Chapter10 Homologous Recombination at the Molecular Level 孙颖 生物科学类 200431110002
Outline • Models for Homologous Recombination • Homologous Recombination Protein Machines • Homologous Recombination in Eukaryotes • Mating-Type Switching • Genetic Consequences of the Mechanism of Homologous Recombination
When homologous chromosomes pair prior to the first nuclear division, genetic exchange between the chromosomes occurs which is termed crossing over, one of the results of homologous recombination. • The frequencyof crossing over between two genes on the same chromosome depends on the physical distance between these genes with long distances giving the highest frequencies of exchanges---genetic maps
Homologous recombination is catalyzed by enzymes synthesized and regulated for these purposes: • Provide genetic variation • Retrieve sequences lost through DNA damage • Provide a mechanism to restart stalled or damaged replication forks • Special types of recombination regulate the expression of some genes • Lead to the development of methods to manipulate genes
Topic1:Models for Homologous Recombination Key steps of homologous recombination shared by these models include: • Alignment of two homologous DNA molecular. • 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
PartⅠ:The Holliday Model Illustrates Key Steps in Homologous Recombination • Initiated by the introduction of a nick in each DNA molecule at an identical location • Requires resolution of the Holliday junction • Two distinct classes of DNA products : crossover product & patch products (non-crossover products) The Double-Strand Break (DSB) Repair Model More Accurately Describes Many Recombination Events---two DSB-repair pathways
PartⅡ:The Double-Strand DNA Breaks Arise by Numerous Means and Initiate Homologous Recombination • An unrepaired nick in o:ne DNA strand will lead to collapse of a passing replication fork • A lesion in DNA will stop a replication fork ----the initiated recombination with a homologous DNA molecule will heal the break
Damage in the DNA template canlead To DSB formation during DNA replication
Topic2:Homologous Recombination Protein Machines • Organisms from all branches of life encode enzymes that catalyze the biochemical steps of recombination • Other recombination steps are catalyzed by different classes of proteins in different organisms but with the same general outcome ----the following section focuses on the RecBCD pathway
PartⅠ:The RecBCD Helicase/Nuclear Processes Broken DNA Molecules for Recombination The RecBCD Enzyme Structure: three subunits & both DNA helicase and nuclease activities ----RecB&RecD subunits are both DNA helicases Function: • processes broken DNA molecules to generate these regions of ssDNA • Help load the RecA strand-exchange protein onto these ssDNA ends • Provide a means for cells to “choose” whether to recombine with, or destroy, DNA molecules that enter a cell Energy: ATP-hydrolysis
Upon encountering the chi sequence, the nuclease activity of the RecBCD enzyme is altered, consequences: ----as a result of this change in activity, a duplex DNA molecule is converted into one with a ssDNA tail ----the structure is ideal for assembly of RecA and initiation of strand exchange
The DNA-degradation activity of RecBCD has multiple consequences: • This degradation is needed to process DNA at a break site for the subsequent steps of RecA assembly and strand invasion. • RecBCD sometimes functions simply to destroy DNA which protect cells from the potentially deleterious consequences of taking up foreign sequences.
PartⅡ:RecA Protein Assembles on Single-Stranded DNA and Promotes Strand Invasion RecA: the member of a family of enzymes called stand-exchange proteins Active Form: a protein-DNA filament ----RecA binding and assembly are much more rapid on single-stranded than on doubled-stranded DNA
PartⅢ:Newly Base –Paired Partners Are Established within the RecA Filament The RecA-ssDNA complex is the active form that participates in the search for a homology. ----The homology search is promoted by RecA because the two distinct DNA-binding sites: a primary site & a secondary site The RecA Homologs Are Present in All Orgnisms
Model of two steps in search for homology and DNA strand exchange within the RecA filament
RuvAB complex specifically recognize Holliday junction and promotes branch migration ----RuvArecognizes and binds to Holliday junctions and recruits the RuvB protein to the site ----RuvB is a hexameric ATPase • RuvC cleaves specific DNA strands at the Holliday junction to finish recombination ----depend on which pair of strands is cleaved by RuvC, the resulting ligated recombination products will be of either the splice or patch type
High resolution structure of the RuvA-DNA complex and schematic model of the RuvAB complex bound to Holliday junction DNA
High resolution structure of the RuvC resolves and schematic model of the RuvC dimer bound to Holliday junction DNA
Topic3:Homologous Recombination In Eukayotes Homologous recombination is also required for DNA repair and the restarting of collapsed replication forks in eukaryotic cells. Additional functions in eukaryotes: homologous recombination is critical for meiosis: • required for proper chromosome pairing and for maintaining the integrity of the genome • ensure variation in the sets of genes passed to the next generation
PartⅠ:Programmed Generation of Double-Stranded DNA Breaks Occurs during Meiosis SPO11: encodes a protein that introduces double-strand breaks in chromosomal DNA to initiate meiotic recombination ----two consequences: • The 5’ ends of the DNA at the site of Spo11 cleavage are covalently bound to the enzyme • The energy of the cleaved DNA phosphodiester bond is stored in the boud protein-DNA linkage. The cleavage mechanism makes the DSB be resealed by the simple reversal of the cleavage reaction.
MRX protein processes the cleaved DNA ends of assembly of the RecA-like strand-exchange proteins • Dmc1is a RecA-like protein that specifically functions in meiotic recombination. Two well-characterized homologs of the bacterial RecA protein:Rad51& Dmc1. • Many protein function together to promote meiotic recombination
Dmc1-dependent recombination occurs preferentially between nonsister homologous chromatids
Co –localizations of the rad51 and Dmc1 proteins to “recombination factories” in cells undergoing meiosis
Topic4:Mating-Type Switching Recombination controls the mating type by switching which mating-type genes are present at a specific location that is being expressed in that organism’s genome.
Haploid S. cerevisiae These HMR and HML loci are known as silent cassettes which provide a “storehouse” of genetic information that can be used to switch a cell’s mating type. This switch requires the transfer of genetic information from the HM sites to the MAT locus via homologous recombination.
Mating-type switching is initiated by a site-specific double-strand break ---- the HO endonucleas , a specialized DNA-cleaving enzyme perform the reaction of mating-type switching • Mating-type switching is a gene conversion event, not associated with crossing over----SDSA (synthesis-dependent strand annealing)
Recombination model for mating-type switching: synthesis-dependent strand annealing
Topic5: Genetic Consequences Of The Mechanism Of Homologous Recombination • Recombination is generally independent of sequence is that the frequency of recombination between any two genes is generally proportional to the distance between those genes • Homologous recombination is what makes it possible to use recombination frequencies to generate useful genetic maps that display the order and spacing of genes along a chromosome
Gene Conversion Occurs because DNA Is Required during Recombination There are two ways that gene conversion can occur : • the gene is very close to the site of the double-strand break • the repair of base pair mismatchesoccur in the recombination intermediates
Comparison of the genetic and physical maps of a typical region of a yeast chromosome
Mismatch repair ofheteroduplex DNA within recombination intermediates can give rise to gene conversion