Site-specific recombination and transposition of DNA 2_1.ppt

Site-specific recombination and transposition of DNA 2 Chapter 11 pp 339-368 DNA transposons DNA transposons transpose by a cut and paste mechanism DNA strand Transfer Transposition leads to a direct target site duplication at the TE integration site 3? A C T T T G C 3? T G A A A C G TGAAACG ACTTTGC ACTTTGC TGAAACG TGAAACG ACTTTGC The non-transferred strand may be cut by alternative mechanisms P element transformation in Drosophila LTR-retrotransposons LTR-retrotransposons and retroviruses move using an RNA intermediate Functional and structural elements of LTR-retrotransposons and retroviruses PBS: primer binding site PPT: polypurine tract GAG: viral capsid proteins ENV: membrane associated envelope proteins POL: encodes for a polyprotein responsible for all enzymatic activities: Protease RT RNase H integrase Integrases promote integration of DNA at the target site and are structurally conserved among transposable elements across TE classes Poly-A retrotransposons lack LTRs All promoter elements are downstream of the transcription initiation site It also encodes for a Reverse transcriptase (RT) but Poly-A retrotransposons require an alternative mechanism of transposition ORF1 encodes Ribonucleoproteins that interact with the RNA ORF2 encodes RT plus endonuclease activity Poly-A or non-LTR retrotransposons move by a target site primed reverse transcription Examples of TEs Tn10 are composite bacterial transposons IS10 elements can transpose independently, but the association with resistance genes provides an advantage to the Tn10 composite Tn10 autoregulates its own transposition by transcribing an antisense RNA Levels of antisense are regulated by the number Tn10 copies in the genome (high number = high levels) Bacteria regulates Tn10 by DNA methylation and Tn10 transposition is coupled to the host DNA replication Tn10 uses IHF from l-phage and transposes only when l-phage integrates in the genome High levels of Mu transposition are possible because of Target Immunity mechanisms Phage Mu is similar to l-phage The Mu genome has more than 35 genes During lytic cycle, Mu can transpose more than 100 times per hour Mu induces high rates of mutations in the host bacteria (Mu stands for Mutator) LTR-Retrotransposons transpose via viral particles similar to those of retroviruses. ?save heavens? in the genome lead to site specific integration of TEs Other examples: Transposable elements integrating in the telomeres in insects Transposable elements integrating in rDNA. Ty1 and Ty3: integrate upstream start sites from promoters initiated by Pol III (tRNA genes) Ty5: integrate in regions that are preferentially silenced (telomeres, rDNA) Integrases promote integration of DNA at the target site and are structurally conserved among transposable elements across TE classes LINES and SINES in vertebrates transpose by a target-site-primed reverse transcription LINE: long interspersed nuclear element (human LINE-1 is the most common) SINE: Sort interspersed nuclear element (alu sequences are the most common) LINES provide the enzymatic machinery for SINE transposition and are the origin of processed pseudogenes in mammals Why LINES do not lead to more unspecific cellular mRNA retrotransposition? LINES and SINES make up more than 30% of the human genome piwiRNAs control TEs transposition in higher eukaryotes TEs may have originated important cell functions example: generation of antibodies in vertebrates V(D)J recombination takes place between V D and J regions in genes encoding antibodies DNA encoding heavy chain The mechanism leading to variable regions in antibodies is know as V(D)J recombination and occurs during differentiation of B-cells DNA encoding light chain Recombination signal sequences (RSS) directly participate in V(D)J recombination Strand cleavages during V(D)J recombination are similar to transposition excision RAG1 and RAG2 are recombinases and they introduce single strand breaks at the junctions between the RSS and the gene segment that will be rearranged. RAG proteins function similar to Hermes transposases and have their origin in the Transib DNA transposon.