Presentation on theme: "DNA Recombination Roles Types Homologous recombination in E.coli"— Presentation transcript:
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 elementDNA 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 frequency1 cM = 1% recombination frequencyChlamydomonas 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 sitesTransposition – DNA element moves from one site to another, usually little sequence similarity involved
7 Holliday ModelR. Holliday (1964)Holliday Junctions form during recombinationHJs can be resolved 2 ways, only one produces true recombinant moleculespatchFig. 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 ExchangingFig 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 ResolutionFig f-h
12 recBCD Pathway of Homologous Rec recBCD Pathway of Homologous Rec. Part II: Branch Migration and ResolutionNicks are sealed Holliday JunctionBranch 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) andDNA-dependent ATPase activityunwinds DNA to generate SS regionsActivity 2 and 3 linked
14 RecA 38 kDa protein that polymerizes onto SS DNA 5’-3’ Catalyzes strand exchange, also an ATPaseAlso binds DS DNA, but not as strongly as SSFig. 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 activity2 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 dimerConsensus 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 theFig a
21 Transposable Elements (Transposons) DNA elements capable of moving ("transposing") around the genomeDiscovered by Barbara McClintock, largely from cytogenetic studies in maize, but since found in most organismsShe was studying "variegation" or sectoring in leaves and seedsShe liked to call them "controlling elements“ because they effected gene expression in myriad ways
22 Mutant Kernel Phenotypes Pigmentation mutantsaffect anthocyanin pathwayelements jump in/out of transcription factor genes (C or R)sectoring phenotype - somatic mutationswhole kernel effected - germ line mutationStarch 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 typeMutantSectoringFig
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 genomeInsertion site of element does not have extensive homology to the transposonTermini are an inverted repeatEncode “transposases” that promote movementA 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 moveElement termini are an imperfect IRAc encodes a protein that promotes movement - TransposaseTransposase 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 mechanismAutonomous and non-autonomous versions; many copies per cellcontains a long terminal IR (~200 bp)transposes via a replicative mechanism, instead of a gain/loss mechanismA “retrotransposon”Similarities with retrovirusesmove via an RNA intermediateencode a reverse transcriptase activity
32 Control of Transposons Autoregulation: Some transposases are transcriptional repressors of their own promoter(s)e.g., TpnA of the Spm elementTranscriptional 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.