Download PowerPoint 프레젠테이션

CHAPTER
19
MECHANISMS OF
RECOMBINATION
Recombination occurs at regions of homology between
chromosomes through the breakage and reunion of DNA
molecules.
Models for recombination, such as the Holliday model, involve the
creation of a heteroduplex branch, or cross bridge, that can
migrate and the subsequent splicing of the intermediate
structure to yield different types of recombinant DNA molecules.
Recombination models can be applied to explain genetic crosses.
Many of the enzymes participating in recombination in bacteria
have been identified.
Basic Crossover Event
Linkage analysis: recombination of genes by cross-over
-> Molecular mechanism of recombination by cross-over
Break
and rejoin
Benzer’s work;
Recombination within the gene
-> should be precise
-> base-pair complementarity
Breakage and reunion of DNA molecules
Direct Proof of chromosome Breakage and Reunion
Lambda phage
++
By Matthew Meselen & Jean weigle, 1961
Grow in 12C, 14N
Infect to bacteria
c mi
Progeny phage
released
Grow in 13C, 15N
CsCl density
gradient centrifuge
of phage DNA
Confirmed by reciprocal cross of heavy + + to light c mi
Recombination event must have occurred through the physical breakage and
reunion of DNA
Chiasmata: the crossover points
Chiasmata Are Actual Site of Crossover
Direct Evidence; Harlequin chromosome
- by C. Tease & G. H. Jones, 1978 (see Ch. 5, 8)
Centromeres are
pulled apart
Indirect Evidence; recombination mapping
 average of one crossover per meiosis produces 50 m.u.
= mean number of chiasmata
Genetic results leading to recombination models
Tetrad analyses in filamentous fungi; Neurospora crassa
(see Ch.6)
Gene conversion
Polarity of conversion frequency
Conversion and crossing-over
Co-conversion
 These crucial findings provided the impetus for the models of intragenic
recombination.
Genetic results leading to recombination models
Gene Conversion during Meiosis
Departures from predicted Mendelian
4:4 segregation
0.1-1.0% in filamentous fungi,
up to 4% in yeast
Gene conversion
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
Genetic results leading to recombination models
Polarity, 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
Genetic results leading to recombination models
Co-conversion
Co-conversion: a single conversion event including several sites at once
- Frequency of co-conversion increases as the distance between alleles decreases.
Holliday Model
Formation of heteroduplex DNA
Branch migration (along the two heteroduplex strands)
Meselson-Radding Model
Heterodplex DNA occurred primarily in only one chromatid
Double-Strand Break-Repair Model
Double strand break, rather than a nick, is the start point
Holliday Model of Recombination
Formation of heteroduplex DNA -> cross bridge -> branch migration -> mismatch repair
-> resolution
Holliday Structure:
partially heteroduplex double helix
Holliday Model of Recombination
Branch Migration; the movement of the crossover point between DNA complexes
Cross
bridge
Holliday Model of Recombination
Resolution of the
Holliday structure
Holliday Model of Recombination
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 Recombination
(a)
(b)
(d)
(c)
Holliday model
Could 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 Recombination
In yeast, induction of double
strand break in plasmid
stimulates 1000-fold of
transformation.
-> J. Szostak, T. Orr-Weaver,
and R. Rothstein
Visualization of recombination intermediates
H. Potter and D. Dressler
Several Genes involved in general recombination in E.coli
recA, recB, recC, recD, SsB(single strand binding protein)
 RecBCD pathway
RecBCD pathway
RecA
RecF pathway
RecE pathway
Minor pathway
Production 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 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 Migration
RuvA and RuvB protein catalyze branch migration
RuvA: bind to crossover point, recruit RuvB
RuvB: ATPase hexameric ring motor
Resolution of Holliday Junction
RuvC: an endonuclease that resolves Holliday junction by symmetric cleavage of
the continuous pair of DNA strands
(b)
180° rotation of
arm I and II
Summary 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 ………..