chapter 19 m echanisms of r ecombination. recombination occurs at regions of homology between...

CHAPTER CHAPTER 1919MMECHANISMS OF ECHANISMS OF

RRECOMBINATIONECOMBINATION

RecombinationRecombination occurs at regions of occurs at regions of homologyhomology between between chromosomes through the chromosomes through the breakage and reunionbreakage and reunion of DNA of DNA molecules.molecules.

Models for recombinationModels for recombination, such as the , such as the Holliday modelHolliday model, involve the , involve the creation of a creation of a heteroduplex branchheteroduplex branch, or cross bridge, that can , or cross bridge, that can migrate and the subsequent splicingmigrate and the subsequent splicing of the intermediate of the intermediate structure to yield different types of recombinant DNA molecules.structure to yield different types of recombinant DNA molecules.

Recombination models can be applied to Recombination models can be applied to explain genetic crossesexplain genetic crosses..

Many of the Many of the enzymes participating in recombinationenzymes participating in recombination in bacteria in bacteria have been identified.have been identified.

Break

and rejoin

Basic Crossover EventBasic Crossover Event

Linkage analysis: recombination of genes by cross-over-> Molecular mechanism of recombination by cross-over

Benzer’s work;Recombination within the gene

-> should be precise-> base-pair complementarity

Direct Proof of chromosome Breakage and Reunion Direct Proof of chromosome Breakage and Reunion

By Matthew Meselen & Jean weigle, 1961

Grow in 13C, 15N

Grow in 12C, 14N+ +

c mi

Infect to bacteria Progeny phage

released

CsCl density gradient centrifuge of phage DNA

Recombination event must have occurred through the physical breakage and reunion of DNA

Breakage and reunion of DNA molecules

Lambda phage

Confirmed by reciprocal cross of heavy + + to light c mi

Chiasmata Are Actual Site of CrossoverChiasmata Are Actual Site of Crossover

Direct Evidence; Harlequin chromosome

- by C. Tease & G. H. Jones, 1978 (see Ch. 5, 8)

Centromeres arepulled apart

Indirect Evidence; recombination mapping

average of one crossover per meiosis produces 50 m.u. = mean number of chiasmata

Chiasmata: the crossover points

Tetrad analyses in filamentous fungi; Tetrad analyses in filamentous fungi; Neurospora crassaNeurospora crassa (see Ch.6)(see Ch.6)

Gene conversionGene conversion

Polarity of conversion frequencyPolarity of conversion frequency

Conversion and crossing-overConversion and crossing-over

Co-conversionCo-conversion

These crucial findings provided the impetus for the models of intragenic recombination.

Genetic results leading to recombination models

Gene Conversion during MeiosisGene Conversion during Meiosis

5:3 or 3:5 ratios

; two different strand of double helix carrying information for two different alleles at the conclusion of meiosis

Mutation

The allele that is converted always changes into the other specific allele taking part in the cross

Departures from predicted Mendelian 4:4 segregation

0.1-1.0% in filamentous fungi, up to 4% in yeast


Gene conversion

Polarity, Conversion and Crossing-overPolarity, Conversion and Crossing-over

Accurate allele maps are available, there is a gradient, or polarity, of

conversion frequencies along the gene

Polarity (gradient): the site closer to one end show higher conversion frequency than do the sites farther away from that end

Meiosis, crossover and gene conversion


Co-conversionCo-conversion

Co-conversion: a single conversion event including several sites at once

- Frequency of co-conversion increases as the distance between alleles decreases.


Holliday ModelHolliday Model

Formation of heteroduplex DNAFormation of heteroduplex DNA

Branch migration (along the two heteroduplex strands)Branch migration (along the two heteroduplex strands)

Meselson-Radding ModelMeselson-Radding Model

Heterodplex DNA occurred primarily in only one chromatidHeterodplex DNA occurred primarily in only one chromatid

Double-Strand Break-Repair ModelDouble-Strand Break-Repair Model

Double strand break, rather than a nick, is the start pointDouble strand break, rather than a nick, is the start point

Holliday Model of RecombinationHolliday Model of Recombination

Formation of heteroduplex DNA -> cross bridge -> branch migration -> mismatch repair-> resolution

Holliday Structure: partially heteroduplex double helix


Branch Migration; the movement of the crossover point between DNA complexes

Cross bridge


Resolution of the Holliday structure


Application of the Holliday model to genetic crosses

Gene conversion & Aberrant ratio; a consequence of mismatch repair

Polarity of gene conversion; in heteroduplex region

Coconversion; both sites within heteroduplex same excision-repair act

Meselson-Radding Model of RecombinationMeselson-Radding Model of Recombination

(d)

(a)

(b)

(c)

Holliday modelCould not explain all of cross

-> aberrant 4:4 ratio very rare 6:2 ratio frequent-> gene conversion in only one chromatid-> Meselson and Radding

Double-Strand Break-Repair Model of RecombinationDouble-Strand Break-Repair Model of Recombination

In yeast, induction of doublestrand break in plasmid stimulates 1000-fold of transformation.

-> J. Szostak, T. Orr-Weaver, and R. Rothstein

Visualization of recombination intermediatesVisualization of recombination intermediates

H. Potter and D. Dressler

Several Genes involved in general recombination in Several Genes involved in general recombination in E.coliE.coli

recrecA, A, recrecB, B, recrecC, C, recrecD, SsB(single strand binding protein)D, SsB(single strand binding protein)

RecBCD pathwayRecBCD pathway

RecBCD pathwayRecBCD pathwayRecBCD pathwayRecBCD pathway

RecF pathwayRecF pathwayRecF pathwayRecF pathway RecE pathwayRecE pathwayRecE pathwayRecE pathway

RecA

Minor pathwayMinor pathway

Production of single-stranded DNAProduction of single-stranded DNA

- RecBCD protein complex have both nuclease (nicking) and helicase activity (unwinding)

- Chi site; 5’- G C TG G T G G -3’

target site for nuclease activity of RecBCD

RecA-protein-mediated Single-Strand ExchangeRecA-protein-mediated Single-Strand Exchange

- RecA protein can bind to single strand forming a nucleoprotein complex, and catalyze single strand invasion of a duplex forming a D loop

Branch MigrationBranch Migration

RuvA and RuvB protein catalyze branch migration

RuvA: bind to crossover point, recruit RuvB RuvB: ATPase hexameric ring motor

Resolution of Holliday JunctionResolution of Holliday Junction

(b)

RuvC: an endonuclease that resolves Holliday junction by symmetric cleavage of the continuous pair of DNA strands

180° rotation of arm I and II

Summary of Resolution PathwaySummary of Resolution Pathway

+ RecA

- RecA

Recombination produces new gene combinations by exchanging homologous chromosomes.

Both genetic and physical evidence has led to several models of recombination

Common features of recombination models

heteroduplex DNA formation

mismatch repair

resolution (splicing)

The process of recombination itself is under genetic control by numerous genes

RecA, B, C, D, E, F and G

RuvA, B and C

Rus AND ………..

chapter 19 m echanisms of r ecombination. recombination occurs at regions of homology between...

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