Genetics: Cre and Transposon Functions

DNA Mobility

• DNA mobility refers to the movement of DNA elements within a genome
• This movement impacts genome structure, function, and evolution
• Some mobile elements use RNA as an intermediate, while others use only DNA
• Mobility can be random or directed
• Mobile elements have been exploited for experimental mutagenesis and gene transfer

Mobile Genetic Elements

• Mobile genetic elements (MGEs) are common in all life forms (prokaryotes, archaea, and eukaryotes)
• They drive genomic plasticity
• MGEs influence genome structure, function, and evolution
• Some MGEs move through RNA intermediates; others only use DNA
• Their movement can be random or programmed
• Scientists use them in experimental mutagenesis and gene transfer

Transposition

• Transposition is the movement of a discrete DNA element to different target sites within a genome
• Two common mechanisms:
  • excision and integration
  • replication
• The movement is facilitated by proteins that are specific to the particular element
• Transposition often involves the duplication of the target DNA sequence, a "target site duplication" (TSD).

Transposable Elements and Genetic Diversity

• Transposable elements (TEs) are major sources of genetic diversity
• TEs disrupt genes within and between chromosomes, potentially altering gene expression
• TEs can alter gene expression by introducing regulatory signals near host genes (promoters, enhancers, splice sites, polyA sites)
• TEs are substrates for homologous recombination, influencing genome dynamics
• Transposable elements are highly dynamic parts of genomes

Transposons and Mutations

• Transposons are a type of transposable element that causes mutations
• Barbara McClintock studied transposons in maize, demonstrating how their movement affects pigment expression

Active Human Transposons

• Researchers identified active human transposons from mutations in patients
• Haig Kazazian identified insertions in the Factor VIII gene in unrelated boys
• The insertions were not in their parents, indicating recent activity of transposable elements
• The inserted sequences were non-LTR LINE elements

Frequency of Transposon Insertion

• Transposon insertion frequency varies across species:
  • 0.3% in humans, where TEs comprise about 45% of the genome
  • 10% in mice, where TEs comprise about 38% of the genome

  • 50% in Drosophila, where TEs comprise about 5.5% of the genome.

• Most insertions in humans and mice are non-coding, relatively inconsequential

Classification of Transposable Elements

• Transposable elements (TEs) are categorized based on various criteria, including structure, transposition mechanism, and effects on the donor site.
• Major groups are defined by structure (DNA-only, RNA intermediates, LTR, non-LTR elements)

Genome Components in Different Organisms

• The slides present a comparative chart reflecting the relative abundance of various transposable elements in bacterial, archaeal, and eukaryotic genomes, in different species (e.g. humans, mice, yeast, maize, Drosophila).

DNA-only Transposons

• DNA-only transposons move via cut-and-paste or replicative mechanisms
• Terminal inverted repeats (TIRs) are essential for their movement

Long Terminal Repeat (LTR) Elements

• These elements exhibit an RNA-DNA intermediate cycle
• LTRs facilitate RNA to cDNA conversion for integration into the host genome
• Retroviruses disseminate extracellularly, using an envelope protein
• Retroviral-like elements replicate intracellularly

Non-LTR Elements

• LINEs (Long Interspersed Elements) are autonomous, possessing their transposing machinery
• SINEs (Short Interspersed Elements) lack their own transposition machinery and rely on LINEs
• Both LINE and SINE elements occupy a significant portion of the human genome

DNA Transposon Superfamilies

• A variety of distinct transposon superfamilies are highlighted, including bacterial (IS elements, Mu, Tn elements) and eukaryotic families (Mutator, Tc1/mariner, hAT, piggyBac).

DNA-only Cut and Paste Elements

• IS elements contain inverted repeats flanking a transposase gene
• Composite transposons comprise multiple IS elements flanking other genes

DNA-only Cut and Paste Transposition Strategy

• The strategy involves transposase binding, DNA cleavage, excision, target joining, gap repair, leading to TSD formations

Excision Pathways and 3' OH generation

• Various pathways by which transposases carry out excision, producing a 3'OH end that's critical for subsequent reactions

DNA breakage by Transposase

• Transposase-mediated DNA breakage occurs in trans, requiring the specific binding and catalytic domains of the transposase.

RAG and VDJ Recombination

• RAG and VDJ recombination systems employ nicking and attack mechanisms to liberate spacers from the intermediate segments generating 3'OH DNA ends, similar to transposon activity.

Replicative Nick and Paste Transposition

• This process involves the replication of the mobile element before inserting a copy at a new site.

Co-integrate Resolution

• Co-integrate resolution involves using conservative site-specific recombination, a specific recombination method using enzymes called resolvases.

LTR Elements

• Retrotransposons (Ty and Gypsy) and retroviruses (MuLV, RSV, HIV) use an RNA intermediate in their transposition cycle.

LTR Retrotransposons – Intracellular Propagation

• The processes involve assembly, translation, reverse transcription, and integration (using integrase) to ensure the propagation of LTR retrotransposons.

Retroviruses – Intercellular Propagation

• Retroviruses use RNA as an intermediate, with processes involving integration and packaging into new viral particles to propagate intercellularly

LTR Elements (cont)

• The RNA intermediate produced during retrotransposition contains information from both 5' and 3' LTRs

Synthesis of Long Terminal Repeat (LTR) Element DNA

• The synthesis of LTR DNA from RNA depends on reverse transcription. This process preserves the integrity of the retroviral genome.

Integration of CDNA Intermediates

• Integrase facilitates the integration of cDNA intermediates into the host genome.

Structural Similarity

• HIV-1 integrase and MuA transposase exhibit structural similarities, particularly in their catalytic cores. Key residues in integrase and transposase catalyze DNA cleavage, which is critical.

Non-LTR Retrotransposons

• LINEs (Long Interspersed Elements) and SINEs (Short Interspersed Elements) are two primary classes of Non-LTR retrotransposons. LINEs are autonomous and SINEs are non-autonomous.

Transposition of Non-LTR elements

• Non-LTR element transposition generally involves RNA intermediates and reverse transcription.

Non-LTR Retrotransposons (cont.)

• Group II introns are self-splicing mobile genetic elements (MGEs).

Conservative Site-Specific Recombination (CSSR)

• This process involves site-specific recombinases without affecting DNA sequence length and relying solely on short DNA sequences and recombinases. Recombinases carry out recombination at specific sites

Recombination

• Recombination mechanisms are illustrated and classified based on the homology (direct or inverted repeats) required between DNA sequences.

The λ Integrase family

• Tyr recombinases of site-specific recombinases, more than 100 such enzymes are known. Examples: bacteriophage λ, P1 phage Cre, and S. cerevisiae Flp systems.

Resolution of P1 Replicative Intermediates

• This process involves resolvase and loxP sites that yield two separate DNA molecules.

A 34-bp loxP site

• This specific site sequence is essential for recombination with Cre recombinase.

Synapsed Substrates for Cre-mediated Recombination

• Graphical representations show Cre-mediated recombination substrates undergoing synapsis. These are the specific substrates forming the structure for Cre-mediated recombination.

Model for Cre-mediated Recombination

• Model illustrating Cre recombination, using Holliday junctions as the intermediate (structural components that facilitate recombination).

Transposase-mediated DNA Fragmentation

• Describes the process of transposase-mediated insertion and DNA fragmentation and the related methodology

ATAC-seq Chromatin Accessibility

• The technique involves Tn5 transposase mediated oligonucleotide insertions for genome-wide mapping of chromatin accessibility
• Graphical representations and related quantitative data are provided.

Nucleosome Mapping

• Shows how analysis of genomic fragments produced by a DNA-fragmenting transposase (from Tn5) helps identify the centers of nucleosomes in the cell.

Transposon Mutagenesis

• Transposon mutagenesis approaches for identifying and characterizing essential genes in various organisms, including mice and Candida glabrata

Cre-activated Transposon Mutagenesis

• An approach for discovering genes involved in cancer, using Cre-mediated transposon insertion for targeted mutagenesis and analysis.