Chapter 12

Question 0

CSSR recombination site mechanism

Why conservative? Intermediates?

Answer 0

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.

Question 1

Turbosina recombinases

Answer 1

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”.

Question 2

Cre lox recombination, what is it

Answer 2

• 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.

Question 3

Cre lox recomb mechanism

Answer 3

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.

Question 4

Biological function of site specific recomb

Answer 4

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.

Question 5

Gamma integrase and mechanism

Answer 5

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).

Question 6

IHF in gamma integrase mech

Answer 6

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.

Question 7

Gin recomb and mech

Answer 7

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.

Question 8

Him recombination’s DNA enhancer

Answer 8

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.

Question 9

Transposons

Answer 9

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

Question 10

Transposons may…

Answer 10

cause mutations.
- increase (or decrease) the amount of DNA in the genome.
- promote genome rearrangements.
- regulate gene expression.
- induce chromosome
breakage and rearrangement.

Question 11

IS insertion seq transposons effect on chromosomal DNA

Answer 11

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.

Question 12

Types of transposons

Answer 12

Poly A retrotransposons, virus retrotransposons, DNA transposons

Question 13

Transposon mechs

Answer 13

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”

Question 14

Three mechanisms for cleaving the nontransferred strand

Answer 14

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.

Question 15

Poly-A retrotransposons (e.g. human LINE element) move by a “reverse splicing” mechanism. Explain

Answer 15

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.

Question 16

Mechanism of retroviral integration and transposition of virus-like retrotransposons

Answer 16

1. Transcription of RNA using a promoter sequence within one of the LTRs;
2. Reverse transcription to generate cDNA (copied DNA);
3. Integrase assembles at the ends of the cDNA and cleaves a few nucleotides off the 3’ end of each strand;
4. 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.
5. 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)

Question 17

BACTERIAL TRANSPOSONS - TYPES
(4)

Answer 17

1. Insertion sequence 2.Composite transposon 3.Tn3-type transposon 4.Transposable phage

Question 18

Bacterial transposons insertion seq of bacterial IS element

Answer 18

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.

Question 19

Tn10 transposons and regulation

Answer 19

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.

Question 20

Antisense regulation of Tn10 expression

Answer 20

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.

Question 21

Mu phage

Answer 21

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

Question 22

Mu phage transposition mech

Answer 22

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.

Question 23

Mu phage target immunity

Answer 23

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.

Question 24

Eukaryotic transposons

Answer 24

Eukaryotic Transposons
• Transposons have powerful selective forces on their side
• Transposons carry genes that are an advantage to their
hosts
– Their host can multiply at the expense of completing organisms
– Can multiply the transposons along with rest of their DNA
• If transposons do not have host advantage, can replicate themselves within their hosts in a “selfish” way

Question 25

Ds Ac system of maize transposons

Answer 25

Ds and Ac of Maize (Corn)
• Mutation was resulted from the insertion of transposable element
{Ds (dissociation)} element into the C gene.
• When Ds is mutated, transposes is out again, it reverts to wild type
• Ds cannot transpose on its own
• Must have help from an autonomous transposon, Ac (for activator)
– Ac supplies transposase
– Ds is an Ac element with most of its middle removed – Ds needs
• A pair of inverted terminal repeats
• Adjacent short sequences that Ac transposase can recognize

Question 26

Immune System Diversity

Answer 26

• Enormous diversity of immune system is generated by 3 basic mechanisms:
– Assembly of genes for antibody light chains (Ƙ and λ) and heavy chains from 2 or 3 component parts
– Joining the gene segments by an imprecise mechanism that can delete bases or add extra bases
– A high rate of somatic mutations, during proliferation of a clone of immune cells (this only applies to the B cell receptor/antibody)

Question 27

Retrotransposon Ty

Answer 27

Target-site preference: Ty1, Ty2, Ty3 and Ty4 insertions are near tRNA genes, which are transribes by the cellular RNA Polymerase III. It is away from the important regions of the genome that are involved directly in coding for proteins. That is important for gene-rich genomes, such as yeast.

Question 28

Retrotransposons

Answer 28

Several eukaryotic transposons transpose in a way similar to retroviruses
– Ty (transposon in yeast) of yeast
– copia of Drosophila
• Start with DNA in the host genome
– Make an RNA copy
– Reverse transcribe it within a virus-like particle into DNA that can
insert into new location
• Humans also have LTR containing retrotransposons, but they lack a functional env gene. HERVs likely transposed in the same way until the ability to transpose was lost
– HERV = human endogenous retroviruses (1-2% of the genome)

Question 29

Non-LTR Retrotransposons

Answer 29

Non-LTR Retrotransposons
• LTR are lacking in most retrotransposons
• Most abundant type lacking LTR are LINEs and
LINE-like elements
– Long interspersed elements
– Encode an endonuclease that nicks target DNA
– Takes advantage of new DNA 3’-end to prime reverse transcriptase of element RNA
– After 2nd strand synthesis, element has been replicated at target site
• New round of transposition begins when the LINE is transcribed
• LINE polyadenylation signal is weak, so transcription of a LINE often includes exons of downstream host DNA

Question 30

LINE

Answer 30

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.

Question 31

Diseases caused by L1-mediated mutations

Answer 31

Diseases caused by L1-mediated mutations
L1 element in:
Blood clotting factor VIII hemophilia A and B
DMD gene Duchenne muscular dystrophy
APC gene adenomatous polyposis coli (Colon Cancer)
Beneficial effect:
There is significant homology between L1 and human telomerase (L1 may have been the origin of the enzyme that maintains the ends of our chromosome).

Question 32

Transposons causing diseases

Answer 32

Transposons causing diseases
• Transposons are mutagens. They can damage the genome of their host cell in different ways:
1. A transposon or a retroposon that inserts itself into a functional gene will most likely disable that gene.
2.After a transposon leaves a gene, the resulting gap will probably not be repaired correctly.
3.Multiple copies of the same sequence, such as Alu sequences can hinder precise chromosomal pairing during mitosis and meiosis, resulting in unequal crossovers, one of the main reasons for chromosome duplication.