1. DNA Recombination Roles Types Homologous recombination in E.coli
Transposable elements

2. Biological Roles for Recombination
Generating new gene/allele combinations (crossing over during meiosis)
Generating new genes (e.g., Immuno- globulin rearrangement)
Integration of a specific DNA element
DNA repair

3. Practical Uses of Recombination
1. Used to map genes on chromosomes
(recombination frequency proportional to distance between genes)
2. Making transgenic cells and organisms

4. Map of Chromosome I of Chlamydomonas reinhardtii
cM = centiMorgan; unit of recombination frequency
1 cM = 1% recombination frequency
Chlamydomonas Genetics Center

5. Types of Recombination
Homologous - occurs between sequences that are nearly identical (e.g., during meiosis)
Site-Specific - occurs between sequences with a limited stretch of similarity; involves specific sites
Transposition – DNA element moves from one site to another, usually little sequence similarity involved

6. Examples of Recombination
Fig. 22.1

7. Holliday Model
R. Holliday (1964)
Holliday Junctions form during recombination
HJs can be resolved 2 ways, only one produces true recombinant molecules
patch
Fig. 22.2

8. EM of a Holliday Junction w/a few melted base pairs around junction
Fig. 22.3

9. The recBCD Pathway of Homologous Recombination
Part I: Nicking and Exchanging
Fig a-e

10. recBCD Pathway of Homologous Recombination Part I: Nicking and Exchanging
A nick is created in one strand by recBCD at a Chi sequence (GCTGGTGG), found every 5000 bp.
Unwinding of DNA containing Chi sequence by recBCD allows binding of SSB and recA.
recA promotes strand invasion into homologous DNA, displacing one strand.
The displaced strand base-pairs with the single strand left behind on the other chromosome.
The displaced and now paired strand is nicked (by recBCD?) to complete strand exchange.

11. recBCD Pathway of Homologous Recombination
Part II: Branch Migration and Resolution
Fig f-h

12. recBCD Pathway of Homologous Rec
recBCD Pathway of Homologous Rec. Part II: Branch Migration and Resolution
Nicks are sealed  Holliday Junction
Branch migration (ruvA + ruvB)
Resolution of Holliday Junction (ruvC)

13. RecBCD : A complex enzyme
RecBCD has:
Endonuclease subunits (recBCD) that cut one DNA strand close to Chi sequence.
DNA helicase activity (recBC subunit) and
DNA-dependent ATPase activity
unwinds DNA to generate SS regions
Activity 2 and 3 linked

14. RecA 38 kDa protein that polymerizes onto SS DNA 5’-3’
Catalyzes strand exchange, also an ATPase
Also binds DS DNA, but not as strongly as SS
Fig. 22.6

15. RecA Function Dissected
3 steps of strand exchange:
Pre-synapsis: recA coats single stranded DNA (accelerated by SSB, get more relaxed structure, Fig. 22.8)
Synapsis: alignment of complementary sequences in SS and DS DNA (paranemic or side-by-side structure)
Post-synapsis or strand-exchange: SS DNA replaces the same strand in the duplex to form a new DS DNA (requires ATP hydrolysis)

16. RuvA and RuvB DNA helicase that catalyzes branch migration
RuvA tetramer binds to HJ (each DNA helix between subunits)
RuvB is a hexamer ring, has helicase & ATPase activity
2 copies of ruvB bind at the HJ (to ruvA and 2 of the DNA helices)
Branch migration is in the direction of recA mediated strand-exchange

17. The RuvA protein binds and forces the holiday junction into a flat planar structure.
The number of RuvA subunits in this figure is not clear. Should be 4 or 8 (see fig in Weaver).
When helicases unwind DNA only one strand of the double-helix passes through the center.
The helicase on the right is actually unwinding both the all yellow and all-blue helices, as is the helicase on the left.
Having the unwound DNA strands (2 of them) pass through the center of the helicase also promotes annealing of the heteroduplex.

19. RuvC : resolvase Endonuclease that cuts 2 strands of HJ
Binds to HJ as a dimer
Consensus sequence: (A/T)TT (G/C)
- occurs frequently in E. coli genome
- branch migration needed to reach consensus sequence!

20. RuvC bound to Holliday junction
The formation of the dimer in this way positions the active sites so they can cut the
Fig a

21. Transposable Elements (Transposons)
DNA elements capable of moving ("transposing") around the genome
Discovered by Barbara McClintock, largely from cytogenetic studies in maize, but since found in most organisms
She was studying "variegation" or sectoring in leaves and seeds
She liked to call them "controlling elements“ because they effected gene expression in myriad ways

22. Mutant Kernel Phenotypes
Pigmentation mutants
affect anthocyanin pathway
elements jump in/out of transcription factor genes (C or R)
sectoring phenotype - somatic mutations
whole kernel effected - germ line mutation
Starch synthesis mutants
- stain starch with iodine, see sectoring in endosperm

23. Some maize phenotypes caused by transposable elements excising in somatic tissues.
Start with mutant kernels defective in starch synthesis (endosperm phenotypes) or anthocyanin synthesis (aleurone and pericarp phenotypes).

24. Somatic Excision of Ds from C
Wild type
Mutant
Sectoring
Fig

25. Other Characteristics of McClintock's Elements
Unstable mutations that revert frequently but often partially, giving new phenotypes.
Some elements (e.g., Ds) correlated with chromosome breaks.
Elements often move during meiosis and mitosis.
Element movement accelerated by genome damage.

26. Molecular analysis of transposons
Transposons isolated by first cloning a gene that they invaded. A number have been cloned this way, via "Transposon trapping“.
Some common molecular features:
Exist as multiple copies in the genome
Insertion site of element does not have extensive homology to the transposon
Termini are an inverted repeat
Encode “transposases” that promote movement
A short, direct repeat of genomic DNA often flanks the transposon : “Footprint”

27. Ac and Ds Ds is derived from Ac by internal deletions
Ds is not autonomous, requires Ac to move
Element termini are an imperfect IR
Ac encodes a protein that promotes movement - Transposase
Transposase excises element at IR, and also cuts the target

28. Structure of Ac and Ds deletion derivatives
Ds is not autonomous, requires Ac to move!
Fig

29. How duplications in the target site probably occur.
Duplication remains when element excises, thus the Footprint.

30. Mutator (A Retrotransposon)
Discovered in maize; differs significantly from Ac by structure and transposing mechanism
Autonomous and non-autonomous versions; many copies per cell
contains a long terminal IR (~200 bp)
transposes via a replicative mechanism, instead of a gain/loss mechanism
A “retrotransposon”
Similarities with retroviruses
move via an RNA intermediate
encode a reverse transcriptase activity

31. Fig in Buchanan et al.

32. Control of Transposons
Autoregulation: Some transposases are transcriptional repressors of their own promoter(s)
e.g., TpnA of the Spm element
Transcriptional silencing: mechanism not well understood but important; correlates with methylation of the promoter

33. Biological Significance of Transposons
They provide a means for genomic change and variation, particularly in response to stress (McClintock’s "stress" hypothesis)
(1983 Nobel lecture, Science 226:792)
or just "selfish DNA"?
No known examples of an element playing a normal role in development.