Transcript Lecture 19-Chap15

3.12 Gene Knockouts and Transgenics
• transgenics – Organisms created by
introducing DNA prepared in test tubes into the
germline.
– The DNA may be inserted into the genome or exist in
an extrachromosomal structure.
Figure 03.27: Transfection can
introduce DNA directly into
the germline of animals.
Photo reproduced from P. Chambon, Sci. Am.
244 (1981): 60-71. Used with permission of
Pierre Chambon, Institute of Genetics and
Molecular and Cellular Biology, College of
France.
3.12 Gene Knockouts and Transgenics
• Embryonic stem (ES) cells that are injected into
a mouse blastocyst generate descendant cells
that become part of a chimeric adult mouse.
– When the ES cells contribute to the germline, the next
generation of mice may be derived from the ES cell.
– Genes can be added to the mouse germline by
transfecting them into ES cells before the cells are
added to the blastocyst.
3.12 Gene Knockouts and Transgenics
Figure 03.29: ES cells can be used to generate mouse chimeras.
3.12 Gene Knockouts and Transgenics
• An endogenous gene can be replaced by a transfected
gene using homologous recombination.
• The occurrence of successful homologous
recombination can be detected by using two selectable
markers, one of which is incorporated with the integrated
gene, the other of which is lost when recombination
occurs.
3.12 Gene Knockouts and Transgenics
• The Cre/lox system is widely used to make
inducible knockouts and knock-ins.
– knockout – A process in which a gene function is
eliminated, usually by replacing most of the coding
sequence with a selectable marker in vitro and
transferring the altered gene to the genome by
homologous recombination.
– knock-in – A process similar to a knockout, but more
subtle mutations are made.
3.12 Gene Knockouts and Transgenics
Figure 03.31: The Cre recombinase catalyzes a site-specific recombination between two
identical lox sites, releasing the DNA between them.
Chapter 15
Homologous and
Site-Specific
Recombination
15.1 Introduction
• Homologous recombination is essential in meiosis for
generating diversity and for chromosome segregation,
and in mitosis to repair DNA damage and stalled
replication forks.
Figure 15.01: No crossing over
between the A and B genes gives
rise to only nonrecombinant
gametes.
15.1 Introduction
• Site-specific recombination involves specific DNA
sequences.
• somatic recombination – Recombination that occurs in
nongerm cells (i.e., it does not occur during meiosis);
most commonly used to refer to recombination in the
immune system.
• Recombination systems have been adapted for
experimental use.
Figure 15.02: Site-specific
recombination occurs
between the circular and
linear DNAs at the boxed
region.
Adapted from B. Alberts, et al. Molecular Biology of the
Cell, Fourth edition. Garland Science, 2002.
15.2 Homologous Recombination Occurs
Between Synapsed Chromosomes in Meiosis
• Chromosomes must synapse (pair) in
order for chiasmata to form where
crossing over occurs.
• The stages of meiosis can be
correlated with the molecular events at
the DNA level.
Figure 15.03: Recombination occurs during
the first meiotic prophase.
15.2 Homologous Recombination Occurs
Between Synapsed Chromosomes in Meiosis
• sister chromatid – Each of two identical copies
of a replicated chromosome; this term is used as
long as the two copies remain linked at the
centromere.
– Sister chromatids separate during anaphase in
mitosis or anaphase II in meiosis.
• bivalent – The structure containing all four
chromatids (two representing each homolog) at
the start of meiosis.
15.2 Homologous Recombination Occurs
Between Synapsed Chromosomes in Meiosis
• synaptonemal complex – The morphological structure
of synapsed chromosomes.
• joint molecule – A pair of DNA duplexes that are
connected together through a reciprocal exchange of
genetic material.
15.3 Double-Strand Breaks Initiate
Recombination
• The double-strand break repair (DSBR) model of
recombination is initiated by making a double-strand
break in one (recipient) DNA duplex and is relevant for
meiotic and mitotic homologous recombination.
• In 5 end resection, exonuclease action generates 3′–
single-stranded ends that invade the other (donor)
duplex.
15.3 Double-Strand Breaks
Initiate Recombination
• When a single strand from one
duplex displaces its counterpart in
the other duplex (single-strand
invasion), it creates a branched
structure called a D loop.
• Strand exchange generates a
stretch of heteroduplex DNA
consisting of one strand from each
parent.
Figure 15.04: Double-strand break-repair model of
homologous recombination.
15.3 Double-Strand Breaks Initiate
Recombination
• New DNA synthesis replaces the
material that has been degraded.
• branch migration – The ability of
a DNA strand partially paired with
its complement in a duplex to
extend its pairing by displacing
the resident strand with which it is
homologous.
Figure 15.06: Branch migration can occur in
either direction when an unpaired single
strand displaces a paired strand.
15.3 Double-Strand Breaks Initiate
Recombination
• Capture of the second DSB end by annealing generates
a recombinant joint molecule in which the two DNA
duplexes are connected by heteroduplex DNA and two
Holliday junctions.
• The joint molecule is resolved into two separate duplex
molecules by nicking two of the connecting strands.
• Whether recombinants are formed depends on if the
strands involved in the original exchange or the other
pair of strands are nicked during resolution.
15.4 Gene Conversion Accounts for
Interallelic Recombination
• Heteroduplex DNA that is created by recombination can
have mismatched sequences where the recombining
alleles are not identical.
• Repair systems may remove mismatches by changing
one of the strands so its sequence is complementary to
the other.
15.4 Gene Conversion Accounts for
Interallelic Recombination
• Mismatch (gap)
repair of heteroduplex
DNA generates
nonreciprocal
recombinant products
called gene
conversions.
Figure 15.07: Spore formation in the ascomycetes
allows determination of the genetic constitution
of each of the DNA strands involved in meiosis.
15.5 The SynthesisDependent StrandAnnealing Model
• The synthesis-dependent
strand-annealing (SDSA)
model is relevant for mitotic
recombination, as it produces
gene conversions from
double-strand breaks without
associated crossovers.
Figure 15.08: Synthesis-dependent strandannealing model of homologous
recombination.
15.6 The Single-Strand
Annealing Mechanism
Functions at Some
Double-Strand Breaks
• Single-strand annealing
(SSA) occurs at doublestrand breaks between direct
repeats.
Figure 15.09: Single strand annealing model of
homologous recombination.
15.6 The Single-Strand Annealing Mechanism
Functions at Some Double-Strand Breaks
• Resection of double-strand break ends results in 3′–
single-stranded tails.
• Complementarity between the repeats allows for
annealing of the single strands.
• The sequence between the direct repeats is deleted after
SSA is completed.
15.7 Break-Induced Replication Can Repair
Double-Strand Breaks
• Break-induced
replication (BIR) is
initiated by a one-ended
double-strand break.
• BIR at repeated
sequences can result in
translocations.
Figure 15.10: Break-induced replication can
result in nonreciprocal translocations.
15.8 Recombining Meiotic Chromosomes Are
Connected by the Synaptonemal Complex
• During the early part of meiosis, homologous
chromosomes are paired in the synaptonemal complex.
• The mass of chromatin of each homolog is separated
from the other by a
proteinaceous
complex.
Figure 15.13: Each pair of sister
chromatids has an axis made of
cohesins.
15.8 Recombining Meiotic Chromosomes Are
Connected by the Synaptonemal Complex
• axial element – A proteinaceous structure around which
the chromosomes condense at the start of synapsis.
• lateral element – A structure in the synaptonemal
complex that forms when a pair of sister chromatids
condenses on to an axial element.
15.8 Recombining Meiotic Chromosomes Are
Connected by the Synaptonemal Complex
• central element – A structure that lies in the
middle of the synaptonemal complex, along
which the lateral elements of homologous
chromosomes align.
– It is formed from Zip proteins.
• recombination nodules (nodes) – Dense
objects present on the synaptonemal complex;
they may represent protein complexes involved
in crossing over.
15.9 The Synaptonemal Complex Forms
After Double-Strand Breaks
• Double-strand breaks that initiate recombination occur
before the synaptonemal complex forms.
• If recombination is blocked, the synaptonemal complex
cannot form.
• Meiotic recombination involves two phases: one that
results in gene conversion without crossover, and one
that results in crossover products.
15.9 The Synaptonemal Complex Forms
After Double-Strand Breaks
Figure 15.15: Model of meiotic homologous recombination.
Adapted from M. J. Neale and S. Keeney, Nature 442 (2006): 153-158.
15.10 Pairing and Synaptonemal Complex
Formation Are Independent
• Mutations can occur in either chromosome pairing or
synaptonemal complex formation without affecting the
other process.
15.11 The Bacterial RecBCD System Is
Stimulated by chi Sequences
• The RecBCD complex has nuclease and helicase
activities.
• RecBCD binds to DNA downstream of a chi sequence,
unwinds the duplex, and degrades one strand from 3′–5′
as it moves to the chi site.
• The chi site triggers loss of the RecD subunit and
nuclease activity.
15.11 The Bacterial RecBCD System Is
Stimulated by chi Sequences
Figure 15.16: RecBCD nuclease approaches a chi sequence from one side, degrading DNA
as it proceeds.
15.12 Strand-Transfer Proteins Catalyze
Single-Strand Assimilation
• RecA forms filaments with single-stranded or duplex
DNA and catalyzes the ability of a single-stranded DNA
with a free 3′ end to displace its counterpart in a DNA
duplex.
• presynaptic filaments – Single-stranded DNA bound in
a helical nucleoprotein filament with a strand transfer
protein such as Rad51 or RecA.
Figure 15.17: RecA promotes the
assimilation of invading single strands
into duplex DNA so long as one of the
reacting strands has a free end.
15.12 Strand-Transfer
Proteins Catalyze SingleStrand Assimilation
Figure 15.19: RecA-mediated strand exchange between
partially duplex and entirely duplex DNA.
15.13 Holliday Junctions Must Be Resolved
• The bacterial Ruv complex acts on recombinant
junctions.
• RuvA recognizes the structure of the junction.
• RuvB is a helicase that catalyzes branch migration.
Figure 15.20: RuvAB is an
asymmetric complex that
promotes branch migration
of a Holliday junction.
15.13 Holliday Junctions
Must Be Resolved
• RuvC cleaves junctions to
generate recombination
intermediates.
• Resolution in eukaryotes is
less well understood, but a
number of meiotic and
mitotic proteins are
implicated.
Figure 15.21: Bacterial enzymes can catalyze all
stages of recombination in the repair pathway.
15.13 Holliday Junctions Must Be Resolved
• patch recombinant – DNA that results from a
Holliday junction being resolved by cutting the
exchanged strands.
– The duplex is largely unchanged, except for a DNA
sequence on one strand that came from the
homologous chromosome.
15.13 Holliday Junctions Must Be Resolved
• splice recombinant – DNA that results from a
Holliday junction being resolved by cutting the
nonexchanged strands.
– Both strands of DNA before the exchange point come
from one chromosome; the DNA after the exchange
point come from the homologous chromosome.
15.14 Eukaryotic Genes Involved in
Homologous Recombination
• The MRX complex, Exo1, and Sgs1/Dna2 in yeast and
the MRN complex and BLM in mammalian cells resect
double-strand breaks.
• The Rad51 recombinase
binds to single-stranded
DNA with the aid of
mediator proteins, which
overcome the inhibitory
effects of RPA.
Figure 15.22: Structure of Rad50 and model for the
MRX/N complex binding to double-strand breaks.
Adapted from M. Lichten, Nat. Struct. Mol. Biol. 12
(2005): 392-393.
15.14 Eukaryotic Genes Involved in
Homologous Recombination
• Strand invasion is
dependent on Rad54 and
Rdh54 in yeast and Rad54
and Rad54B in mammalian
cells.
• Yeast Sgs1, Mus81/Mms4
and human BLM,
MUS81/EME1 are
implicated in resolution of
Holliday junctions.
Figure 15.23: Double Holliday junction
dissolution by the action of a DNA helicase
and topoisomerase.
15.15 Specialized Recombination Involves
Specific Sites
• Specialized recombination
involves reaction between
specific sites that are not
necessarily homologous.
• recombinase – Enzyme that
catalyzes site-specific
recombination.
• Phage lambda integrates into
the bacterial chromosome by
recombination between a site
on the phage and the att site
on the E. coli chromosome.
Figure 15.24: Circular phage DNA is
converted to an integrated prophage
by a reciprocal recombination
between attP and attB.
15.15 Specialized Recombination Involves
Specific Sites
• core sequence – The segment of DNA that is
common to the attachment sites on both the
phage lambda and bacterial genomes.
– It is the location of the recombination event that
allows phage lambda to integrate.
• The phage is excised from the chromosome by
recombination between the sites at the end of
the linear prophage.
• Phage lambda int codes for an integrase that
catalyzes the integration reaction.
15.16 Site-Specific Recombination Involves
Breakage and Reunion
• Cleavages staggered by 7 bp are made in both attB and
attP, and the ends are joined crosswise.
Figure 15.26: Staggered cleavages in the common core sequence of attP and attB.
15.17 Site-Specific
Recombination Resembles
Topoisomerase Activity
• Integrases are related to
topoisomerases, and the
recombination reaction resembles
topoisomerase action except that
nicked strands from different
duplexes are sealed together.
• The reaction conserves energy by
using a catalytic tyrosine in the
enzyme to break a phosphodiester
bond and link to the broken 3′ end.
Figure 15.27: Integrases catalyze
recombination by a mechanism
similar to that of topoisomerases.
15.17 Site-Specific Recombination
Resembles Topoisomerase Activity
• Two enzyme units bind to each recombination site and
the two dimers synapse to form a complex in which the
transfer reactions occur.
Figure 15.28: A synapsed loxA recombination
complex has a tetramer of Cre recombinases,
with one enzyme monomer bound to each
half site.
15.18 Lambda Recombination Occurs in an
Intasome
• Lambda integration takes place
in a large complex that also
includes the host protein IHF.
• The excision reaction requires
Int and Xis and recognizes the
ends of the prophage DNA as
substrates.
Figure 15.30: Multiple copies of Int protein
organize attP into an intasome, which
initiates site-specific recombination by
recognizing attB on free DNA.
15.19 Yeast Can Switch Silent and Active
Loci for Mating Type
Figure 15.31: Changes of mating type
occur when silent cassettes replace
active cassettes of opposite genotype.
• The yeast mating type locus
MAT, a mating type
cassette, has either the
MATa or MATα genotype.
• Yeast with the dominant allele
HO switch their mating type
at a frequency ~10–6.
• The allele at MAT is called
the active cassette.
• There are also two silent
cassettes, HMLα and HMRa.
15.19 Yeast Can Switch Silent and Active
Loci for Mating Type
• Switching occurs if
MATa is replaced by
HMRα or MATα is
replaced by HMRa.
Figure 15.32: Silent cassettes have the
same sequences as the corresponding
active cassettes.
15.20 Unidirectional Gene
Conversion Is Initiated by
the Recipient MAT Locus
• Mating type switching is
initiated by a double-strand
break made at the MAT locus
by the HO endonuclease.
• The recombination event is a
synthesis-dependent strandannealing reaction.
Figure 15.34: Cassette substitution is initiated by a
double-strand break in the recipient (MAT) locus.
15.21 Antigenic Variation in Trypanosomes
Uses Homologous Recombination
• Variant surface glycoprotein
(VSG) switching in
Trypanosoma brucei evades
host immunity.
• VSG switching requires
recombination events to
move VSG genes to
specific expression
sites.
Figure 15.35: Switching mechanisms in
trypanosome antigenic variation.
Reprinted from Trends Genet., vol. 22, J. E. Taylor and G.
Rudenko, Switching trypanosome coats..., pp. 614-620.
Copyright 2006, with permission from Elsevier
[http://www.sciencedirect.com/science/journal/01689525].
15.22 Recombination Pathways Adapted for
Experimental Systems
• Mitotic homologous recombination allows for targeted
transformation.
• The Cre/lox and Flp/FRT systems allow for targeted
recombination and gene knockout construction.
15.22 Recombination Pathways Adapted for
Experimental Systems
Figure 15.36: Using Cre/lox to make cell-type specific gene knockouts in mouse.
Adapted from H. Lodish, et al. Molecular Cell Biology, Fifth
edition. W. H. Freeman & Company, 2003.
15.22 Recombination
Pathways Adapted for
Experimental Systems
• The Flp/FRT
system has been
adapted to
construct recyclable
selectable markers
for gene deletion.
Figure 15.37: Using FLP/FRT to make
homozygous recessive cells by
homologous recombination.