12. Site Specific Recombinations

site specific recombination occurs between sequences with a

limited stretch of similarity; involves specific sites (recombination sites)

regions with similar sequence will “flank” DNA

that can be moved or rearranged

three types/functions of site specific recombination

1. inversion

2. insertion

3. deletions

inversion e.g

expression of alternative genes - inversion of DNA sequence/gene by DNA invertases

Insertion e.g

infection - insertion of bacteriophages in the bacterial genome by DNA integrase

Deletions e.g

reversal of insertion, mediation of programmed DNA rearrangement during embryogenesis

components of the site-directed recombination are

1. rearrangement enzymes

2. recombination site

rearrangement enzymes - recombinases (in some cases accessory proteins are required0

site specific recombinases cleave and rejoin DNA using a covalent protein-DNA intermediate

a) site specific endonuclease activity (cleavage)

b) DNA ligase activity (rejoining)

Recombination site (specific sequence) places where DNA exchange will occur

c) recombinase recognizes sequence

d) non-palindromic (asymmetric) sequences

recombination sites in SAME ORIENTATION, TWO different DNA molecules

insertion (integration; lambda integrase)

recombinatino sites in SAME orientation, same DNA moolecule

deletion (excision)

Recombination sites in INVERTED orientation, same DNA moelcule

Inversion* (DNA invertase)

DNA deletions and insertions used ro recycle genes

• recombination sites are recognized by specific recombinase

• recombination sites are flanking the gene (e.g antibiotic resistance gene) in direct orientation and on the same DNA molecule

steps for DNA deletions and insertions

1. recombination sites align next to each other

2. recombinase resolves the structure and cuts out the insert gene

both the insertion of the lambda phage DNA into bacterial genome and its deletion from it are accomplished by

site-specific recombination event, catalyzed by the lambda integrase enzymel insertion invovles some bacterial enzyme as well

when the lambda DNA enters the cell, the ends join to form a circular DNA molecule, it can go in two pathways

1. prophase pathway

   insert its DNA into bacterial genome and enter a latent prophage state

2. lytic pathway

   lambda bacteriophage can multiple in in E.coli, cell lysis releases a large number of new viruses and destroys cell

prophage can exit the host chromosome and shift to

lytic growth (induction)

DNA invertases

• recombination sites are repeated sequences but in inverted orientation on the same molecule DNA molecule

• recombination sites flank the DNA sequence which is to be inverted

STEPs for DNA inversion

1. recombination sites align next to each other

2. cross-over and resolution by recombinase → DNA sequence is inverted

Hin recombinase in Salmonela - invertase inverts segment of the bacterial genome (around 1kb)

• controls expression of two alternative sets of genes that code for two alternative forms of protein flagellin

• recombination sites hixL and hixR

nonhomologous recombination

transposable (mobile, transposon) elements insert into DNA that has no sequence homology with the transposon

two sites in transposition

1. donor site

   contains a transposable element (transposon)

2. target site

   usually random, however, there are hot spots; preferred sequences that are targeted

autonomous

encode all the enzymes necessary to move

nonautonomous

have no coding capacity (their mobility depends on the enzymatic machinery of their autonomous “relatives”)

non-replicative transposition

“cut and paste”

both strands of original transposon DNA move together from 1 place to another without replicating

replicative transposition

• copy and paste

• involves DNA replication phase → 1 copy of a transposon remains at original site

• new copy inserts at the new site

transposons can cause genetic changes

(horizontal transfer - contributions to the evolution of genomes)

transposons insert

• into genes/coding sequences

• regulatory sequences (induce change in gene expression)

transposons can also form

chromosomal rearrangements and relocate genes

insertion of transposon in protein-coding region

disrupts the protein sequence

transposon element insertions represent the major source of

spontaneous mutations in drosophila melanogaster

ultrabithorax mutant

double thorax/wing mutation is due to insertion of Doc transposable element into the first exon of the Ubx gene

in humans, transposon elements

1. Haemophilia A: insertions of L1 element into exon 14 of the factor VIII gene in two of 240 unrelated patients with haemophilia A

2. Primary breast cancer: insertion of Alu in BRCA2 gene

however, possible positive effects of transposition - domestication of genes carried by transposons

systems with transposon- derived genes thru domestication

1. initiation of meiotic recombination (possible role remodelling)

2. regulation apoptosis

3. control of cell cycle

4. defense from transposon invasion

5. regulation of transcription

DNA-only transposons

short inverted repeats at each end

retroviral-like retrotransposons

directly repeated long terminal repeats (LTRs) at each end

nonretroviral retrotransposons

poly A at 3’ end of RNA transcript; 5’ end is often truncated

LTR

long terminal repeats exist in retroviruses; substitute “LTR” with retroviral-like

DNA-only Transposons: Insertion Sequence (IS) elements

1. simplest type of transposable element found in bacterial genomes and plasmids

2. encode only genes for mobilization and insertion (host replication machinery used for replication)

3. transposon size from 768 bp to 5 kb

4. end of all IS elements show Inverted Terminal Repeats (ITRs)

Retroviruses:

exist as a ss RNA genome packed into capsid, together with reverse transcriptase (protein) and the dsDNA form has terminal repeat sequences (LTR = direct long terminal repeats)

retroviral like

1. LTRs

2. RNA (intermediary)

3. codes for enzymes

Retroviral-like retrotransposons - cannot move from cell to cell; moves DNA to new location on chromosome

Terminal repeat sequences for recombination, R (binding of integrase) are embedded within LTRs (long direct terminal repeats) on the ends of the element

in retroviral-like retrotransposons: transcription and translation of transposon DNA starts at

promoters (P) in LTR: transcripts (RNA) are templates for cDNA that will be integrated AND codes for enzymesL reverse transcriptase (rvt) and integrase (int)

LINEs (long-interspersed sequences)

• up to 6.5 kb sequences, repeated 50,000-100,000X (~5% of genome)

• presumable transcribed from internal (downstream) RNAP II promoter (do not have LTRs)

majority LINEs encode two ORFs whicha re transcribed as a

bicistronic mRNA composed of ORF1 (RNA binding protein, etc.) and ORF2 (endonuclease and reverse transcriptase activities)

• code for proteins necessary to reverse transcribe and integrate SINEs as well

first: transcription of the transposon (L1: LINE) DNA into L1RNA then

L1RNA translated into L1 reverse transcriptase/endonuclease

Transposition in nonretroviral retrotransposons or Poly-A retrotransposons

1. L1 reverse transcriptase/endonuclease attaches to teh L1 RNA

2. complex makes a nick in the target DNA at the point of the future insertion

3. release of a 3’OH DNA end int he target DNA will be used as a primer for the reverse transcription, does “target site primed reverse transcription” of itself

4. single-stranded DNA copy of the transposon still directly linked to the target DNA

5. generation of a new dsDNA copy of the L1 element that is inserted at teh site where the initial nick was made

SINEs (short-interspersed sequences)

• ~ 300 bp long, repeated 300,000 - 500,000X

• flanked by 7-20 bp direct repeats

• do not code for proteins so they cannot transpose by themselves

• smoe are transcribed into RNA intermediate (contain poly-A tails for priming reverse-transcription, but reverse transcriptase/endonuclease machinery used is from LINEs)

evolutionary origin of SINEs

tRNA gene or other small RNA genes

humane genome: Active SINE element is called Alu

during the cellular heat shock response, human Alu RNA blocks transcription by binding to RNAP II; it enters into transcriptional machinery complexes at promoters in human cells in vitro - acts as a transacting, promoter specific, transcriptional repressor

transposon elements found in bacteria:

1. transposase gene: encoding enzymes for DNA breakage and joining

2. red segment: DNA sequences as a recognition sites for enzymes

3. yellow segments: antibiotic resistance genes

barbara mcClintock

nobel prize in physiology and medicine 1983: for her discovery of mobile genetic elements

• studied transposable elements in corn

McKlintock’s discvery

muation and its reversion result from Ds (dissociation) element

• transposes into the C gene

• mutates pigment gene (yellow)

• transposes out again; color reverted to wild type (purple)

Ds cannot transpose by itself, must be a

non-autonomous transposon, must have help from an autonomous transposon, Ac (for activator)

Ac carries transposase, Ds is an AC element with most of its middle removed

DS has only

• pair of inverted terminal repeats (insertion into C)

• adjacent short sequences that Ac transposase can recognize

spotting in the maize kernels

multiple reversions of an unstable mutation in the C locus (responsible for purple kernel colour)