chapter10 homologous recombination at the molecular level 200431760044 顾辉辉...
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CHAPTER10
Homologous Recombination at the Molecular Level
200431760044 顾辉辉生物科学第四组
Outline Models for homologous recombinationHomologous recombination protein
machinesHomologous recombination in eukaryotesMating-type switchingGenetic consequences of the mechanism
of homologous recombination
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
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 canlead To DSB formation during DNA replication
Homologous recombination protein machines
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 DNA molecules with single-stranded DNA ext
ensions 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
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
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
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
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
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
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
Homologous recombination is required for chromosome segregation during meiosis
DNA dynamics during meiosis
Cytological view of crossing over
Programmed generation of double –stranded DNA breaks occurs during meiosis
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
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
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
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
Mating-type switching is initiated by a site-specific 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
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 mating-type information
Mating-type switching is a gene conversion event, not associated with crossing over
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 A corollary of the fact that recombination is g
enerally 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