Chapter 12 Flashcards
CSSR recombination site mechanism
Why conservative? Intermediates?
The serine residue within the protein’s active site attacks a specific phosphodiester bond in the recombination site. The liberated OH group on the broken DNA can then reattack the protein-DNA covalent bond to reverse the cleavage reaction, reseal the DNA, and release the protein.
It is conservative because every DNA bond that is broken during the reaction is resealed by the recombinase. No energy is required. Protein-DNA covelent intermediates also exist in DNA topoisomerase and Spo11.
Turbosina recombinases
R1 and R3 subunits cleave the DNA; the protein is linked to the cut DNA by a 3’-tyrosine bond;
(2) Exchange of the first pair of strands occurs when the first 5’-OH groups at the break sites each attack the protein-DNA bond on the other DNA molecule. (forms a Holliday junction);
(3) The second-strand exchange occurs by the same mechanism ,using the R2 and R4 subunits, which undoes the “Holliday junction”.
Cre lox recombination, what is it
• Cre is an enzyme encoded by phage P1, which functions to circularize the linear phage genome during infection. The recombination sites on the DNA, where Cre acts, are called lox sites.
• LoxP site:
• Cre-lox is a simple example of recombination by the tyrosine recombinase family; only the Cre protein and the lox sites are needed for complete recombination.
• Cre is also widely used as a tool in genetic engineering.
Cre lox recomb mechanism
Sequential “one strand at a time” mechanism:
At a first glance, the structures appear to have fourfold symmetry, this is not really the case. (two pairs of the adjacent DNA arms are closer than the other two pairs. 105A vs 95A) Only the green subunits in the figure are in the active conformation.
The pair of subunits in the active conformation switches as reaction processes. The switching is critical for controlling the progression of recombination and ensuring the sequential “one strand at a time” exchange mechanism.
Biological function of site specific recomb
Cells and viruses use conservative site-specific recombination for a wide variety of biological functions.
• Many phage insert their DNA into the host chromosome during infection using the recombination mechanism.
• Site-specific recombination can be used to alter gene expression.
Gamma integrase and mechanism
λ integrase promotes the integration and excision of a viral genome into the host-cell chromosome
For bacteriophage λ to integrate to cells, the λ integrase (int) catalyzes recombination between two specific sites (att or attachment site): attp on phage DNA and attB on bacteria.
λint is a tyrosine recombinase. It is unique that it can have one domain binding to the arm recombination recognition sites and one binding to the core recombination recognition sites.
The arms of attP carry sites bound by several architectural proteins. They govern the directionality and the efficiency of recombination.
There are additional protein binding sites flanking the core λint-binding sites. The regions are called P arm and P’ arm.
P1, P2, P1’ are λint binding sites; Hs are integration host factor (IHF) binding sites. X site binds Xis. F is bound by Fis (another architectural protein).
IHF in gamma integrase mech
IHF bends DNA to bring DNA-binding sites together
IHF binds to the H’ site, which bends DNA to allow one molecule of λint to bind both the P1’ and C’ (core). This allows the λint to find the weak core sites and to catalyze recombination.
Gin recomb and mech
The Hin recombinase inverts a segment of DNA allowing expression of alternative genes.
The inverted segment carries the gene encoding Hin, HixL and hixR (Hin recognition sites) as well as a promoter, which is positioned to express the genes located outside of the invertible segments directly adjacent to the HixR site.
• When the invertible segment is in the “ON” orientation, these adjacent genes are expressed, whereas when the segment is flipped into the “OFF” orientation, the genes cannot be transcribed, because they lack a functional promoter.
• fljB: encodes H2 flagellin. FljA encodes a transcriptional repressor for H1 flagellin. This mechanism controls H1-H2 switch.
Him recombination’s DNA enhancer
A short (~60bp) sequence enhancer stimulates the rate of recombination ~1000 fold.
The sequence can be located quite a distance from the recombination sites. Enhancer function requires Fis (factor for inversion) protein. Like IHF, Fis is a site-specific DNA-bending protein (factor for inversion stimulation). For Fis activation of Hin, the three DNA sites (HixL, HixR and enhancer) need to come together.
Another bacterial architectural protein HU, also facilitates the assembly of this
invertasome complex. HU is a structural homolog of IHF, but it binds to DNA in a sequence- independent manner.
Transposons
A transposable element moves from one DNA address to another. (jumping genes, mobile elements, selfish DNA, molecular parasites).
• The term “selfish DNA” implies that insertion sequences and other transposons replicate at the expense of their hosts, providing no value in return. They can disrupt gene function or promoter activity.
• Some transposons do carry genes that are valuable to their hosts, antibiotic resistance is among the most familiar
Transposons may…
cause mutations.
- increase (or decrease) the amount of DNA in the genome.
- promote genome rearrangements.
- regulate gene expression.
- induce chromosome
breakage and rearrangement.
IS insertion seq transposons effect on chromosomal DNA
Process of integration of an IS element into chromosomal DNA. As a result of the integration event, the target site becomes duplicated, producing direct target repeats. Thus, the integrated IS element is characterized by its inverted repeat (IR) sequences, flanked by direct target-site duplications. Integration involves making staggered cuts in the host target site. After insertion of the IS, the gaps that result are filled in with DNA polymerase and DNA ligase.
Types of transposons
Poly A retrotransposons, virus retrotransposons, DNA transposons
Transposon mechs
Transposons are sometimes called “jumping genes”, DNA doesn’t always leave one place for another
• Whenitdoes,nonreplicativetransposition
– “Cut and paste”
– Both strands of original DNA move together from 1 place to another without replicating
• Transposition frequently involves DNA replication
– 1 copy remains at original site
– New copy inserts at the new site – Replicative transposition
– “Copy and paste”
Three mechanisms for cleaving the nontransferred strand
Three mechanisms for cleaving the nontransferred strand
(a) An enzyme other than transposase is used. (TnsA: it has a structure similar to that of a restriction endonuclease)
(b) The transposase (Tns10, Tns5) catalyzes the attack of one DNA strand on the opposite strand to form the DNA-hairpin intermediate. The two hairpin ends are subsequently hydrolyzed by the transposase. Once this is complete, the 3’- OH ends of the element DNA are ready to be joined to a new target DNA by DNA strand transfer reaction.
(c) The Hermes transposon uses a second mechanism of second-strand cleavage by hairpin formation. In this case, cleavage of the top strand (nontransferred strand) occurs first, and the hairpins are generated on the original insertion site DNA, rather than the transposon ends.
Poly-A retrotransposons (e.g. human LINE element) move by a “reverse splicing” mechanism. Explain
Poly-A retrotransposons (e.g. human LINE element) move by a “reverse splicing” mechanism
(a) A cellular RNA Polymerase initiates transcription of an integrated LINE sequence
(b) The resulting mRNA is translated to produce the products of the two encoded ORFs that then bind to the 3’ end of their mRNA
(c) The complex moves to the nucleus and binds to T-rich site in the target DNA and forms a RNA:DNA hybrid
(d) The 3’-OH end of the target DNA serves as a primer for reverse transcription of the element RNA to produce cDNA
(e) Second strand DNA synthesis and DNA joining and repair to create a newly inserted LINE element.
Mechanism of retroviral integration and transposition of virus-like retrotransposons
- Transcription of RNA using a promoter sequence within one of the LTRs;
- Reverse transcription to generate cDNA (copied DNA);
- Integrase assembles at the ends of the cDNA and cleaves a few nucleotides off the 3’ end of each strand;
- Integrase catalyzes the insertion of these cleaved 3’-ends into a DNA target site in the host-cell genome using a DNA strand transfer reaction.
- Host cell gap repair proteins fill the gaps at the target site.
The RNA doesn’t carry the entire LTR sequence. A special mechanism is needed to regenerate the full LTR sequence.
Reverse transcription involves two internal priming and two strand switches, resulting the duplication of the sequences at the ends of the cDNA. (Fig. 23.23 in Weaver’s book)
BACTERIAL TRANSPOSONS - TYPES
(4)
- Insertion sequence 2.Composite transposon 3.Tn3-type transposon 4.Transposable phage
Bacterial transposons insertion seq of bacterial IS element
Bacterial IS element
Central region encodes for one or two enzymes required for transposition. It is flanked by inverted repeats of characteristic sequence.
The 5’ and 3’ short direct repeats are generated from the target-site DNA during the insertion of mobile element.
The length of these repeats is constant for a given IS element, but their sequence depends upon the site of insertion and is not characteristic for the IS element.
Tn10 transposons and regulation
9kb element, contains its own transposase and genes imparting resistance
to antibiotics tetracycline. It carries two “minitransposons” at its termini. Both transposon can transpose (but only the right one is autonomous since the left one encodes a defective transposase gene. White triangles show the inverted repeats at the end of the IS elements, the 4 copies are not exact the same in sequence, all are recognized by the Tn10 transposase and are used in recombination sites.
Antisense regulation of Tn10 expression
Antisense regulation of Tn10 expression
(A) A map of overlapping promoter region. It generates two transcripts with 36 nt complementary to each other. The anti-sense RNA is longer- lived than the transposon mRNA
(B) In cells having high copy number of Tn10, the RNA:RNA pairing occurs frequently and block the translation of the transposase mRNA
(C) In cells having low copy number of Tn10, RNA:RNA pairing is rare, the translation of transposase mRNA is efficient, and the copy number in cells is increased.
Mu phage
Transposable phage Mu is ~40kb, carries more than 35 genes, only two encode proteins (MuA and MuB) with dedicated roles in transposition.
ITR - inverted terminal repeats
Mu phage transposition mech
Overview of the early steps of Mu transposition
MuA is the transposase and is a member of the DDE protein family. MuB is an ATPase that stimulates MuA activity and controls the choice of DNA target site.
A. 4 subunits of MuA assemble on the ends of Mu DNA.
B. MuB binds to ATP and then to any DNA sequence.
C. A protein-protein interaction between MuA and MuB brings the MuA DNA-transposome complex to a new DNA target site.
Mu phage target immunity
Mu uses target immunity to avoid transposing into its own DNA
Mu, like many transposons, show very little sequence preference at it target sites. How does Mu avoid transposing into its own DNA?
Transposition Target immunity: DNA sites surrounding a copy of the Mu element, including the element’s own DNA, are rendered very poor targets for a new transposition event.
MuA inhibits MuB from binding to nearby DNA sites. (MuA stimulates ATP hydrolysis by MuB and the disassociation of MuB from this DNA)
MuB helps MuA find a target site for transposition.
