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Chapter 28
DNA Metabolism: Replication,
Recombination, and Repair
Biochemistry
by
Reginald Garrett and Charles Grisham
Garrett and Grisham, Biochemistry, Third Edition
Essential Question
• How is this genetic information in the form of
DNA replicated, how is the information
rearranged, and how is its integrity maintained
in the face of damage?
Garrett and Grisham, Biochemistry, Third Edition
Outline
•
•
•
•
•
•
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?
Special Focus: Gene Rearrangements and
Immunology – Is It Possible to Generate Protein
Diversity Using Genetic Recombination?
Garrett and Grisham, Biochemistry, Third Edition
28.1 – How Is DNA Replicated?
• DNA replication is semiconservative
• DNA replication is bidirectional
• Replication requires unwinding of the DNA
helix
• DNA replication is semidiscontinuous
• The lagging strand is formed from Okazaki
fragments
Garrett and Grisham, Biochemistry, Third Edition
The Dawn of Molecular Biology
April 25, 1953
• Watson and Crick: "It has not escaped our
notice that the specific (base) pairing we have
postulated immediately suggests a possible
copying mechanism for the genetic material."
• The mechanism: Strand separation, followed
by copying of each strand.
• Each separated strand acts as a template for
the synthesis of a new complementary strand.
Garrett and Grisham, Biochemistry, Third Edition
Figure 28.1
Watson and Crick’s
famous paper, in its
entirety. (Reprinted
with permission from
Watson,J.D., and
Crick,F.H.C., Molecular
structure of nucleic acid,
1953. Nature 171:737738. Copyright 1953
Macmillan Publishers
Ltd.)
Figure 28.2
Untwisting of DNA strands exposes their
bases for hydrogen bonding. Base
pairing ensures that appropriate
nucleotides are inserted in the correct
positions as the new complementary
strands are synthesized. By this
mechanism, the nucleotide sequence of
one strand dictates a complementary
sequence in its daughter strand. The
original strands untwist by rotating about
the axis of the unreplicated DNA double
helix.
DNA Replication
The Semiconservative Model
• Matthew Meselson and Franklin Stahl showed
that DNA replication results in new DNA duplex
molecules in which one strand is from the
parent duplex and the other is completely new
• Study Figure 30.4 and understand the density
profiles from ultracentrifugation experiments
• Imagine and predict the density profiles that the
conservative and dispersive models would
show
Garrett and Grisham, Biochemistry, Third Edition
Figure 28.3
Three models of DNA replication prompted by Watson and Crick’s double helix structure of
DNA. (a) Conservative: Each strand of the DNA duplex is replicated, and the two newly
synthesized strands join to form one DNA double helix while the two parental strands remain
associated with each other. The products are completely new DNA duplex and the original DNA
duplex. (b) Semiconservative: The two strands separate, and each strand is copied to generate
a complementary strand. Each parental strand remins associated with its newly synthesized
complement, so
each DNA duplex
contains one
parental strand
and one new
strand. (c)
Dispersive: This
model predicts that
each of the four
strands in the two
daughter DNA
duplexes contains
both newly
synthesized
segments and
segments derived
from the parental
strands.
Figure 28.4
The Meselson and Stahl experiment
demonstrating that DNA replication is
semi-conservative. On the left are
densitometric traces made of UV
absorption photographs taken of the
ultracentrifugation cells containing DNA
isolated from E. coli grown for various
generation time after 15N-labeling. The
photographs were taken once the
migration of the DNA in the density
gradient had reached equilibrium.
Density increases from left to right. The
peaks reveal the positions of the
banded DNA with respect to the density
of the solution. The number of
generations that the E. coli cells were
grown (following 14 generations of 15N
density-labeling) is shown down the
middle. A schematic representation
interpreting the pattern expected of
semiconservative replication is shown
on the right side of this figure. (Adapted
from Meselson,M., and Stahl,F.W., 1958. The
replication of DNA. Proeceedings of the
National Academy of Sciences, U.S.A.
44:671-682.)
Features of DNA Replication
• DNA replication is bidirectional
– Bidirectional replication involves two
replication forks, which move in opposite
directions
• DNA replication is semidiscontinuous
– The leading strand copies continuously
– The lagging strand copies in segments
(Okazaki fragments) which must be joined
Garrett and Grisham, Biochemistry, Third Edition
Figure 28.5
Bidirectional
replication of the
E. coli
chromosome. (a)
If replication is
bidirectional, autoradiograms of
radioactively
labeled replicating
chromosomes
should show two
replication forks
heavily labeled
with radioactive
thymidine. (b) An
autoradiogram of
the chromosome
from a dividing E.
coli cell shows
bidirectional
replication. (Photo
courtesy of David M.
Prescott, University
of Colorado.)
The Enzymology
of DNA Replication
• If Watson and Crick were right, then there
should be an enzyme that makes DNA
copies from a DNA template
• In 1957, Arthur Kornberg and colleagues
demonstrated the existence of a DNA
polymerase - DNA polymerase I
• Pol I needs all four deoxynucleotides, a
template and a primer - a ss-DNA (with a
free 3'-OH) that pairs with the template to
form a short double-stranded region
Garrett and Grisham, Biochemistry, Third Edition
28.2 – What Are the Properties of
DNA Polymerases?
• E. coli cells have several DNA polymerases
• E. coli DNA polymerase I has three active sites
• E. coli DNA polymerase I is its own proofreader
and editor
• E. coli DNA polymerase III holoenzyme
replicates the E. coli chromosome
• A DNA polymerase III holoenzyme sits at each
replication fork
Garrett and Grisham, Biochemistry, Third Edition
DNA Polymerase I
•
•
•
•
•
Replication occurs 5' to 3'
Nucleotides are added at the 3'-end of the
strand
Pol I catalyzes about 20 cycles of
polymerization before the new strand
dissociates from template
20 cycles constitutes moderate "processivity"
Pol I from E. coli is 928 aa (109 kD) monomer
In addition to 5'-3' polymerase, it also has 3'-5'
exonuclease and 5'-3' exonuclease activities
Garrett and Grisham, Biochemistry, Third Edition
Figure 28.6 The semidiscontinuous model for DNA replication. Newly synthesized DNA is shown
as red. Because DNA polymerases only polymerize nucleotides 5  3, both strands must be
synthesized in the 5  3 direction. Thus, the copy of the parental 3  5 strand is
synthesized continuously; this newly made strand is designated the leading strand. (a) As the helix
unwinds, the other parental strand ( the 5 3, strand) is copied in a discontinuous fashion
through synthesis of a series of fragments 1000 to 2000 nucleotides in length, called the Okazaki
fragments;
the strand constructed from the
Okazaki fragments is called the
lagging strands. (b) Because both
strands are synthesized in
concert by a dimeric DNA
polymerase situated at the
replication fork, the 5 3
parental strand must warp around
in trombone fashion so that the
unit of the dimeric DNA
polymerase replicating it can
move along it in the 3 5
direction. This parental strand is
copied in a discontinuous fashion
because the DNA polymerase
must occasionally dissociate from
this strand and rejoin it further
along. The Okazaki fragments
are then covalently joined by DNA
ligase to form an uninterrupted
DNA strand.
More on Pol I
Why the exonuclease activity?
• The 3'-5' exonuclease activity serves a
proofreading function! It removes
incorrectly matched bases, so that the
polymerase can try again
• See Figures 30.9 and 30.10! Notice how
the newly-formed strand oscillates
between the polymerase and 3'exonuclease sites,adding a base and then
checking it
Garrett and Grisham, Biochemistry, Third Edition
Figure 28.7
The chain elongation
reaction catalyzed by DNA
polymerase. DNA
polymerase I joins
deoxynucleoside
monophosphate units to
the 3´-OH end of the
primer, employing dNTPs
as substrates. The 3´-OH
carries out a nucleophilic
attack on the a-phosphoryl
group of the incoming
dNTP to form a
phosphoester bond, and
PPi is released. The
subsequent hydrolysis of
PPi by pyrophosphatase
renders the reaction
effectively irreversible.
Figure 28.8
The 3´5´ exonuclease activity of DNA polymerase I removes nucleotides from the 3’-end of
the growing DNA chain.
Even More on Pol I
Nicks and Klenows....
• 5'-exonuclease activity, working together
with the polymerase, accomplishes "nick
translation"
• Hans Klenow used either subtilisin or
trypsin to cleave between residues 323
and 324, separating 5'-exonuclease (on 1323) and the other two activities (on 324928, the so-called "Klenow fragment”)
• Tom Steitz has determined the structure of
the Klenow fragment - see Figure 30.9
Garrett and Grisham, Biochemistry, Third Edition
Figure 28.9
(a) Ribbon diagram of the b-subunit dimer of the DNA polymerase III holoenzyme on B-DNA.,
viewed down the axis of the DNA. One monomer of the b-subunit dimer is colored red and the
other yellow. The centrally located DNA is mostly blue. (b) Space-filling model of the b-subunit
dimer of the DNA polymerase III holoenzyme on B-DNA. One monomer is shown in red, the
other in yellow. The B-DNA has one strand colored white and the other blue. The hole formed
by the b-subunits (diameter 3.5 nm) is large enough to easily accommodate DNA (diameter
2.5nm) with no steric repulsion. The rest of polymerase III holoenzyme (“core” polymerase +
g-complex) associates with this sliding clamp to form the replicative polymerase (not shown).
(Adapted from Kong,X-P., et al, 1992.Three-dimensional structure of the beta subunit of E.coli DNA
polymerase III holoenzyme: a sliding DNA clamp. Cell 69:425-437; photos courtesy of John Kuriyan of the
Rockefeller University)
Features of Replication
•
•
•
•
•
•
Mostly in E. coli, but many features are general
Replication is bidirectional
The double helix must be unwound - by
helicases
Supercoiling must be compensated - by DNA
gyrase
Replication is semidiscontinuous
Leading strand is formed continuously
Lagging strand is formed from Okazaki
fragments - discovered by Tuneko and Reiji "O"
Garrett and Grisham, Biochemistry, Third Edition
More Features of Replication
• Read page 994 on chemistry of DNA
synthesis
• DNA Pol III uses an RNA primer
• A special primase forms the required primer
• DNA Pol I excises the primer
• DNA ligase seals the "nicks" between Okazaki
fragments (See Figure 30.14 for mechanism)
• See Figure 30.15 for a view of replication fork
Garrett and Grisham, Biochemistry, Third Edition
Figure 28.10
General features of a replication fork. The DNA duplex is unwound by the action of DNA
gyrase and helicase, and the single strands are coated with SSB (ssDNA-binding protein).
Primase periodically primes synthesis on the lagging strand. Each half of the dimeric
replicative polymerase is a “core” polymerase bound to its template strand by a b-subunit
sliding clamp. DNA polymerase I and DNA ligase act downstream on the lagging strand to
remove RNA primers, replace them with DNA, and ligate the Okazaki fragments.
DNA Polymerase III
•
•
•
•
•
•
•
The "real" polymerase in E. coli
At least 10 different subunits
"Core" enzyme has three subunits - a, , and 
Alpha subunit is polymerase
Epsilon subunit is 3'-exonuclease
Theta subunit is involved in holoenzyme
assembly
The beta subunit dimer forms a ring around
DNA
Enormous processivity - 5 million bases!
Garrett and Grisham, Biochemistry, Third Edition
28.3 – How Is DNA Replicated in
Eukaryotic Cells?
• The cell cycle controls the timing of DNA
replication
• Eukaryotic cells contain a number of
different DNA polymerases
• DNA polymerase d is the principal DNA
replicase
Garrett and Grisham, Biochemistry, Third Edition
Figure 28.11
A replication factory
“fixed” to a cellular
substructure extrudes
loops of newly
synthesized DNA as
parental DNA duplex is
fed in from the sides.
Parental DNA strands are
blue; newly synthesized
strands are green; small
circles indicate origins of
replication. (Adapted from
Cook,P.R., 1999. The
organization of replication
and transcription. Science
284:1790-1795.)
Figure 28.12
The eukaryotic cell cycle.
The stages of mitosis and
cell division define the M
phase (M for mitosis). G1
(G for gap, not growth) is
typically the longest part of
the cell cycle; G1, is
characterized by rapid
growth and metabolic
activity. Cells that are
quiescent, that is, not
growing and dividing (such
as neurons), are said to be
in G0. The S phase is the
time of DNA synthesis. S
is followed by G2, a
relatively short period of
growth when the cell
prepares for cell division.
Cell cycle times vary from
less than 24 hours (rapidly
dividing cell such as the
epithelial cells lining the
mouth and gut) to
hundreds of days.
28.4 – How Are the Ends of
Chromosomes Replicated?
• Telomeres, the structures at the ends of
eukaryotic chromosomes, consist of 5-8 bp
tandemly repeated G-rich nucleotide
sequences
• Telomeres are 1-12 kbp long
• Telomeres are replicated by an RNAdependent DNA polymerase called
telomerase
Garrett and Grisham, Biochemistry, Third Edition
Eukaryotic DNA Replication
•
•
•
•
•
Like E. coli, but more complex
Human cell: 6 billion base pairs of DNA to
copy
Multiple origins of replication: 1 per 3- 300 kbp
Several known animal DNA polymerases see Table 30.4
DNA polymerase alpha - four subunits,
polymerase (processivity = 200) but no 3'exonuclease
DNA polymerase beta - similar to alpha
Garrett and Grisham, Biochemistry, Third Edition
Figure 28.13
Model for initiation of the
DNA replication cycle in
eukaryotes. ORC is
present at the replicators
throughout the cell cycle.
The pre-replication
complex (pre-RC) is
assembled through the
sequential addition of
Cdc6p and MCM proteins
during a window of
opportunity defined by
the state of the cyclinCDKs. After initiation, a
post-RC state is
established. (Adapted from
Figure 2 in Stillman,B., 1996.
Cell cycle control of DNA
replication. Science
274:1659-1663.)
Mechanism of Replication
in E. coli
• The replisome consists of: DNA-unwinding
proteins, the priming complex (primosome)
and two equivalents of DNA polymerase III
holoenzyme
• Initiation: DnaA protein binds to repeats in
ori, initiating strand separation and DnaB,
a helicase delivered by DnaC, further
unwinds the DNA. Primase then binds and
constructs the RNA primer
Garrett and Grisham, Biochemistry, Third Edition
Figure 28.14
Structure of the PCNA homotrimer. Note that the trimeric PCNA ring of eukaryotes is
remarkably similar to its prokaryotic counterpart, the dimeric b2-sliding clamp (Figure 28.9).
(a) Ribbon representation of the PCNA trimer with an axial view of a B-form DNA duplex in its
center. The molecular mass of each PCNA monomer is 37 kD. (b) Molecular surface of the
PCNA trimer with each monomer colored differently. The red spiral represents the sugarphosphate backbone of a strand of B-form DNA. (Adapted from Figure 3 in Krishna,T.S., et at.,
1994. Crystal structure of the eukaryotic DNA polymerase processivity factor PCNA. Cell 79:1233-1243.
Photos courtesy of John Kuriyan of the Rockefeller University.)
Replication Mechanism II
Elongation and Termination
• Elongation involves DnaB helicase unwinding,
SSB binding to keep strands separated, and
DNA polymerase replicating each strand
• Termination: the "ter" locus, rich in Gs and Ts,
signals the end of replication. A Ter protein is
also involved. Ter protein is a contrahelicase
and prevents unwinding
• Topoisomerase II (DNA gyrase) relieves
supercoiling that remains
Garrett and Grisham, Biochemistry, Third Edition
Figure 28.15
Telomere replication. (a) In replication of the lagging strand, short RNA primers are added
(pink) and extended by DNA polymerase. When the RNA prirmer at the 5-end of each strand
is removed, there is no nucleotide sequence to read in the next round of DNA replication.
The result is a gap (primer gap) at the 5-end of each strand (only one end of the chromosome
is shown in this figure). (b) Asterisks indicate sequences at the 3-end that cannot be copied
by conventional DNA replication. Synthesis of telomeric DNA by telomerase extends the 5ends of DNA strands, allowing the strands to be copied by normal DNA replication.
28.5 – How Are RNA Genomes
Replicated?
• Many viruses have genomes composed of
RNA
• DNA is an intermediate in the replication of
RNA viruses
• The viral RNA serves as a template for
DNA synthesis
• The RNA-directed DNA polymerase is
called Reverse Transcriptase
Garrett and Grisham, Biochemistry, Third Edition
Figure 28.16
The structures of AZT (3-azido-2,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.
28.6 – How Is the Genetic Information
Shuffled by Genetic Recombination?
• Genetic recombination rearranges genetic
information, creating new associations
• 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
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
• Primer required, but a strange one - 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
• All RNA tumor viruses contain a reverse
transcriptase
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
• HIV therapy: AZT (or 3'-azido-2',3'dideoxythymidine) specifically inhibits RT
Garrett and Grisham, Biochemistry, Third Edition
More Eukaryotic polymerases
• DNA polymerase gamma - DNA-replicating
enzyme of mitochondria
• DNA polymerase delta has a 3'exonuclease as well as proliferating cell
nuclear antigen (PCNA)
• PCNA give delta unlimited processivity and
is homologous with prokaryotic pol III
• DNA polymerase epsilon - highly
processive, but does not have a subunit
like PCNA
Garrett and Grisham, Biochemistry, Third Edition
Figure 28.17
Meselson and Weigle’s experiment
demonstrated that a physical exchange
of chromosome parts actually occurs
during recombination. Density-labeled,
“heavy” phage, symbolized as ABC
phage in the diagram, were used to
coinfect bacteria along with ”light” phage,
the abc phage. The progeny from the
infection were collected and subjected to
CsCl density gradient centrifugation.
Parental-type ABC and abc phage were
well separated in the gradient, but
recombinant phage (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.
Figure 28.18
The generation of progeny bacteriophage of two different genotypes from a single phage
particle carrying a heteroduplex DNA region within its chromosome. The heteroduplex DNA is
composed of one strand that is genotypically XYZ (the + strand), and the other strand that is
genotypically XyZ (the - strand). That is, the genotype of the two parental strands for gene Y
is different (one is Y, the other y).
Figure 28.19
The Holliday model for
homologous
recombination. The +
signs and – signs label
strands of like polarity.
For example, assume
that the two strands
running 5´3´ as read left
to right are labeled +; and
the two strands running
3´5´ as read left to right
are labeled-. Only
strands of like polarity
exchange DNA during
recombination. (See text
for detailed description.)
Figure 28.20
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. (b)
As it unwinds the DNA, SSB ( and some RecA) bind
to the single-stranded 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. (c) When
RecBCD encounters a properly oriented c site, the
3-terminal strand is cleaved just below the 3-end of
c. (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. (Adapted
from Figure 2 in Eggleston,A.D., and West,S.C., 1996.
Exchanging partners: recombination in E.coli. Trends in
Genetics 12:20-25; and Figure 3 in Eggleson,A.K. and
West,S.C., 1997. Recombination initiation: Easy as A,B,C,D
….c? Current Biology 7:R745--R749.)
Figure 28.21
The structure of RecA, a
352-residue, 38-kD protein.
(a) Ribbon diagram of the
RecA monomer. Note the
ADP bound at the site near
helices C and D. (b) RecA
filament. Four turns of a
helical filament that has six
RecA monomers per turn. A
RecA monomer is
highlighted in red. (Adapted
from Figures 2 and 3 in Roca,
A.I., and Cox,M.M., 1997. RecA
protein: Structure, function, and
role in recombinational DNA
repair. Progress in Nucleic Acid
Research and Molecular Biology
56:127-223. Photos courtesy of
Michael M. Cox, University of
Wisconsin.)
Figure 28.22
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.
Figure 28.24
The typical transposon has
inverted nucleotide-sequence
repeats at its termini,
represented here as the 12-bp
sequence ACGTACGTACGT
(a). It acts at a target sequence
(shown here as the sequence
CATGC) within host DNA by
creating a staggered cut (b)
whose protruding singlestranded 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.
28.7 – Can DNA Be Repaired?
A fundamental difference from RNA, protein,
lipid, etc.
• All these 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
• Two principal mechanisms: mismatch repair
and methods for reversing chemical
damage
Garrett and Grisham, Biochemistry, Third Edition
Figure 28.23
Mismatch Repair
• Mismatch repair systems scan DNA
duplexes for mismatched bases, excise
the mispaired region and replace it
• Methyl-directed pathway of E. coli is an
example
• Since methylation occurs post-replication,
repair proteins identify methylated strand
as parent, remove mismatched bases on
other strand and replace them
Garrett and Grisham, Biochemistry, Third Edition
Figure 28.25
UVA 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.
Reversing Chemical Damage
• 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
Figure 28.26
Base excision repair. A damaged base (■) is
excised from the sugar-phosphate backbone by
DNA glycosylase, creating an 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.
28.8 – 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
Garrett and Grisham, Biochemistry, Third Edition
Figure 28.27 Point mutations due to base mispairings. (a) An example based on tautomeric
properties. 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 medicated by a water molecule.
Figure 28.28
5-Bromouracil usually favors the keto tautomer that mimics the base-pairing properties of
thymine, but it frequently shifts to the enol form, whereupon it can base-pair with guanine,
causing a T-A to C-G transition.
Figure 28.29
(a) 2-Aminopurine normally
base-pairs with T but (b) may
also pair with cytosine through
a single hydrogen bond.
Figure 28.30
Oxidative deamination of
adenine in DNA yields
hypoxanthine, which basepairs with cytosine,
resulting in an A-T to G-C
transition.
Figure 28.31
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 O6-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, N-methylN-nitro-N-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.
Gene Rearrangements and
Immunology
• Cells active in the immune response are
capable of gene rearrangement
• IgG molecules, the major class of
circulating antibodies, are encoded by
rearranged genes
• DNA rearrangements assemble an L-chain
gene from 3 separate genes
• DNA rearrangements assemble an Hchain gene from 4 separate genes
Garrett and Grisham, Biochemistry, Third Edition
Figure 28.32
Diagram of the
organization of the IgG
molecule. Two identical L
chains are joined with two
identical H chains. Each L
chain is held to an H chain
via an interchain disulfide
bond. The variable regions
of the four polypeptides lie
at the ends of the arms of
the Y-shaped molecule.
These regions are
responsible for the antigen
recognition function of the
antibody molecules. The
actual antigen-binding site
is constituted from
hypervariable residue
within the VL and VH
regions. For purposes of
illustration, some features
are shown on only one of
the other L chain or H
chain, but all features are
common to both chains.
Figure 28.33
The characteristic
“collapsed b-barrel
domain” known as
the immunoglobulin
fold. The b-barrel
structures for both (a)
variable and (b)
constant regions are
shown. (c) A
schematic diagram of
the 12 collapsed bbarrel domains that
make up an IgG
molecule. CHO
indicates the
carbohydrate
addition site; Fab
denotes one of the
two antigen-binding
fragments of IgG,
and Fc, the
proteolytic fragment
consisting of the
pairs of CH2 and CH3
domains.
Figure 28.34
Organization of human immunoglobulin gene segments. Green, orange, blue or purple colors
indicate the exons of a particular VL or VH gene. (a) L-chain gene assembly: During Blymphocyte maturation in the bone marrow, one of the 40 V genes combines with one of the 5
J genes and is joined with a
C gene. During the
recombination process, the
intervening DNA between
the gene segments is
deleted (see Figure 28.36).
These rearrangements
occur by a mostly random
process, giving rise to many
possible light-chain
sequences from each gene
family. (b) H-chain gene
assembly: H chains are
encoded by V,D,J. and C
genes. In H-chain gene
rearrangements, a D gene
joins with a J gene and then
one of the V genes adds to
the DJ assembly. (Adapted
from Figure 2b and c in Nossal,
G.J.V., 2003. The double helix
and immunology. Nature
421:440-444.)
Figure 28.35
Consensus elements are located above and below germline variable-region genes that
recombine to form genes encoding immunoglobulin chains. These consensus elements are
complementary and are arranged in a heptamer-nonamer,12-to 23-bp spacer pattern. (Adapted
from Tonewaga,S.,1983. Somatic generation of antibody diversity. Nature 302:575.)
Figure 28.36 Model for V(D)J recombination. A RAG1: RAG2 complex is assembled on DNA in
the region of recombination signal sequences (a), and this complex introduces double-stranded
breaks in the DNA at the borders of protein-coding sequences and the recombination signal
sequences (b). The products of RAG1:RAG2 DNA cleavage are novel: The DNA bearing the
recombination signal sequences has blunt ends, whereas the coding DNA has hairpin ends. That
is, the two strands of the V and J coding DNA segments are covalently joined as result of
transesterification reactions catalyzed by RAG1:RAG2. To complete the recombination process,
the two RSS ends are precisely joined to make a covalently closed circular dsDNA, but the V and
J coding ends undergo further processing before they are joined (c). Coding-end processing
involves opening of the V and J hairpins and the addition or removal of nucleotides from the
strands. This processing means
that joining of the V and J coding
ends is imprecise, providing an
additional means for introducing
antibody diversity. Finally, the V
and J coding segments are then
joined to create a recombinant
immunoglobulin-encoding gene
(d). The processing and joining
reactions require RAG1:RAG2,
DNa-dependent protein kinase
(DNA-PK, which consists of three
subunits-Ku70,Ku80, and DNAPKCS), and DNA ligase. (Adapted
from Figure 1 in Weaver,D.T., and
Alt,F.W., 1997. From RAGs to stitches.
Nature 388:428-429.)
Figure 28.37
Recombination between the VK
and JK genes can vary by
several nucleotides, giving rise
to variations in amino acid
sequence and hence diversity
in immunoglobulin L chains.