Download Models for homologous recombination

CHAPTER10
Homologous Recombination
at the Molecular Level
200431760044 顾辉辉
生物科学第四组
Outline
Models for homologous recombination
 Homologous recombination protein
machines
 Homologous recombination in eukaryotes
 Mating-type switching
 Genetic consequences of the mechanism
of homologous recombination
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Models for homologous
recombination
Key steps of homologous recombination shared
by different models
 Alignment of two homologous DNA molecules
 Introduction of breaks in the DNA
 Formation of initial short regions of base
pairing between the two recombining DNA
molecules
 Movement of the Holliday junction
 Cleavage of the Holliday junction
The Holliday model illustrates key
steps in homologous
recombination
The Holliday model very well illustrates the
DNA strand invasion, branch migration,
and ,Holliday junction resolution processes
central to homologous recombination
Holliday model through the steps
of branch migration
Holliday junction cleavage
Double –stranded DNA breaks
arise by numerous means and
initiate homologous recombination
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Double-straned breaks in DNA arise quite
frequently
In bacteria, the major biological role of
homologous recombination is to repair DSBs
In addition to repairing DSBs in chromosomal
DNA, homologous recombination promotes
genetic exchange in bacteria
In eukaryotic cells, homologous
recombination is critical for repairing DNA
Damage in the DNA template can
lead To DSB formation during DNA
replication
Homologous recombination
protein machines
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The recBCD helicase nuclease processes broken
DNA molecules for recombination
RecA protein assembles on single-stranded DNA
and promotes strand invasion
Newly base-paired partners are eatablidshed
within the recA filament
RuvAB complex specifically recognizes Holliday
junctions and promotes branch migration
RuvC cleaves specific DNA strands at the Holliday
junction to finish recombination
The recBCD helicase nuclease
processes broken DNA molecules
for recombination
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DNA molecules with single-stranded DNA
extensions or tails are the preferred substrate
for initiating strand exchange between
regions of homologous sequence
The RecBCD enzyme processes broken DNA
molecules to generate these regions of
ssDNA
RecBCD provide a means for cells to choose
Steps of DNA processing
By RecBCD
Polar action of chi
RecA protein assembles on
single-stranded DNA and
promotes strand invasion
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RecA is the central protein in homologous
recombination
RecA makes DNA pairing and strand exchange
The active form of RecA is a protein-DNA
filaments
Examples of DNA paring and
strand -exchange reaction
Three view of the RecA filament
Polarity of RecA assembly
Newly base-paired partners are
established within the RecA
filament
RecA-catalyzed strand exchange can be
divided into different stages
 cross-section of single DNA strand bound to
RecA protein
 DNA in secondary site is testes for
complementarity
 Base -paring between strands is switched
Model of two steps in search for
homology and DNA strand exchange
within the RecA filament
RecA homologous are present
in all organisms
Strand-exchange proteins of the
RecA family are present in all forma
of life
RecA-like protein in three
branches of life
RuvAB complex specifically
recognizes Holliday junctions and
promotes branch migration
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RuvA protein is a Holliday junction specific
DNA-binging protein that recognizes the
structure of the DNA junction
RuvB is a hexametric ATPase
High resolution structure of the
RuvA-DNA complex and schematic
model of the RuvAB complex bound
to Holliday junction DNA
RuvC cleaves specific DNA
strands at the Holliday junction to
finish recombination
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Completion of recombination requires that the
Holliday junction between the two
recombining DNA molecules be resolved
RuvC specifically nicks two of the
homologous DNA strands that have the same
polarity
RuvC cleaves DNA with modest sequence
specificity
High resolution structure of the RuvC
resolves and schematic model of the
RuvC dimer bound to Holliday
junction DNA
Homologous recombination in
eukaryotes
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Homologous recombination has additional functions in
eukaryotes
Homologous recombination is required for chromosome
segregation during meiosis
Programmed generation of double –stranded DNA breaks
occurs during meiosis
MRX protein processes the cleaved DNA ends for
assembly of the RecA-like strand –exchange proteins
Dmc1 is a RecA-like protein that specifically functions in
meiotic recombination
Many protein function together to promote meiotic
recombination
Homologous recombination has
additional functions in eukaryotes
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Homologous recombination can repair
double –stranded breaks in DNA , to restart
collapsed replication forks, and to allow a
cell’s chromosomal DNA to recombine with
DNA that enters via phage infection or
junction
During meiosis, homologous recombination is
required for proper chromosome pairing
Homologous recombination is
required for chromosome
segregation during meiosis
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Recombination must be complete before the
first nuclear division to allow the homologs to
properly align and then separate
The improper segregation of chromosomes,
called nondisjunction, leads to a large
number of gametes without the correct
chromosome complement
The homologous recombination events that
occur during meiosis are called meiotic
recombination
DNA dynamics during meiosis
Cytological view of crossing over
Programmed generation of double
–stranded DNA breaks occurs
during meiosis
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The spo11protein cuts the DNA at many
chromosomal locations
The mechanism of DNA cleavage
The cleavage involves a covalent protein –
DNA complex has two consequences
Mechanism of cleavage by spo11
MRX protein processes the
cleaved DNA ends for assembly of
the RecA-like strand –exchange
proteins
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The DNA a5 5he site Spo11-cataluzed
double-strand break is processed to generate
dingle-stranded regions needed for assembly
aof the RecA-like strand-exchange protein
Processing of the DNA at the break site
occurs exclusively on the DNA strand that
terminates with a 5’ end
Overview of meiotic
recombination pathway
Dmc1 is a RecA-like protein that
specifically functions in meiotic
recombination
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Eukaryotes encode two well-characterized
homologs of the bacterial RecA protein:
Rad51 and Dmc1
Dmc1 is expressed only as cells enter
meiosis
strand exchange during meiosis occurs
between a particular type of homologous
DNA partner
Dmc1-dependent recombination
occurs preferentially between
nonsister homologous
chromatids
Many protein function together to
promote meiotic recombination
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Some large protein-DNA complexes, known
as recombination factories
Rad51, Dmc1, Rad52……
Co –localizations of the rad51 and
Dmc1 proteins to “recombination
factories” in cells undergoing meiosis
mating-type switching
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Mating-type switching is initiated by a sitespecific double-strand break
Mating-type switching is a gene conversion
event, not associated with crossing over
Mating-type switching is initiated
by a site-specific double-strand
break
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Mating-type switching is initiated by the
introduction of a DSB at the MAT locus
This reaction is performed by a specialized
DNA-cleaving enzyme, called the HO
endonuclease
Mating-type switching is unidirectional
Genetic loci encoding matingtype information
Mating-type switching is a gene
conversion event, not associated
with crossing over
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Models for recombination that do not involve
Holliday junction intermediates better explain
mating-type switching
Synthesis-dependent strand annealing
Completing recombination requires that the
other “old” DNA strand present at MAT be
removed
Recombination model for mating-type
switching: synthesis-dependent
strand annealing
Genetic consequences of the
mechanism of homologous
recombination
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A corollary of the fact that recombination is
generally independent of sequence is that the
frequency of recombination between any two
genesis generally proportional to the distance
between those genes
A region of DNA does not have the “average”
probability of participation in recombination
Gene conversion occurs because DNA is
repaired duringrecombination
Comparison of the genetic and
physical maps of a typical region
of a yeast chromosome
Mismatch repair of heteroduplex
DNA within recombination
intermediates can give rise to gene
conversion