module 17

Holliday Junctions and Homologous Recombination

  • Definition: A Holliday junction is a cross-shaped DNA structure formed during homologous recombination, when two DNA molecules exchange strands.

  • Process of Formation:

    • 1.** Strand break and invasion**: One DNA strand is cut. It invades a homologous DNA molecule and pairs with its complementary strand.

    • 2. Formation of the Holliday junction:

    • The strands from the two DNA duplexes become interconnected.

    • This creates a 4-stranded cross structure known as the Holliday junction.

    • DNA strands are physically exchanged zwischen molecules.

  • Branch migration:

    • The junction can move along the DNA.

    • This movement extends the region of strand exchange.

  • Resolution (Critical Part):

    • The junction is cut by enzymes in two possible ways:

    • Cut pattern 1 (Non-crossover): DNA returns mostly to its original configuration. Only a small patch of genetic material is exchanged.

    • Cut pattern 2 (Crossover): Chromosomes exchange large segments. This path leads to genetic recombination.

Homologous Recombination Repair (HRR)

  • Overview: Homologous Recombination Repair (HRR), also called Homology Directed Repair (HDR), fixes double-strand breaks (DSB) using a sister chromatid.

  • Key Requirements and Mechanics:

    • It requires a non-damaged DNA fragment to serve as a template.

    • Short segments of both DNA strands at the break site are digested.

    • There is an invasion and exchange of strands between the broken and unbroken sister chromatids.

    • The unbroken strands are used as templates for DNA synthesis.

    • Finally, the crisscrossed strands (Holliday junctions) are resolved (broken and rejoined).

  • Timing in the Cell Cycle:

    • The sister chromatid used as a template is available only during the SS and G2G2 phases of the cell cycle.

  • Step-by-Step HRR Process:

    • Step 1: Double-strand break: DNA is broken on both strands.

    • Step 2: End resection: Ends are trimmed to form single-stranded overhangs.

    • Step 3: Strand invasion: The broken strand invades a similar DNA (sister chromatid) and uses it as a template.

    • Step 4: DNA synthesis: DNA polymerase copies the missing DNA.

Pathways of Homologous Recombination Repair

  • Pathway 1: SDSA (Synthesis-dependent strand annealing):

    • The strand returns to the original DNA.

    • No stable junction is formed.

    • This pathway always results in a non-crossover.

  • Pathway 2: DSBR (Double-strand break repair) with two Holliday Junctions:

    • Two DNA molecules form two Holliday junctions (X-shaped structures).

    • DNA strands from each molecule are temporarily exchanged.

    • Resolution Outcomes:

    • Non-crossover: Occurs if both junctions are cut in the same orientation (represented by black arrowheads at both junctions). The result is DNA returning to its original arrangement with only a small copied region.

    • Crossover: Occurs if the junctions are cut in different orientations (e.g., cut at green arrowheads at one junction and black arrowheads at the other). The result is the exchange of DNA arms between chromosomes.

Detailed Step-by-Step Resolution of Holliday Junctions

  • 1. Double-strand break (DSB): Initiating cut in the DNA.

  • 2. End resection: Formation of 3′3' single-stranded DNA.

  • 3. Strand invasion and D-loop formation: A D-loop (displacement loop) is formed when a single DNA strand invades a double helix and displaces one of its strands. The invading strand pairs with homologous DNA.

  • 4. DNA synthesis: The missing DNA sequence is copied.

  • 5. Second end capture: Holliday junctions (cross-shaped DNA structures) form.

  • 6. Resolution: The junctions are cut leading to either non-crossover or crossover.

Transposable Elements (TEs): Overview and Significance

  • Definition: Transposable elements, or "jumping genes," are DNA sequences that can move within genomes, moving within and between chromosomes.

  • Prevalence: Mobile elements are present in all organisms, from bacteria to humans, and often constitute large portions of the genome.

  • Impact on Genome:

    • TEs act as naturally occurring mutagens.

    • Coding region insertion: Can alter the reading frame or introduce stop codons.

    • Regulatory region insertion: Can disrupt proper gene expression.

    • Chromosomal damage: Can cause double-stranded breaks, inversions, and translocations.

  • Human Genome Composition (Approximate percentages):

    • Exons (protein-coding): 2%2\%

    • Introns: 24%24\%

    • Unique sequences not in genes: 15%15\%

    • Repetitive DNA sequences: 59%59\% (Nearly 50%50\% of the human genome consists of sequences derived from TEs).

Categories of Transposable Elements

1. DNA Transposons (Class II)

  • Mechanism: "Cut-and-paste." They move directly as DNA without an RNA intermediate.

  • Key Enzyme: Transposase (excises and reinserts the element).

  • Subtypes:

    • Autonomous elements: Encode their own transposase and can move independently.

    • Non-autonomous elements: Do not encode transposase and depend on autonomous elements for movement (e.g., the DsDs element in maize).

  • Structural Features:

    • Open Reading Frame (ORF): Encodes the transposase enzyme.

    • Inverted Terminal Repeats (ITR): Short sequences at both ends of the element recognized by transposase.

    • Direct Repeats (DR): Short, identical DNA sequences flanking the TE, created during insertion (not part of the transposon itself).

2. Retrotransposons (Class I)

  • Mechanism: "Copy-and-paste." They move via an RNA intermediate and increase copy number through reverse transcription.

  • Subtypes:

    • LTR Retrotransposons: Contain Long Terminal Repeats (LTRs); similar to retroviruses.

    • Non-LTR Retrotransposons:

    • LINEs (Long Interspersed Elements): Autonomous, encode reverse transcriptase (Example: LINE−1LINE-1 or L1L1).

    • SINEs (Short Interspersed Elements): Non-autonomous, do not encode enzymes, use LINE machinery (Example: AluAlu elements).

Mechanisms and Examples of DNA Transposons

  • Cut-and-Paste Process Steps:

    • 1. Transposase cleaves DNA at ITRs.

    • 2. Transposase makes staggered cuts at the target site in chromosomal DNA.

    • 3. Transposase inserts the transposon at the target site.

    • 4. Gaps in the target site are filled by DNA polymerase and DNA ligase, creating new Direct Repeats (DRs).

  • Examples:

    • Insertion element: Simplest form; contains transposase gene and Inverted Repeats (IRs).

    • Simple transposon: Larger; contains transposase and often antibiotic-resistance genes.

    • Ac-Ds System in Maize (Barbara McClintock):

    • Ac (Activator): Autonomous; encodes functional transposase.

    • Ds (Dissociation): Non-autonomous; has a 194 bp194\,bp deletion. It moves only in the presence of AcAc.

    • Effect on phenotype: DsDs insertion near or into gene WW (responsible for kernel color) inhibits expression. If DsDs excises, expression is restored (reversion).

  • Scientific Application: Transgenic Mice:

    • The Sleeping Beauty transposon system is used to insert genes (like GFP) into the mouse genome.

    • Transposase mRNA/DNA is co-injected into a fertilized egg (zygote) to achieve stable integration.

Mechanisms and Examples of Retrotransposons

  • Reverse Transcription Process:

    • Uses an RNA template to synthesize cDNA (complementary DNA).

    • Enzyme: Reverse Transcriptase (RT).

    • Result: Double-stranded DNA derived from RNA.

  • cDNA Library Generation:

    • Studied to analyze mRNA pools or tissue-specific protein expression.

    • Steps: RNA isolation rightarrow\\rightarrow cDNA synthesis by RT rightarrow\\rightarrow Restriction digestion rightarrow\\rightarrow Ligation to vectors rightarrow\\rightarrow Transformation into bacteria.

  • Retrotransposons in Humans:

    • Account for approximately 42%42\% of the human genome.

    • LINEs (Non-LTR): 11 to 10 kb10\,kb long; ~850,000850,000 copies; 21%21\% of the genome.

    • SINEs (Non-LTR): 100100 to 500 bp500\,bp long; ~1,500,0001,500,000 copies; 13%13\% of the genome (e.g., AluAlu family).

    • LTR elements: ~65 kb65\,kb; ~443,000443,000 copies; 8%8\% of the genome.

  • Steps of LINE-1 (L1) Retrotransposition:

    • 1. Transcription of LINE−1LINE-1 into RNA with a poly(A) tail.

    • 2. Target DNA is cut by Endonuclease (EN), exposing a 3′ OH3'\,OH group.

    • 3. RNA binds to the target site via poly(A) tail and T-rich DNA.

    • 4. Target-Primed Reverse Transcription (TPRT) synthesizes cDNA directly at the target site.

    • 5. Second DNA strand is cut and synthesized; RNA is degraded.

    • 6. Repair and integration by DNA polymerase and ligase results in a fully integrated copy flanked by Target Site Duplications (TSDs).

Case Studies and Evolutionary Impacts

  • Copia Elements in Drosophila:

    • Causes the White-apricot mutation, changing eye color from red to orange-yellow.

    • An insertion of copiacopia in the second intron leads to premature termination of transcription.

  • Alu Elements:

    • ~10%10\% of human genome; derived from the ancestral 7SL7SL RNA gene (part of the signal recognition particle).

  • X-linked Hemophilia Case (L1 Insertion):

    • A son presented with hemophilia caused by a LINE−1LINE-1 (L1) insertion in the F8F8 gene on the X-chromosome.

    • The insertion was a de novo mutation; the mother did not have the insertion on her X-chromosomes, but both parents had the specific L1L1 on chromosome 22, indicating transposition from chromosome 22 to the X chromosome in the mother's gamete-forming cells.

  • Evolutionary Function:

    • TEs can create beneficial mutations.

    • In Drosophila, LINE-like elements function as telomeres, maintaining chromosome length.

    • Roughly 0.2%0.2\% of human mutations are due to TE insertions, compared to 10%10\% in mice and 50%50\% in Drosophila.