2. 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.
3. Break
and rejoin
Basic 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
4. 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
5. 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
Chiasmata: the crossover points
6. 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
7. Gene 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
Genetic results leading to recombination models
Gene conversion
8. 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
9. Co-conversion
Co-conversion: a single conversion event including several sites at once
- Frequency of co-conversion increases as the distance between alleles decreases.
Genetic results leading to recombination models
10. 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
11. Holliday Model of Recombination
Formation of heteroduplex DNA -> cross bridge -> branch migration -> mismatch repair
-> resolution
Holliday Structure: partially heteroduplex double helix
12. Holliday Model of Recombination
Branch Migration; the movement of the crossover point between DNA complexes
Cross
bridge
13. Holliday Model of Recombination
Resolution of the
Holliday structure
14.
15.
16. 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
17. Meselson-Radding Model of Recombination
(d)
(a)
(b)
(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
18. 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
21. Several Genes involved in general recombination in E.coli
recA, recB, recC, recD, SsB(single strand binding protein)
RecBCD pathway
RecBCD pathway
RecF pathway RecE pathway
RecA
Minor pathway
22. 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
24. Branch Migration
RuvA and RuvB protein catalyze branch migration
RuvA: bind to crossover point, recruit RuvB
RuvB: ATPase hexameric ring motor
25. Resolution 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
27. 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 ………..
