| M W F; 10:10 - 11:00 pm |
Molecular Biology |
Douglas W. Smith |
| York 2722 |
BIMM 100 |
5254 Muir Biology Building |
| Fall, 2000 |
x42620; dsmith@ucsd.edu |
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Readings: Brown, 13: 353-357.
Outline:
Homologous Recombination: recombination between duplex DNA molecules of identical sequence
Occurs in Eucaryotes mainly
during Meiosis but also during Mitosis
Occurs widely in Procaryotes and Viruses
A. Breakage and Reunion ... Crossing Over as the basic mechanism ...
physical exchange of parts of the 2 DNA molecules ...
General Model: Holliday ...
[Brown, Fig 13.24]
Mechanism: ... essentially that of Radding and Meselson: ... [Brown, Fig 13.25B]
1. Nick introduced in one of one, or both, DNA molecules ...
2. Strand Assimilation
/ Exchange: nicked strand(s) exchange between the
duplexes
If only one nick were introduced, have D loop formation
...
3. Crossover Point can move up and down the joined duplexes: Branch Migration
This 4-stranded structure is a Holliday Structure, and the Crossover Point is called a Holliday Junction or Chi Form ... [Brown, Fig 13.24]
4. Branch Migration of the Holliday Structure ... [Brown, Fig 13.27]
5. Resolution of the 4-stranded structure by nicks in one strand of each of the two duplex molecules ... [Brown, Fig 13.24, 13.27]
If nicks here are in same strands as first nicks, then get recombination only in region of Branch Migration: Heteroduplex region ... [Brown, Fig 13.24]
If nicks here are in the other DNA strands, get Reciprocal Recombination, again with some Heteroduplex Regions depending on where nicks occurred and how much Repair DNA Synthesis takes place. ... [Brown, Fig 13.24]
Web animation of Holiday Junctions:
A nice Holiday Junction
animation is available on the Web here.
Animation of RuvA:
This Holiday Junction animation site also provides a link to representations and animations
of some of the proteins involved, in particular the E. coli
Ruv proteins (see below).
Evidence suggests that Double Strand Breaks in one DNA duplex (timing during Zygotene of appearance and disappearance of Double Strand Breaks) may initiate recombination events in Eukaryotes ... one model of such is given in Brown, Fig 13.29
This model (Brown, Fig 13.29) exemplifies how easy one can generate mechanistic models, given:
1. Strand Assimilation; 2. Repair DNA synthesis; 3. Branch Migration
B. Enzymes from E. coli
... [Brown,
Fig 13.26]
E. coli enzymes are known which can catalyze all of the above steps for Homologous Recombination via Breakage and Reunion ...
RecBCD - ExoV invades linear dsDNA at ends, separates strands as a Helicase in an ATP-dependent reaction ... until a Chi site is encountered ... here a nick is generated ...
Strand Assimilation catalyzed by Rec A protein ...
All that is needed is a nick in one of two DNA duplex molecules
Pol I for needed Repair DNA synthesis ... [eg Brown, Fig 13.20, 13.21]
RuvAB to promote Branch Migration ... [Brown, Fig 13.27]
RuvC to cleave the Holliday Junction ... [Brown, Fig 13.27]
Note how the Meselson-Radding
mechanism can account for
Post-Replication
Recombination-Mediated Repair
C. Gene Conversion
... [Brown, Fig
13.28, last parts of Fig 13.29]
The Heteroduplex Region probably generated in Recombination events can account for Gene Conversion:
Gene Conversion: proportions of alleles differ from ideal 1:1 or 2:2 or 4:4 ratios ...
A Heteroduplex Region, by definition, contains Mismatched Base Pairs.
These are subject to repair by Mismatch Repair systems ... [Brown, Fig 13.22, 13.23]
In meiosis, prior to cell divisions, one has four chromosomes. Recombination, when it occurs, usually occurs between two of these, yielding the following in a heteroduplex region containing a gene with alleles D and d on the two DNA strands in the heteroduplex region:
d:d ... D:d ... d:D ... D:D
Depending on which strands are repaired, different Segregation Ratios are obtained, as shown in ... [last parts of Brown, Fig 13.29]:
1. If no repair occurs, then one gets the normal 2:2 ratio.
2. If one mismatch is repaired, one gets 5:3 ratios:
d:d ... d:d ... d:D ... D:D
If one mismatch is repaired, and then the other is repaired, the ratios depend on the alleles which result from the repair process. If the first repair is as above (D -> d):
d:d ... d:d ... d:D ... D:D
then:
3. If the second repair is d -> D, the two repair processes "balance out" and one again gets a 2:2 ratio:
d:d ... d:d ... D:D ... D:D
4. If however the second repair is the same as the first, i.e. D -> d, the first repair process is actenuated and on gets a 3:1 ratio:
d:d ... d:d ... d:d ... D:D
Note: 2:2 is the same as 4:4 ... and 3:1 is the same as 6:2 ...
Hence, the common ratios of 3:1 and 5:3 seen in Gene Conversion can be accounted for by invoking Mismatch Repair in heteroduplex regions resulting from Branch Migration during Homologous Recombination ... an excellent example of a molecular explanation for a biological process ...
| BIMM100 | Syllabus
| Sections / Off Hrs | Grading
Policy | DNASYSTEM
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| Lectures | Journal
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If you have problems or comments, send email to Doug Smith