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DNA Recombination. Roles Types Homologous recombination in E.coli Transposable elements. Biological Roles for Recombination. Generating new gene/allele combinations (crossing over during meiosis) Generating new genes (e.g., Immuno- globulin rearrangement)
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DNA Recombination • Roles • Types • Homologous recombination in E.coli • Transposable elements
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 (or virus) • DNA repair
Practical Uses of Recombination • Used to map genes on chromosomes - recombination frequency proportional to distance between genes 2. Making transgenic cells and organisms
Map of Chromosome I of Chlamydomonas reinhardtii cM = centiMorgan; unit of recombination frequency 1 cM = 1% recombination frequency Chlamydomonas Genetics Center
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
Holliday Model • R. Holliday (1964) • Holliday Junctions form during recombination • HJs can be resolved 2 ways, only one produces true recombinant molecules patch
EM of a Holliday Junction w/a few melted base pairs around junction Fig. 22.3
The recBCD Pathway of Homologous Recombination Part I: Nicking and Exchanging Fig. 22.2 a-d
recBCD Pathway of Homologous Recomb. 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.
recBCD Pathway of Homologous Recombination Part II: Branch Migration and Resolution Fig. 22.5 f-h
recBCD Pathway of Homologous Recom. Part II: Branch Migration and Resolution • Nicks are sealed Holliday Junction • Branch migration (ruvA + ruvB) • Resolution of Holliday Junction (ruvC)
RecBCD : A Complex Enzyme • RecBCD has: • Endonuclease subunits (recBC) that cut one DNA strand close to Chi sequence. • DNA helicase activity (recD subunit) and a DNA-dependent ATPase activity • unwinds DNA to generate the 3’ SS tails
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
RecA binds preferentially to SS DNA and will catalyze invasion of a DS DNA molecule by a SS homologue. Important for many types of homologous recombination, such as during meoisis (in yeast). Fig. 6.19 in Buchanan et al.
RecA Function Dissected • 3 steps of strand exchange: • Pre-synapsis: recA coats single-stranded DNA (accelerated by SSB, so get more relaxed structure). • 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).
RuvA and RuvB • DNA helicase that catalyzes branch migration • RuvA tetramer binds to HJ (each DNA helix between subunits), forces it into square planar conformation • 2 copies of RuvB bind at the HJ (to RuvA and 2 of the DNA helices) • RuvB is a hexamer ring, has helicase & ATPase activity • Branch migration is in the direction of recA mediated strand-exchange
RuvA/RuvB/DNA Complex RuvB RuvA Shows RuvB encircling DNA duplexes
RuvB RuvA RuvA removed for visual purposes only Similar to Figure 22.13 Model based on EM images.
RuvC : resolvase • Endonuclease that cuts 2 strands of HJ • Binds to HJ as a dimer (that already has RuvA/RuvB) • Consensus sequence: (A/T)TT (G/C) - occurs frequently in E. coli genome - branch migration needed to reach consensus sequence!
RuvC bound to a HJ Fig. 22.16
A model for binding of RuvA, RuvB, and RuvC to a HJ. Fig. 22.17b
Meiotic Recomb. in Yeast- is initiated by a double-strand break (DSB) Fig. 22.18
Repair of double-strand breaks (DSBs)in non-dividing or mitotic cells DSBs probably most severe form of DNA damage, can cause loss of genes or even cell death (apoptosis) DSBs caused by: - ionizing radiation - certain chemicals - some enzymes (topoisomerases, endonucleases) - torsional stress
2 general ways to repair DSBs: • Homologous recombination (HR) - repair of broken DNA using the intact homologue, very similar to meiotic recombination. Very accurate. • Non-homologous end joining (NHEJ) - ligating non-homologous ends. Prone to errors, ends can be damaged before religation (genetic material lost) or get translocations. (Mechanism in Fig 20.38) Usage: NHEJ >> HR in plants and animals