet & Pratt 2013 Table 25.1
β-Clamp of E. Coli DNA Polymerase III
around a DNA Molecule and Processivity
Processivity: the number
of consecutive residues an
enzyme can synthesize.
The β-clamp of the
polymerase III
holoenzyme keeps the
enzyme synthesizing for
>5,000 residues versus
12 in its absence for the
Polymerase III core
enzyme.
DNA molecule
Voet, Voet & Pratt 2008 Fig 24.16b
Leading & Lagging Strand DNA
Synthesis I
helicase
Topoisomerase II
not shown
Lehninger (Nelson & Cox) 2005 Fig 25.14a
Leading & Lagging Strand
DNA Synthesis II
Lehninger (Nelson & Cox) 2005 Fig 25.14b
Leading & Lagging Strand
DNA Synthesis III
Lehninger (Nelson & Cox) 2005 Fig 25.14c
Leading & Lagging Strand
DNA Synthesis IV
Lehninger (Nelson & Cox) 2005 Fig 25.14d
Leading & Lagging Strand DNA
Synthesis V
Lehninger (Nelson & Cox) 2005 Fig 25.14e
Simplified Schematic Overview of the Overall
DNA Replication Process
•The helicase unwinds the two
parental strands
•SSB proteins prevent the parental
strands from rejoining
•The primase puts on an RNA
primer; multiple primers on the
lagging strand on the right.
•DNA polymerase IIIs on the
replicase complex synthesize the
leading and lagging strands
•DNA polymerase I on the
lagging strand removes the RNA
primer and replaces it with DNA
•DNA ligase seals the nick left by
the DNA polymerase I on the
lagging strand.
Berg, Tymoczko & Stryer 2002 Fig 27.32
E. Coli Chromosome with
Termination Sites
Replication terminus - A
350 kb region flanked by 7
nearly identical
nonpalindromic terminator
sites ~25 bp each.
Termination sites act as one
way valves – insures
bidirectional replication
forks will meet in
replication terminus
TUS protein specifically
binds to a Ter site and
prevents strand separation
by Dna B helicase
Counter clockwise
clockwise
Voet, Voet & Pratt 2013 Fig 25.19
Accurate Replication is Important
•Cells maintain a balance of dNTP’s
•Polymerase itself uses a two stage process. dNTP
complementary binding followed by polymerization
•3’→5’ polymerase (I & III) exonuclease activity detects and
removes occasional errors.
•Other cellular repair mechanisms for errors in new DNA or later
damage to DNA exist.
•Using an RNA primer that is later replaced with DNA reduces
errors that can arise when the amount of bases for cooperative
base-pairing is low.
EUKARYOTIC
REPLICATION
Eukaryotic DNA Polymerases - Properties
PCNA proliferating cell nuclear antigen – a sliding clamp protein
Polymerases and names are different in eukaryotes.
Voet, Voet & Pratt 2008 Table 25.2
Eukaryotic Initiation & Elongation
Multiple origins in eukaryotic chromosomes, but each replicon is
replicated only once during a cell cycle
Eukaryotic DNA is packaged in nucleosomes (more complex
chromosome structure) – histones disassemble immediately ahead
of replication fork then reassociate with daughter duplexes
STEPS:
•Helicase needed to prepare DNA for replication
•Separated strands are coated with protein to prevent reassociation
replication protein A (viz. SSB).
•Along with accessory proteins DNA Pol α/Primase start new strand.
•Pol δ replace Pol α and extends the strand until another replication fork
is met (no termination sequence viz. Ter).
Eukaryotic
Primer
Removal
Two enzymes involved in
primer removal:
RNase H1
Flap endonuclease-1 (FEN-1)
Voet, Voet & Pratt 2013 Fig 25.23
Avoiding a Bad End: Telomeres
and Telomerase
Problem: DNA polymerase cannot
synthesize the extreme 5’ end of
lagging strand. Could result in
shorter chromosome each
replication cycle
Solution: Special enzyme,
telomerase, to synthesize DNA at
end of chromosome, the region
called a telomere.
Telomeric DNA – 1000+ tandem repeats
of short G-rich sequence on 3’ ending
strand of each chromosome end.
Voet, Voet & Pratt 2013 Fig 25.25
Synthesizing Telomeric DNA
Telomerase – a ribonuclear protein
-Telomerase contains an RNA
sequence complementary to the
repeating DNA sequence (i.e. its own
template)
-Repeatedly translocates to the new 3’
end of the DNA – adds multiple
telomeric sequences.
-Normal cellular “machinery” for
lagging strand synthesizes the
complementary DNA strand to the
telomeric sequence leaving a 3’
overhang on the G-rich strand
Voet, Voet & Pratt 2013 Fig 25.26
Extension of Chromosome Ends
by Telomerase and Polymerase
a) Telomerase hybridizes with G-rich 3’
end of telomere strand.
b) Adds TTG (complmentary to AAC on
telomerase) to 3’ end of daughter strand
c) Then add GGGTTG sequence using
RNA template to 3’ end.
{Steps a – c can repeat many times}
d) When 3’ strand much longer a RNA
primer is synthesized by primase
complementary to G-rich strand
e) DNA pol. Uses primer to fill remaining
gap in C-rich strand
f) Primer removed leaving 12-16 nt Grich strand 3’ overhang
Weaver 2005 Fig 21.34
NEED FOR DNA REPAIR
Undesirable changes to DNA if not fixed can become part of the
permanent genome.
Mutation – a heritable alteration of the genetic information. Notable
when they occur in germ-line cells of multicellular organism
Changes result from:
•Point mutations

Transition: a purine (or a pyrimidine) replaced by
another

Transversion: a purine replaced by a pyrimidine or
vice versa
•Insertion/Deletion mutations: one or more nucleotide
pairs are inserted or deleted.
Types of Repair
•Direct Reversal
There are several enzymes that recognize and repair
several types of DNA damage – DNA photolyases for pyrimidine
dimers. Alkyltransferases for base methylation.
•Base Excision Repair (BER) – remove bases that cannot be
directly repaired, e.g. by DNA glycosylases.
•Nucleotide Excision Repair (NER) – correct pyrimidine dimers
and other DNA lesions where bases are displaced from their
normal position or have bulk substituents.
•Mismatch Repair (MMR) - correct replication mispairing and
insertions or deletions up to 4 nucleotides. Bad MMR system
then higher incidence of cancer!
DNA Repair by Base-Excision
Repair Pathway
•A glycosylase cleaves
the glycosidic bond of
corresponding type of
altered nucleotide
removing the base
•An AP endonuclease
removes the apurinic or
apyrimidinic site.
•DNA Pol I –
synthesizes replacement
DNA
•DNA ligase seals nick
Voet, Voet, &Pratt 2013 Fig 25.33
Lehninger (Nelson & Cox) 2005 Fig 25.23
Nucleotide Excision Repair of
Pyrimidine Dimers
Repair prompted by distortion
of the helix structure. Pathway
similar in all organisms.
In E. Coli.
ATP-dependent process
cleaves 11nt sequence.
Cleaved nt displaced by
UvrD (Helicase II)
Pol I and DNA ligase
repair
Voet, Voet & Pratt 2013 Fig 25.35
Mismatch Repair (MMR) - E. Coli
MutS binds to mismatched pair or
unpaired base.
MutS dimer then binds MutL.
MutS2MutL2 complex translocates
along DNA in both directions to
form DNA loop.
Parent strand distinguishable due to
existing methylation that occurs later
to new daughter strand.
MutH recruited and nicks strand.
UvrD helicase separates strands.
DNA polymerase III replaces
daughter strand sequence.
Voet, Voet & Pratt 2013 Fig 25.36
DNA Methylation
Voet, Voet & Pratt 2013 Box 25.4
End of Lectures