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 Top map is a map of molecular markers, bottom one is a map of phenotypic markers on the same chromosome 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

Examples of (mostly) Homologous Recombination Fig. 22.1 Crossovers involving circular chromosomes can give different outcomes than the linear DNAs in (a).

Holliday Model R. Holliday (1964) Holliday Junctions form during recombination HJs can be resolved 2 ways, only one produces true recombinant molecules Fig. 22.2 in 2nd edition 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 From 1st or 2nd edition, Fig. in 3rd edition slightly different 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 Nicks are sealed by DNA ligase. Fig. 22.5 f-h

recBCD Pathway of Homologous Recom 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 Homologues found in eukaryotes (Rad51, etc.), mitochondria, and chloroplasts. Figure not in 3rd edition.

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). RecA is the bacterial gene, Rad51 is the nuclear homologue in eukaryotes. 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 Shows RuvB encircling DNA duplexes

RuvB RuvA Similar to Figure 22.13 Model based on EM images. 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 The formation of the dimer in this way positions the active sites so they can cut the 2 strands shown. The yellow balls in figure b are the phosphates on each strand that could be attacked by RuvC. 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) Spo11 makes the Double-strand break 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