Comprehensive Notes on Replication, Recombination, and Transposable Elements

Replication

DNA Replication

• DNA replication relies on the complementarity of DNA strands.
  • A pairs with T, and C pairs with G (AT/GC rule).
• The process involves:
  • The two complementary DNA strands coming apart.
  • Each strand serving as a template for the synthesis of new complementary DNA strands.
  • The newly-made DNA strands are called daughter strands, while the original strands are parental strands.

Replication Initiation

• "Initiation" involves:
  • Finding the start site.
  • Assembling the machinery.

Initiation and Replication

• The entire genome must be replicated precisely, once for every cell division.
• The duplicate genomes are segregated, one to each daughter cell.

Semiconservative Replication

• DNA strands separate at the origin of replication.
• Each parental strand serves as a template for the synthesis of a complementary strand.

Bacteria Replication Initiation

• Three types of DNA sequences in oriC (origin of chromosome replication) are functionally important:
  • DnaA boxes: sites for the binding of DnaA protein.
  • AT-rich regions: sites where the DNA strands separate.
  • GATC methylation sites: sites that help to regulate DNA replication.

E. Coli OriC

• OriC is a type of DNA element containing sequences that certain proteins can specifically bind to (i.e., recognize) that sequence.
• E. coli OriC is approximately 245 base pairs long and contains two types of sequences:
  • DnaA binding sites: dnaA boxes (DnaA is the protein; dnaA box is the DNA sequence). DnaA is the replication initiator, recruiting replication machinery.
  • GATC/CTAG repeats that are methylated on adenine on both strands: can overlap with dnaA boxes, and methylation is required for DnaA binding.

Role of GATC Methylation Sites in Replication

• GATC methylation sites regulate replication.
• DNA adenine methyltransferase (Dam) methylates the Adenine on both strands.
  • Sites are fully methylated prior to replication.
  • Replication only occurs efficiently if DNA is fully methylated.
  • Immediately after replication, the daughter strand is not methylated.
  • This prevents a second round of initiation from occurring too soon.

E. Coli OriC: Methylation

• Two functions of methylation during bacteria replication:

  1. Methylation on both strands is required for DnaA binding and, therefore, is required for replication initiation. Newly synthesized hemimethylated DNA cannot be replicated to prevent premature replication.
  2. DNA methylation also allows DNA mismatch recognition proteins to distinguish the old template from the new strand. Repair will be made based on methylated strand.

• Only methylated origins are functional.

DNA Polymerases

• DNA polymerases are the enzymes that catalyze the attachment of nucleotides during the synthesis of new DNA strands.
• In E. coli, there are five proteins with polymerase activity:
  • DNA pol I and III: Normal replication.
  • DNA pol II, IV, and V: DNA repair and replication of damaged DNA.

Structure of DNA Polymerase III

• Structure resembles a human right hand with thumb, palm, and fingers.
• Template DNA is threaded through the 'palm'.
• Thumb and fingers are wrapped around the DNA.
• The catalytic subunit of all DNA polymerases is similar.

Features of DNA Polymerase

• DNA polymerases cannot initiate DNA synthesis de novo; primers are required.
  • Primase synthesizes RNA primers (~10 nucleotides).
  • A single primer is used on the leading strand, while multiple primers are used on the lagging strand.
• DNA polymerases can attach nucleotides only in the 5' to 3' direction, but the two strands are anti-parallel and go in opposite directions.
• Since strands are antiparallel, synthesis of new strands happens both toward and away from the replication fork.

DNA pol I

• DNA synthesis:
  • Removes RNA primers (5' to 3' exonuclease activity).
  • Fills the gap with DNA (5' to 3' polymerase activity).
• DNA ligase catalyzes the formation of a covalent (ester) bond to connect the DNA backbones.

Replication Complexes

• Primosome: A complex formed by DNA helicase and primase.
  • Moves along the lagging strand, separating DNA strands and adding primers.
• Replisome: A complex of the Primosome with two DNA polymerase holoenzymes.

Termination Sequences

• On the opposite side of the chromosome to oriC is a pair of termination sequences called ter sequences.
  • These are designated T1 and T2 (T1 stops counterclockwise forks, T2 stops clockwise forks).
• The protein tus (termination utilization substance) binds to the ter sequences. Tus bound to the ter sequences stops the movement of the replication forks.

Termination of Replication

• DNA replication often results in two intertwined molecules, termed catenanes.
• These are separated by the action of topoisomerase II, a process called decatenation.

Replication Forks and Directions

• Replication is unidirectional when a single replication fork is created at an origin.
• Replication is bidirectional when an origin creates two replication forks that move in opposite directions.
• There is no difference in the appearance of the replication fork/bubble.

Bacterial DNA Replication: Chemistry and Accuracy

• Step 1: An incoming nucleotide base pairs with a complementary base on the template strand.
• Step 2: The 3'OH on the end of the growing strand reacts with the phosphate group attached to the sugar of the incoming nucleotide.
• Step 3: A covalent (ester) bond is formed, and pyrophosphate (PP) is released (reaction is exergonic).
  O − P − O − P − O − P − O − CH_2

DNA Polymerase III: A Highly Processive Enzyme

• In E. coli, DNA pol III attaches approximately 750 nucleotides per second.
• DNA polymerase III remains attached to the template as it synthesizes the daughter strand.
  • This is referred to as processivity i.e., when an enzyme catalyzes consecutive reactions without releasing its substrate (in this case, no dissociation after forming a covalent bond).
• DNA pol III holoenzyme subunits contribute to processivity:
  • Clamp Protein: The β subunit forms a dimer in the shape of a ring around template DNA. Once bound, the β subunits slide freely along dsDNA and promote the association of the holoenzyme with DNA.
  • Clamp loader: Allows DNA pol III to initially clamp onto the DNA.

DNA Polymerase III: A Processive Enzyme 2

• How processive is DNA pol III?
  • In the absence of the B subunit:
    • DNA pol III would fall off the DNA template after about 10 nucleotides have been polymerized. The rate is approximately 20 nucleotides per second.
  • In the presence of the B subunit:
    • DNA pol III remains on the DNA template and polymerizes approximately 500,000 nucleotides. The rate is approximately 750 nucleotides per second.

DNA Polymerases Activity: Proofreading

• The enzyme adds a base to the growing strand.
• Some polymerases (e.g., E. coli Pol I) have a second enzymatic activity:
  • 3' to 5' exonuclease activity (opposite direction of synthesis) for the removal of bases.
  • Substrate: Mismatched base pair (leading to structural changes in DNA).
  • Proofreading: Error control system.

Fidelity of Replication

• Preventing substitution:
  1. The catalytic center only allows Watson-Crick pairs to form: The geometry of an A-T base pair is very similar to that of a G-C base pair but different from A-C/A-G/G-T/T-C.
  2. Polymerases with high fidelity have a tightly fitted active site so that only an incoming dNTP that base pairs properly with the template nucleotide fits in the active site, whereas mismatches such as A-C or A-A have the wrong geometry to fit into the active site.
     • Low fidelity polymerases (error-prone polymerases, e.g., translesion pol) have a more open active site and therefore can fit non-Watson-Crick base pairs.
  3. Proofreading allows wrong base-pairs to be removed.

Replication II

The Pre-replication Complex

• Origin Recognition Complex (ORC).
• Cdc6/Cdt1 Binding and MCM Recruitment.
• Pre-replication Complex.

Eukaryotic DNA Polymerases

• Mammalian cells have more than a dozen different DNA polymerases.
• Four polymerases alpha (α), delta (δ), epsilon (ɛ) and gamma (γ) have the primary function of replicating DNA.
  • α, 8 and ɛ - Nuclear DNA
  • Y - Mitochondrial DNA

Telomeres and DNA Replication

• Linear eukaryotic chromosomes have telomeres at both ends.
  • Telomere-region of repetitive DNA sequences at the end of a chromosome.
  • Telomeres protect the ends of the chromosomes.
• Telomeric sequences contain moderately repetitive tandem arrays.
  • 3' overhang that is 12 to 16 nucleotides long.
  • May be repeated up to hundreds of times (varies with species).
  • Telomeric sequences tend to contain guanines and many thymines.

Replication Problem at the Ends of Linear Chromosomes

• DNA polymerases possess two unusual features that contribute to the problem at the ends of the strands.
  • Polymerases synthesize DNA only in the 5' to 3' direction.
  • They cannot initiate DNA synthesis on a bare (unprimed) DNA strand.
• Hence, at the 3' ends of linear chromosomes the end of the strand cannot be replicated because a primer cannot be added upstream at the very 3' end of the strand.

Enzymatic Action of Telomerase

• Three steps of telomere lengthening:
  • Step 1: Binding. The repeat sequence in the RNA of the telomerase binds to the complementary sequence at the end of the telomere.
  • Step 2: Polymerization. The remaining RNA serves as the template for the addition of a 6-nt sequence, carried out by telomere reverse transcriptase (enzyme, which uses RNA template to synthesize DNA).
  • Step 3: Translocation. These steps are repeated many times to lengthen one strand (multiple 6-nt sequences are added). Finally, the complementary strand is synthesized using primase, DNA polymerase, and ligase.

Two Types of DNA Synthesis

• Synthesis involved in genome replication.
• Synthesis involved in DNA repair.
• Implication: different polymerases are responsible for different types of DNA synthesis.

DNA Polymerases Classification

• Eukaryote DNA polymerases:
  • Nuclear genome replication.
  • Repair.
  • Translesion replication.
  • Organelle genome replication.
  • More split of labor or specialist.

DNA Polymerases Classification (E. Coli)

• E. Coli DNA polymerases:
  • Enzyme
  • Gene
  • Function
  • Major repair enzyme Most abundant

DNA Polymerases Activity

• Activities common to both prokaryotic and eukaryotic polymerases.
• One fundamental enzymatic activity shared by all DNA polymerases:
  • Catalyzing the formation of a phosphodiester bond between the 3' -OH of one nucleotide and the 5'-triphosphate of the next nucleotide.
  • Requires a primer (existing 3' -OH from a chain): primer is RNA. Hence the need for telomerase sequence).
  • Direction: 5' to 3' grows longer from 3' (same as how we write down a DNA sequence).

DNA Polymerase Common Structure

• Bacterial DNA polymerases have a large cleft composed of three domains that resemble a hand: palm, fingers, and thumb.
• DNA lies across the “palm" in a groove created by the “fingers” and “thumb."
  • Palm: catalytic active site.
  • Fingers: positions template DNA (and probably incoming nucleotide) in the active site.
  • Thumb: has a role in processivity, translocation, and positioning of the DNA (DNA binding).

Eukaryote vs Prokaryotic Replication

• Similarity in functions at the replication fork:
  • E. coli
  • Eukaryote
  • Phage T4

Recombination I

Recombination

• Recombination is a process by which pieces of DNA are broken and recombined to produce new combinations of alleles.
  • Recombination creates genetic diversity at the level of genes.
• Recombination: the rearrangement of genetic material, especially by crossing over in chromosomes (in the same cell) or by the artificial joining of segments of DNA from different organisms.

The Roles of Homologous Recombination

1. Contribute to genetic diversity through allele shuffling. Double-strand break repair (DSBR) between two homologous chromosomes at the equivalent locus does not lead to new mutations.
2. A mechanism of precise repairs of DNA double-strand break.

Recombination

• Generalized recombination occurs between two homologous DNA duplexes and may occur at any point along their length.
  • Crossover point at which each becomes joined.
  • The overall organization of the DNA does not change.
  • Products retain the same structure as parents.
  • Parents and products are homologous.

Sister Chromatid Exchange (SCE)

• Sister chromatid exchange (SCE) is the reciprocal exchange of chromatin between two identical sister chromatids. SCE possibly occurred during DNA synthesis either due to some replication error or due to inhibition of DNA replication

Recombination Hotspots

• Hotspots: 1- to 2-kb genomic regions where meiotic recombination occurs.
• Positions and activities are determined by PRDM9, a DNA-binding histone methyltransferase.
• The binding of PRDM9 to specific DNA sequences targets the initiation of recombination at specific locations in the genome.
• Induction of DSBs is a highly regulated process and occurs in specific regions of the chromosomes called hotspots.

The Holliday Model for Homologous Recombination

• The Holliday model is an early molecular model that described the recombination process.
  • It was deduced from genetic crosses in fungi, and electron micrograph data came later.
  • The structure has been called a chi (X) form.
• Chi sites (Chi = crossover hotspot instigator) are known hot spots for genetic recombination.

Holliday Junctions

• Two homologous chromatids align.
• A break or nick occurs in one strand of each of the two chromatids.
• Strands invade opposite helices and base pair with complementary strands.
• Covalent linkage creates a Holliday junction.
• The next step is branch migration.
• Heteroduplex regions may have a few mismatches since these chromosomes are not identical (similar but not identical).

Resolution of Holliday Junctions

• Involves breakage and rejoining of two DNA strands, resulting in two separate chromosomes.
• In this next step, the figure is simply redrawn by bending the ends labeled A and B upward and bending the ends labeled a and b downward, making it look more like a true Holliday junction. The Holliday junction is viewed in two different planes.

Recent Models Refining Recombination Steps

• More recently, the Holliday model has been refined.
  • Model describes modified initiation phase of recombination.
• Modifications to the Holliday model:
  • Two nicks in the same location on two strands is unlikely.
  • It is more likely for nicks to occur in both strands of one chromatid.
  • DSB recombination: Genetic exchange is initiated by a Double-Strand Break.
  • The mechanism requires DNA gap repair synthesis.

Double Strand Break Recombination

• Occurs in meiosis I during spermatogenesis or oogenesis between non-sister chromatids (one maternal and one paternal: homologous chromosome)
• Homologous chromosomes can be observed to pair with each other: they are physically next to each other and in the same orientation.

Double Strand Break Model

• Step 1: DS break in the top chromatid.
• Step 2: degradation of small region near the break.
• Step 3: newly formed single-stranded segment invades intact bottom chromatid; displacement loop forms.
• Step 4: After D-loop formation, DNA synthesis occurs to fill in the gap (DNA gap repair synthesis).
• Step 5: After gap repair, two Holliday junctions are made.
• Step 6: Resolution of Holliday junctions results in either recombinant or nonrecombinant chromosomes (each with a short heteroduplex).

Double Strand Break Repair (DSBR) Mechanism

• DSB is actively produced by the endonuclease called Spoll in the “recipient” duplex. Ends of DSBs are covalently bound to Spo11 to protect them.

• 5'-end Resection

  • Exonucleases and endonuclease digest 5' end of the break, creating a 3' free end or overhang. The reason is 3' but not 5' overhang.

• Second strand invasion/capture:

  • The second resected end in the recipient strand anneals to the donor strand.
  • The recipient strand is extended to complete the second recipient strand.
  • D-loop is converted into two Holliday Junctions.

• Resolution of Holliday junctions:

  • If both junctions “cut” in the same way (both vertical/red or both horizontal/blue)? Noncrossover DNA results.
  • If two junctions are resolved in opposite ways (one blue + one red)? Crossover/recombinant DNA results.

Synthesis-dependent Strand-Annealing (SDSA)

• During mitotic homologous recombination, DSB repair events are not typically associated with crossing over.
  • The break is repaired using the homologous sequence as a template but does not involve crossing over.

Consequences of Heteroduplex DNA

• Heteroduplex DNA: the regions where strands from non-sister chromatids anneal to each other (blue and red = green boxes). These are NOT always perfectly complementary to each other.
• Mismatches will be repaired using the mismatch repair systems.
• The repair could change the DNA sequence in either allele, either maintaining the gene sequence or changing it. If the allele sequence changes, this is referred to as "Gene Conversion."

Gene Conversion

• Gene conversion: when one allele (recipient) is converted/changed to the allele on the homologous chromosome.
• Sequence is copied from a homologous chromosome (donor) to the recipient, which is a unidirectional transfer of genetic information.
• Often occurs in a region where crossover has taken place.
• Hence, homologous recombination can lead to gene conversion via DNA mismatch repair or DNA gap synthesis.

Gene Conversion

• General recombination that happens as a one-way transfer of genetic information.
• Results in an allele of a gene on one chromosome being changed to the allele on the homologous chromosome.
• 
  In this recombination between two homologous chromosomes A+B+C+ and A-B-C- can result in a new arrangement, A-B+C-, without a change in the parental A+B+C+ without a reciprocal change in the other chromosome.

Repair of Double Stranded Breaks

• Double-strand breaks (DSBs) are particularly deleterious DNA lesions for cells.
  • Repair is critical for the cell to maintain genomic integrity.
  • Cells have developed multiple mechanisms for repair.
  • If not accurately repaired, DSBs can lead to cell death, chromosomal rearrangements, and loss of genetic material.

Outcomes of DSB Repair

• HR restores the genetic information at the site of the DSB.
• Both Non-homologous end joining (NHEJ) and Single-strand annealing (SSA) result in potentially mutagenic outcomes because of end processing and/or loss of genetic information at the site of the break.
• Pathway choice depends on the organism, cell-cycle phase, and cellular context of the lesion.

Models of DSB Repair

• DSB
  • 5' to 3' resection
    • NHEJ (end joining)
    • HR (strand invasion, repair synthesis, D-loop formation)
    • SSA (Annealing of direct repeats)

Recombination II

Proteins Involved in Homologous Recombination

• Homologous recombination is found in all species.
• Meiotic recombination in Eukaryotic cells has been studied in Saccharomyces cerevisiae.
• The molecular details of homologous recombination are best understood in E. coli.
• Note: The term Rec indicates that the proteins function in recombination.

DSBR Model of Homologous Recombination

• Four steps in the reaction:
  • Step 1. End processing/presynapsis: = 5' resection = generating ss 3'
  • Step 2. Synapsis =3' invasion
  • Step 3. DNA heteroduplex extension and branch migration (DNA synthesis, branch migration, holiday junction formation)
  • Step 4. Resolution of holiday junctions

RecA Protein: Single Strand Invasion/Synapsis

• RecA protein
  • Polymerizes along single-strand DNA to form a nucleofilament (ssDNA associated with RecA proteins).
  • Catalyzes single-strand invasion/exchange: promotes base pairing between a single strand of DNA and its complement in a duplex molecule (three-strand intermediate).
• Homologs of RecA exist in every kingdom of living systems:
  • RecA is the first to be identified; Rad51 in eukaryotes. *Strand exchange occurs while RecA remains bound to the strand

Holiday Junction Formation Following Strand Invasion

• A reaction catalyzed by RecA between two duplex molecules (two invasions) leads to the formation of a holiday junction.
• 5' resection produces the free 3' end and the single strand.

Branch Migration by RuvAB

• Ruv genes (named from “repair of UV")
• Recombination is needed in the repair of DNA double-strand breaks because recombination mutants are often sensitive to agents that cause DSB.
• RuvA: recognizes Holliday structure and binds to all four strands.
• RuvB: hexameric ATP dependent helicase for branch migration (10-20 bp/s). RuvB hexamer binds as a ring around DNA.

Resolution of Holliday Junctions by RuvC

• RuvC: endonuclease that specifically recognizes and cleaves Holliday junctions.
• RuvABC complex of three proteins mediates branch migration and resolves the Holliday junction created during homologous recombination in bacteria.

RecA vs. Ruv Genes in Bacteria

• RecA is a recombinase protein that has a central role in DNA repair.
  • Promotes DNA strand exchange reactions.
  • Repairs stalled replication forks.
  • Involved in double-strand break repair.
• Ruv genes are involved in DNA recombination and repair.
  • RuvA, RuvB, and RuvC work together to process Holliday junctions created by RecA and resolve them.

Summary In Bacterial Cells

• Rec system: (RecA, RecBC)
  • End processing = 5' resection/generating 3' overhand
  • RecBCD: helicase and nuclease
  • Chi site
  • Strand invasion
  • RecA: 1) polymerize around DNA into nucleofilaments; 2) generate holiday junctions
• Ruv system: (RuvA, RuvB, and RuvC)
  • HJ recognition: RuvA
  • Branch migration: RuvB helicase
  • HJ resolution: RuvC endonuclease

Eukaryote Recombination Genes

• Most eukaryote HR genes are called RAD genes because they are first isolated in screens for mutant genes in yeast sensitive to X-ray irradiation.
  • X-ray irradiation leads to DSB.
  • If the dose is low, wild-type cells can repair the damages and recover to proliferate.
• Mutations in genes involved in DSB repair or recombination lead to failure in DSB repair. Therefore, these individuals are more likely to get ill or die when hit by X-rays (= sensitive).

DNA Repair

DNA Repair

• Mutations and DNA damage are potentially harmful to the cell.
• DNA repair is vital to the cell's survival.
• Several DNA repair systems exist.
• In most cases, DNA repair is a multi-step process:
  • Detection: An irregularity in DNA structure is detected.
  • Removal: The abnormal DNA is fixed or removed.
  • Normal DNA is synthesized.

Direct Repair

• An enzyme repairs an altered base by returning it to its correct structure without removing it.
• Alkyltransferase is an enzyme that repairs alkylated bases by transferring the methyl or ethyl group from the base to a cysteine side chain within the alkyltransferase protein.

Base Excision Repair

• In both prokaryotes and eukaryotes, the first step is the removal of a damaged base.
• Base removal triggers further removal and replacement of one or a stretch of nucleotides.
  • The process is similar to nucleotide excision but involves different complexes (involve DNA synthesis).
• Glycosylases remove the base. Some glycosylases are also lyases that further open the deoxyribose ring.

Excision Repair

• Many excision repair systems exist, differing in specificity (targeting different lesions) and enzymes involved.
• All share the feature of removing and resynthesizing DNA using the complementary strand as a template.
• Base excision, Nucleotide excision, Mismatch excision

Nucleotide Excision Repair (NER)

• A general DNA repair process that removes damaged DNA segments.
• Able to repair many types of damage, including:
  • Thymine dimers and chemically modified bases.
  • Missing bases.
  • Some types of crosslinks.
• Found in all eukaryotes and prokaryotes, and the mechanism is better understood in prokaryotes.

Nucleotide Excision Repair in Bacteria

• Recognition: UvrAB recognizes a pyrimidine dimmer or other bulk lesion, then UvrA dissociates; UvrB remains bound, and UvrC joins.
• Incision: UvrC makes cuts in the DNA at both sides of the damaged DNA.
• Excision: Helicase UvrD recognizes the region, separates the strands, and the damaged segment is released.
• Synthesis: Pol I uses the intact strand as the template and fills in the gap.
• Ligation: Using DNA ligase.

Mismatch Repair Systems Recognize and Correct a Base Pair Mismatch

• A key characteristic of Muth is that it can distinguish between the parental strand and the daughter strand.
• Prior to replication, both parental strands are methylated.
• Immediately after replication, the parental strand is methylated, whereas the newly made daughter strand is not (hemimethylation).
• The strand that lacks methylation is excised.

Non-Homologous End Joining

• Broken ends are pieced back together.
• Broken ends are recognized by end-binding proteins.
• Additional proteins bind to these and form a crossbridge.
• DNA processing proteins digest parts of the strands; may result in the deletion of a small region.
• Gaps are then filled in by polymerase and joined with ligase.
• Not error-free (error-prone).

Transposable Elements

Transposable Elements = Transposon

• Part of the genome: the DNA region that is mobile.
• Transposition: Process in which a DNA segment is inserted into a new location in the genome.

TEs Account for Almost Half of the Human Genome

• Most transposons in the human genome have lost their mobility.
• They are "Ancestral traces" of TE invasion.
• Most do not contribute to cell activities and are mostly non-coding sequences in heterochromatin (not transcriptionally active).
• Many are moderately repetitive sequences, and they can be a source of mutations and polymorphisms.

Biological Significance of Transposons 1

• Two schools of thought exist regarding the biological significance of transposons in evolution:
  1. TEs exist because they simply can! They can proliferate within the host as long as they do not harm it to the extent that they significantly disrupt survival. This has been termed the selfish DNA theory.
  2. TEs exist because they offer some advantage, e.g., in prokaryotes, bacterial TEs carry antibiotic-resistance genes.

Biological Significance of Transposons

• TEs may cause greater genetic variability through recombination.
• TEs may cause the insertion of exons into the coding sequences of protein-encoding genes (Exon shuffling), which may lead to the evolution of genes with more diverse functions.

Transposons and Mutagenesis

1. Disruption of a single gene by inserting into a functional gene.
2. Imprecise excision can result in exon shuffling.
3. Chromosome rearrangement: recombination between different copies of the transposons (homologous recombination or single-strand annealing recombination).

Transposons and Chromosome Rearrangement

• Alignment between copies in the same chromosome can occur.
• Two transposons of the same orientation (Direct repeats) lead to the deletion of the region between repeats.
• Although similar consequences, this is homologous recombination but not site-specific recombination.

Transposons and Chromosome Rearrangement

• Alignment between copies in the same chromosome can lead to inversion of the region between repeats.
  • Two copies orientation = Inverted repeats inversion

Mechanisms of Transposition

1. Non-replicative (Cut and paste): Results in a single copy.
2. Replicative (Copy and paste): Elements end up with more copies. It requires both transposase and a resolvase.

Classes of Transposon

• Class I elements (Retrotransposons)
• Class II elements

DNA Transposon: Bacteria IS Insertion Element (IS)

• Simplest DNA (class II) transposons, found in bacterial chromosomes and plasmids.
• E. coli likely contains several (~10) copies of different IS elements (numbered IS1, IS2, etc.).
• Autonomous units have one open reading frame for transposase.
• Target sequence: some are random, and some show preferences (overall specificity is low).

Class II Transposons: Mechanism of Insertion

• Staggered nicks are made at the target site in the recipient DNA, and the transposon is joined to the single-stranded ends.
• Gaps at the target site are filled in and sealed, leading to duplication of the target site.

Formation of Direct Repeats at the Insertion Site

• At the target DNA site, transposase makes a straggered cut in the insertion site.
• Gaps in the DNA are filed in resulting in direct repeats on each side of the inserted TE carried out via a gap repair synthesis mechanism.

Retroelements/Class I

• Retrotransposons (9.1%): There are a variety of forms of non-LTR retrotransposons and endogenous retroviruses (ERVS) (which have long terminal repeats (LTRS).
• Long nuclear elements (LINES): LINES make up the majority of non-LTR retrotransposons in the human genome (20.4%).
• Short-interspersed elements (SINES) - (13.1%)

Retrotransposons Use Reverse Transcriptase for Retrotransposition

• Retrotransposons use an RNA intermediate in their transposition mechanism.
• LTR retrotransposon movement requires two key enzymes: reverse transcriptase and integrase.
  Step 1. Ty is transcribed into RNA
  Step 2. DS DNA molecule is synthesized. using reverse transcriptase
  Step 3. Integrase recognizes LTRs and catalyzes insertion into the genome

Model for Integration of Non-LTR Transposons

• Step 1: Retrotransposon is transcribed into RNA with a 3' polyA tail.
• Step 2: A consensus sequence on the target DNA is recognized by an endonuclease (the retrotransposon may code for the endonuclease), and it cuts one of the DNA strands.
• Step 3: PolyA tail binds to the nicked site.
• Step 4: Reverse transcriptase uses the target DNA of the primer and makes a DNA copy of the RNA.
• Step 5: Endonuclease makes a second cut in the other strand several base pairs away from the first cut.
• Step 6: The retrotransposon is integrated into the target DNA strand (probably by NEJ).