DNA Metabolism: Replication,
Recombination, and Repair
Chapter 28
Biochemistry
by
Reginald Garrett and Charles Grisham
Igor Chesnokov
Department of Biochemistry and Molecular Genetics
Office Phone # 934-6974
E-mail: ichesnokov@uab.edu
Garrett and Grisham, Biochemistry, Third Edition
Outline
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How Is DNA Replicated?
What Are the Properties of DNA Polymerases?
How Is DNA Replicated in Eukaryotic Cells?
How Are the Ends of Chromosomes Replicated?
How Are RNA Genomes Replicated?
How Is the Genetic Information Shuffled by Genetic
Recombination?
• Can DNA Be Repaired?
• What Is the Molecular Basis of Mutation?
Garrett and Grisham, Biochemistry, Third Edition
How Are RNA Genomes
Replicated?
• Many viruses have genomes composed of
RNA
• Can viral RNA serve as a template for
DNA synthesis?
• What enzyme could mediate such
process?
Garrett and Grisham, Biochemistry, Third Edition
Another Way to Make DNA
RNA-Directed DNA Polymerase
• 1964: Howard Temin notices that DNA
synthesis inhibitors prevent infection of cells
in culture by RNA tumor viruses. Temin
predicts that DNA is an intermediate in RNA
tumor virus replication
• 1970: Temin and David Baltimore
(separately) discover the RNA-directed DNA
polymerase - aka "reverse trascriptase"
Garrett and Grisham, Biochemistry, Third Edition
Reverse Transcriptase
• All RNA tumor viruses contain a reverse
transcriptase
• Unusual primer is required - a tRNA
molecule that the virus captures from the
host
• RT transcribes the RNA template into a
complementary DNA (cDNA) to form a
DNA:RNA hybrid
Garrett and Grisham, Biochemistry, Third Edition
Reverse Transcriptase Activities
• Three enzyme activities
– RNA-directed DNA polymerase
– RNase H activity - degrades RNA in the
DNA:RNA hybrids
– DNA-directed DNA polymerase - which
makes a DNA duplex after RNase H
activity destroys the viral genome
Garrett and Grisham, Biochemistry, Third Edition
The structures of AZT (3-azido2,3-dideoxythymidine).
This nucleoside was the first
approved drug for treatment of
AIDS. AZT is phosphorylated in
vivo to give AZTTP (AZT 5triphosphate), a substrate analog
that binds to HIV reverse
transcriptase, HIV reverse
transcriptase incorporates
AZTTP into growing DNA chains
in place of dTTP. Incorporated
AZTMP blocks further chain
elongation because its 3-azido
group cannot form a
phosphodiester bond with an
incoming nucleotide.
Host cell DNA polymerases have
little affinity for AZTTP.
Genetic recombination;
major types
• Homologous recombination involves similar
DNA sequences
• Non-homologous recombination – when very
different nucleotide sequences recombine,
occurs at low frequency
• Transposition – enzymatic insertion of
transposon (mobile segment of DNA)
• Nonhomologous recombination and
transposition play significant evolutionary role
Garrett and Grisham, Biochemistry, Third Edition
Homologous recombination
• Recombination involving similar DNA
sequences is called homologous recombination
• Homologous recombination is achieved by the
process of general recombination
• General recombination requires the breakage
and reunion of DNA strands
• The proteins responsible include RecA,
RecBCD, RuvA, RuvB, & RuvC
Garrett and Grisham, Biochemistry, Third Edition
Meselson and Weigle’s experiment
demonstrated that a physical exchange of
chromosome parts actually occurs during
recombination.
Density-labeled, “heavy” phage, (ABC),
were used to co-infect bacteria along with
”light” (abc) phage. The progeny from the
infection were collected and subjected to
CsCl density gradient centrifugation.
Parental-type heavy (ABC) and light (abc)
phage were well separated in the gradient,
but recombinant phage particles (ABc,Abc,
aBc,aBC, and so on ) were distributed
diffusely between the two parental bands
because they contained chromosomes
constituted from fragments of both
“heavy” and “light” DNA.
These recombinant chromosomes formed
by breakage and reunion of parental
“heavy” and “light” chromosomes.
Mechanism of Recombination
• Any pair of homologous DNA segments
can be used as substrates
• In 1964, Robin Holliday proposed a
model involving single-stranded nicks at
homologous sites
• Duplex unwinding, strand invasion and
ligation create a Holliday junction
Garrett and Grisham, Biochemistry, Third Edition
The Holliday model for
homologous recombination.
(A) Two homologous DNA duplexes
are aligned – synapsis.
(B) Recombination begins with the
introduction of single-stranded
nicks at homologous sites on
two chromosomes
(C) Strand invasion occurs through
partial unwinding and basepairing with the intact strand in
the other duplex
(D) Free ends from different
duplexes are ligated resulting in
cross-stranded intermediate –
Holliday junction
(E) Branches can migrate by
unwinding and rewinding of two
duplexes
The Holliday model for homologous recombination.
Branch migration (E) results in strand exchange. Another pair of
nicks must be introduced to resolve Holliday junction into two DNA
duplex molecules. Nicks take place either at E an W (- strands) or at N
and S (+ strands) resulting in “patch” or “splice” recombinant
heteroduplexes.
Garrett and Grisham, Biochemistry, Third Edition
Enzymology of Recombination
• RecBCD initiates recombination in E.coli
• RecA forms nucleoprotein filament for
strand invasion and homologous pairing
• RuvA, RuvB, RuvC drive branch migration
and help to resolve the Holliday junction
into recombination products
• Eukaryotic systems are probably similar,
homologous proteins are identified in
eukaryotes.
Garrett and Grisham, Biochemistry, Third Edition
Model of RecBCD-dependent initiation of recombination.
RecBCD consists of three subunits and has both helicase and
nuclease activities.
c site – recombinational “hotspot” (5-GCTGGTGG-3), more than 1000
in E.coli.
Garrett and Grisham, Biochemistry, Third Edition
Model of RecBCD-dependent initiation of recombination.
(a) RecBCD binds to a duplex DNA end, and its helicase activity begins to
unwind the DNA double helix. “Rabbit ears” of ssDNA loop out from
RecBCD because the rate of DNA unwinding exceeds the rate of ssDNA
release by RecBCD.
Garrett and Grisham, Biochemistry, Third Edition
Model of RecBCD-dependent initiation of recombination.
(b) As it unwinds the DNA, SSB ( and some RecA) bind to the singlestranded regions; the RecBCD endonuclease activity randomly
cleaves the ssDNA, showing a greater tendency to cut the 3’-terminal
strand rather that the 5’-terminal strand.
Garrett and Grisham, Biochemistry, Third Edition
Model of RecBCD-dependent initiation of recombination.
(c) When RecBCD encounters a properly oriented c site, the 3terminal strand is cleaved just below the 3-end of c.
Garrett and Grisham, Biochemistry, Third Edition
Model of RecBCD-dependent initiation of recombination.
(d) RecBCD now directs the binding of RecA to the 3-terminal strand, as
RecBCD endonuclease activity now acts more often on the 5-terminal strand.
(e) A nucleoprotein filament consisting of RecA-coated 3’-strand ssDNA is
formed. This nucleoprotein filament is capable of homologous pairing with a
dsDNA and strand invasion.
Garrett and Grisham, Biochemistry, Third Edition
The RecA Protein –
recombinase.
• 38 kD enzyme that catalyzes ATPdependent DNA strand exchange,
leading to formation of Holliday junction
• RecA forms a helical filament with a
groove to accommodate DNA
• RecA:ssDNA complex binds dsDNA at
secondary site and searches for regions
homologous with the bound ssDNA,
then forms the desired duplex
Garrett and Grisham, Biochemistry, Third Edition
The structure of
RecA, a 352residue, 38-kD
protein.
(a) Ribbon diagram of
the RecA monomer.
Note the ADP bound
at the site near
helices C and D.
(b) (b) RecA filament.
Four turns of a helical
filament that has six
RecA monomers per
turn. A RecA
monomer is
highlighted in red.
RecA filament can
bind multiple DNA
strands!
Model for homologous
recombination as
promoted by RecA
enzyme.
(a) RecA protein (and SSB)
aid strand invasion of the
3’-ssDNA into a
homologous DNA duplex,
(b) forming a D-loop.
(c) The D-loop strand, that
has been displaced by
strand invasion, pairs
with its complementary
strand in the original
duplex to form a Holiday
junction as strand
invasion continues.
Resolving Holliday Junctions
• Ruv proteins resolve the junction into
recombination products
• RuvA and RuvB act as a helicase that
dissociates the RecA filament and
catalyzes branch migration
• RuvC is an endonuclease that binds at the
junction and cuts pairs of DNA strands of
similar polarity. Both splice and patch
recombinants can be produced.
Garrett and Grisham, Biochemistry, Third Edition
Model for resolving Holliday junction.
(left) RuvA tetramer fits snugly within the Holliday junction point.
(center) RuvB hexameric rings assemble on opposite sides of DNA
heteroduplexes and act as motors to promote branch migration by driving
the passage of the DNA duplexes through themselves.
(right) RuvC resolvase binds to the Holliday junction and cuts it by its
nuclease activity.
Knockout Mice: A Method to Investigate the Essentiality of a Gene
based on homologous recombination.
Garrett and Grisham, Biochemistry, Third Edition
Transposons
• In 1950, Barbara McClintock showed
that activator genes in corn could move
freely about the genome.
• This was at first viewed as heresy
• Molecular biologists in the late 1970s
confirmed what McClintock discovered
• She received a MacArthur Award in
1981 and a Nobel Prize in 1983
Garrett and Grisham, Biochemistry, Third Edition
The typical transposon has
inverted nucleotide-sequence
repeats at its termini,
represented here as the 12-bp
sequence ACGTACGTACGT
(a). Transposon acts at a
target sequence (shown here
as the sequence CATGC)
within host DNA by creating
a staggered cut (b) whose
protruding single-stranded
ends are then ligated to the
transposon (c). The gaps at
the target site are then filled
in, and the filled-in strands
are ligated (d). Transposon
insertion thus generates
direct repeats of the target
site in the host DNA, and
these direct repeats flank the
inserted transposon.
Can DNA Be Repaired?
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A fundamental difference from RNA, protein or lipid
All the others can be replaced, but DNA must be
preserved
Cells require a means for repair of missing, altered
or incorrect bases, bulges due to insertion or
deletion, UV-induced pyrimidine dimers, strand
breaks or cross-links
The human genome has about 150 genes
associated with DNA repair
DNA repair systems include: direct reversal
damage repair, single-strand damage repair, double
strand break repair, and translesion DNA synthesis
Garrett and Grisham, Biochemistry, Third Edition
DNA repair systems
• Double-strand breaks (DSBs) are a particular threat
to genome stability, because lost sequence
information cannot be recovered from the same DNA
• Chemical reactions that reverse the damage,
returning DNA to its proper state, are direct reversal
repair systems.
• Single-strand damage repair relies on the intact
complementary strand to guide repair
• Systems repairing single-strand breaks include:
– Mismatch repair (MMR)
– Base excision repair (BER)
– Nucleotide excision repair (NER)
Garrett and Grisham, Biochemistry, Third Edition
Can DNA Be Repaired?
DSB repair through
nonhomologous DNA end
joining (NHEJ). Ku70/80 binds
the ends and recruits a set of
proteins that juxtaposes the
broken ends. Processing of the
ends to generate proper
substrates for DNA ligase IV
then occurs, followed by DNAligase-mediated end joining.
Double-strand breaks that arise
during the S phase of the cell
cycle can be repaired through
homologous recombination.
Garrett and Grisham, Biochemistry, Third Edition
Can DNA Be Repaired?
DSB repair through homologous DNA
recombination. The orange-red pair
of lines symbolizes the doublestranded DNA with a DSB; the blackblue pair represents the sister
chromatid. Homologous
recombination creates a D-loop (c),
and sister chromatid-directed DNA
replication restores the information
content of the damaged duplex (d-f).
Depending on how the Holliday
junctions are resolved, the products
(g) are either (left) non-crossover or
(right) crossover recombinants.
Garrett and Grisham, Biochemistry, Third Edition
Can DNA Be Repaired?
Restarting a stalled replication fork through
homologous DNA recombination. A lesion
in the DNA is symbolized by a circle; in this
case, the lesion is in the leading-strand
template (a). Leading-strand synthesis
halts because of the lesion (b). Laggingstrand synthesis (red) continues, and the
Okazaki fragments are ligated (c). When
the leading strand invades the new DNA
duplex formed by lagging-strand synthesis,
a D-loop is formed and strand exchange
occurs. Using the lagging strand as a
template, synthesis of the leading strand
(black) resumes (d), and the replication fork
is reestablished (e).
Garrett and Grisham, Biochemistry, Third Edition
Mismatch Repair corrects errors
introduced during DNA
replication
• Mismatch repair systems scan DNA duplexes
for mismatched bases, excise the mispaired
region and replace it
• Example is a Methyl-directed pathway of E. coli
• Since methylation occurs post-replication,
repair proteins (MutS, MutH, MutL) identify
methylated strand as parent, remove
mismatched bases on the other strand and
replace them
• http://en.wikipedia.org/wiki/Mismatch_repair
Garrett and Grisham, Biochemistry, Third Edition
Reversing Chemical Damage
(excision repair)
• Pyrimidine dimers can be repaired by
photolyase
• Excision repair: DNA glycosylase removes
damaged base, creating an "AP site"
• AP endonuclease cleaves backbone,
exonuclease removes several residues
and gap is repaired by DNA polymerase
and DNA ligase
Garrett and Grisham, Biochemistry, Third Edition
UV irradiation causes dimerization of adjacent thymine bases. A cyclobutyl
ring is formed between carbons 5 and 6 of the pyrimidine rings. Normal
base pairing is disrupted by the presence of such dimers. Photolyase can
break cyclobutyl ring.
Base excision repair.
A damaged base (■) is excised from the
sugar-phosphate backbone by DNA
glycosylase, creating an apurinic acid
(AP) site. Then, an
apurinic/apyrimidinic endonuclease
severs the DNA strand, and an excision
nuclease removes the AP site and several
nucleotides. DNA polymerase I and
DNA ligase then repair the gap.
What Is the Molecular Basis of
Mutation?
• Point mutations arise by inappropriate
base-pairing
• Mutations can be caused by base analogs
• Chemical mutagens react with bases in
DNA
• Insertions and deletions result in frameshift
mutations
Garrett and Grisham, Biochemistry, Third Edition
Types of mutations
Large chromosomal deletions
Translocations (chromosome segments
swapped)
Point mutations:
-Transition - Pu-Pu or Py-Py
-Transversion - Py-Pu or Pu-Py
-Mis-sense
1 base for another
-Frameshift
Alters the reading frame of the gene
must be in protein coding region
insertion or deletion
Garrett and Grisham, Biochemistry, Third Edition
Examples of point mutations due to base mispairings in unusual circumstances. (A) The
rare imino tautomer of adenine base pairs cytosine rather than thymine. (1) The normal
A-T base pair. (2) The A*-C base pair is possible for the adenine tautomer in which a
proton has been transferred from the 6-NH2 of adenine to N-1. (3) Pairing of C with the
imino tautomer of A (A*) leads to a transition mutation (A-T to G-C) appearing in the
next generation. (B) A in the syn conformation pairing with G (G is in the usual anti
conformation). (C) T and C form a base pair by H-bonding interactions mediated by a
water molecule.
(a) 2-Aminopurine
(adenine analog)
normally base-pairs with
T but (b) may also pair
with cytosine through a
single hydrogen bond.
Another example of
base analogs.
Oxidative deamination
of adenine in DNA
yields hypoxanthine,
which base-pairs with
cytosine, resulting in
an A-T to G-C
transition.
Examples of Chemical mutagens. (a)
HNO2 (nitrous acid) converts cytosine to
uracil and adenine to hypoxanthine. (b)
Nitrosoamines, organic compounds that
react to form nitrous acid, also lead to the
oxidative deamination of A and C. (c)
Hydroxylamine (NH2OH) reacts with
cytosine, converting it to a derivative that
base-pairs with adenine instead of
guanine. The result is a C-G to T-A
transition. (d) Alkylation of G residues to
give methylguanine, which base-pairs with
T. (e) Alkylating agents include
nitrosoamines, nitrosoguanidines,
nitrosoureas, alkyl sulfates, and nitrogen
mustards.
Note that nitrosoamines are mutagenic in
two ways: They can react to yield HNO2,
or they can act as alkylating agents. The
nitrosoguanidine, is a very potent mutagen
used in laboratories to induce mutations in
experimental organisms such as
Drosophila melanogaster. Ethylmethane
sulfonate (EMS) and dimethyl sulfate are
also favorite mutagens among geneticists.
Example of a frameshift
met phe gln gln
phe
ATG TTT CAG CAA TTT
met val ser ala
ile
ATG GTT TCA GCA ATT T
Garrett and Grisham, Biochemistry, Third Edition
Diseases of DNA repair
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Ataxia-Telangiectasia
Bloom Syndrome
Cockayne Syndrome
Fanconi Anemia
Xeroderma Pigmentosum
Hereditary non-polyposis colorectal
cancer
http://www.dnalc.org/resources/3d/index.html
Garrett and Grisham, Biochemistry, Third Edition
Review
• Nucleotides and Nucleic Acids
- Structure and functions of nucleotides
- Important discoveries which helped
solving DNA structure
- Major features of the DNA double helix
Garrett and Grisham, Biochemistry, Third Edition
Review
• Structure of DNA
- ABZs of DNA structure
- Primary,
- Secondary and
- Tertiary structure of DNA
Garrett and Grisham, Biochemistry, Third Edition
Review
• DNA replication
- Major features of DNA replication
- Enzymology of DNA replication
- Eukaryotic DNA replication
Garrett and Grisham, Biochemistry, Third Edition
Review
• DNA recombination and repair
- Enzymology of DNA recombination
- Major types of DNA recombination and
repair
- Molecular basis of mutations
Garrett and Grisham, Biochemistry, Third Edition