Lecture 9 Site Specific Recombination and Transposition

Quiz 5 due today at 4 PM

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Examples of site specific recombination and transposition

Different than homologous recombination

“jumping gene”

• Responsible for important DNA rearrangements

CSSR = recombination between 2 defined sites

CSSR can be used to control gene expression.DNA inversion can allow an alternative gene to be expressed.

Transposition = recombination between specific sequences and non-specific DNA sites

Transposition is a (the) major source of spontaneous mutation.

Nearly half the human genome is transposon-derived sequences.

Conservative site specific recombination (CSSR) and transposition

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Certain viruses use site specific recombinationto integrate into the host chromosomeExample: lambda phage

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3 Types of conservative site specific recombination (CSSR)

A, B, X, and Y denote specific genes.The recombinase recognition sequences are dark orange and blue.Gray regions with white arrows are the crossover regions.

Direct repeats Direct repeats Inverted repeats

2 sites on different DNA molecules 2 sites on the same DNA molecule

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Structures involved in CSSR

Example of an insertion(orange DNA is usually circular)

Recombinase recognitionsequences are symmetric

Crossover region is assymetric

4 subunits of recombinase bind

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Synaptic complex

2 types of recombinasesSerine recombinase and tyrosine recombinase

Both use a covalent protein-DNA intermediate

Conservative in CSSR refers to energy; No ATP needed

Recombinasebinds 5’- phosphate

Leaves free 3’-OH tobind another DNA strand

Links DNA to3’-OH on new strand

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Mechanism of serine recombinase

Makes double stranded breakthat is staggered by 2 bases

All 4 strands cleaved beforestrand exchange

5’- phospho-serine and free 3’-OH formed.

R2 segment recombines withR3 segment

R4 segment recombines withR1 segment

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Mechanism of tyrosine recombinase

Break and rejoin one DNA strand at a time. R1 and R3 break and rejoin segments first.

3’- phospho-tyrosine and free 5’-OH formed.

A Holliday junction is formed.

R2 and R4 segments then recombine using the same mechanism to resolve junction.

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Phage P1 Cre recombinaseis a tyrosine recombinasesometimes used in genetic engineering

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Homologous recombination duringDNA replication of circular chromosomescan generate circular multimers.

CSSR is needed to resolve these multimersback into the monomers.

These recombinases are called resolvases.

XER tyrosine recombinase (resolvase)is an example from bacteria

Animation

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Transposable elements or transposons

Little selectivity in site selection. Can insert in the middle of genes. Less than 2% of human transposons code for proteins.

Non-replicative (cut & paste) Replicative (copy & paste)

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Some organisms (yeast, fruitflies, and E. Coli) contain few transposons.

Occurrence of transposons (green) in genomes

12_Figure18.jpg 3 classes of transposons

(LTR retrotransposons)

(Long terminal repeat)(Reverse transcriptase)

RNA bindingenzyme

RT & endonuclease/ Rnase H

Non-viralretrotransposons

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DNA transposons: Cut &

paste mechanism of transposition

Old host site is grayNew host site is blueDouble strand break atold site must be repaired

Staggered cleavage ofnew target site DNA

New DNA synthesis givesrise to target site duplication

(transpososome)

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DNA strandtransfer3’-OH of transposon attacksphosphate in DNA to leave anew 3’-OH for polymeraseto add onto for the gap to befilled. Then ligase seals nick.

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Transposase only cleaves one strand. 3 ways to cleave nontransferred strand (5’-end) before strand transfer

A different endonuclease(TnsA)

3’-OH on trnspsn 3’-OH on host DNA

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DNA transposons: Replicative “copy & paste” transposition mechanism

Transposase nicks DNA to createthe 3’-OH at each end for strand transfer(same as in cut & paste)

3’-OH on each end cleavesand binds target DNA

DNA replication machinery then useseach 3’-OH on target DNA as a primerand copies the transposon

This leaves a large circular DNAmolecule containing 2 copies ofthe transposon

Animation

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Virus-like retrotransposonsuse an RNA intermediatebut otherwise use the samemechanism as in DNA transposition

Integrated virus

Virus uses an integrase related to DNA transposases.Integrase cleaves 2 nucleotides from the 3’-end, which is then used for DNA strand transfer.

Viruses need to use a special mechanismto regenerate the ends of the LTRs so they can be recognized by integrase.

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Target-site-primed reverse transcriptionof Poly-A (non-LTR) retrotransposonsHuman transposon LINEs (long interspersednuclear elements) use this mechanism.

LINE DNA transcribed. mRNA exported from nucleus.ORF1 and ORF2 proteins are translated from mRNA,remain attached to 3’-end of transcript, and transport mRNA back into the nucleus where itassociates with T- rich DNA by use of the poly-A tailTo form a DNA:RNA hybrid.

ORF2 (RT and endonuclease) nicks DNA generatinga 3’-OH for reverse transcription of RNA back into DNA that integrates into host DNA. DNA joining and repair establish the newly produced LINE element.

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Genetic organization of a LINE and SINE

20 % of the human genome is LINE elements (example L1).LINEs donate the proteins to replicate SINEs (100-400 bpsequences that make up 13 % of human genome). The Alu sequence is a SINE. Pseudogenes can be made by LINE proteins mistakingly binding and reverse transcribing normal mRNA.

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