M2C: Recombination And Genome Rearrangements

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Double Stranded Breaks (DSB)

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Can cause rearrangements of chromasomal pieces including translocations, duplications/amplifications, insertions, deletions, and fusions. Repair for DSB can also cause these problems.

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Sources of DSB that generate genome rearrangements

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1. Intentionally made by the cell for normal development. Ex. meiosis, V(D)J rearrangement.
2. From exogenous DNA damage. Are often repaired, but abberrant repair for these DSBs can cause rearrangements that lead to cell death or cancer.
3. Integrations and excisions of mobile DNA elements.

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DSB Repair

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1. DSB is made
2. At the region of the break, align the equivalent unbroken region from either the intact homologous chromosome, or the intact sister chromatid.
3. Use the intact homolog or sister chromatid as a template for repair.

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In Meiotic Cells: Programmed rearrangement for genetic viariability.

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Parental (diploid) cells undergo meiosis to create four daughter (haploid) cells each with half the number of chromosomes, and mixed genetic information from mom/dad genome.

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Meiosis

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Diploid proginator undergoes DNA replication creating sister chromatids (46-->92). These sister chromatids they undergo homologous recombination (one side of mom and dad line up and share info). The cell then undergoes two divisions without DNA replication to create four haploid and unique daughter cells. These are germ cells.

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Homologous Recombination

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In meiosis, "non-productive" recombination is blocked. CAN have gene conversion or cross over.

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Gene Conversion

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During homologous recombination, one chromatid undergoes DSB and is repaired using homologous chromatid as a template. Original remains unchanged while new strand has a "patch" of new DNA.

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Cross Over Recombination

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DSB is introduced and the two homologous chromatids exchange arms during repair.

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Hereditary Breast Cancer

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Caused in part by defects in gene required for recombinational repair (BRCA2).

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Mitotic Recombination

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Chromosome repair after damage requires recombination between sister chromatids. Usually conservative as sister chromatids are identical, not homologous (like in meiosis). They can accidentally line up (due to many repetitive sequences), however, causing mismatch of homologous areas and unequal sister chromatid exchange. This leads to the duplication of the region between the repetitive elements in one chromatid and the deletion of this area in the other.

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End Joining

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Refers to method of repair for DSB in which broken DNA ends are simply joined back together. Nucleases get rid of damaged ends, polymerases make the ends compatible, and ligases reattach backbones. This is an inaccurate method as this method always involves loss of sequence.

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Homologous Recombination vs. End Joining

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While end joining is inaccurate, it is fast and efficient and can be done in any genome at any time. While more accurate, homologous recombination can only occur during S-phase of cell cycle and requires a lot of synthesis and "things coming together just right".

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Transposons

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Mobile elements within the host genome that can directly relocate themselves to other locations within the genome. DNA excises from one area and integrates in another. Each transposon has a transposase gene encoding for proteins that help with the insertion. Excision is targeted by signals and is specific while integration is much more random. Integration can also occur within gene or near transcription regulators. Excision and integration create DBS on either side. This is dangerous and unless properally repaired can lead to cell death or recombination.

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Retro-elements

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Mobile elements that have an obligatory RNA intermediate. Integrate into human genome in the same way as transposons. Do not excise in same way. Use host transcription machinery to create RNA copy, which is then converted to a DNA copy by reverse transcription. This piece of DNA can then integrate anywhere in the host genome.

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Retroviral Elements

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Can be packaged into infectious virus, and thus have an extracellular phase in their life cycle.

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Retroposons

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Activity limited to intracellular. High number of these (1/4 of our genome) in eukaryotes. Mostly in non-coding regions.

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Insertional Mutagenesis

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Retroelements or transposons insert into the coding region or promotor region of a gene. Many congenital diseases are caused by retroposons inserting into an important gene in the germ cell. Retroviruses like to insert near actively transcribing genes (non-random insertion) and carry with them strong transcriptional enhancers. Can cause over or anomalous expression of nearby genes.

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Pathogenic Retroviruses

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In addition to "accidental" interference with host cell growth, some cells specifically carry genes to disrupt normal cell growth.

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Retroviral transduction of host sequences and non-autonomous viruses

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Retroviruses can also acquire growth disrupting capabilities by recombining with host genome or aberrant splicing of the viral RNA into the host genome. Produces a hybrid viral genome. Hybrid RNA can include sequences for packaging and integration, but often has lost some viral genes and therefore its infectious capabilities. This virus is non-autonomous (not effective on its own). Missing functions can be provided in "trans" by a helper virus present in same cell. Sometimes host/viral hybrid is enough to cause cancer as in v-src, a sarcoma.

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Adaptive Immune System

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Subset of cells can respond SPECIFICALLY to (and protect us from) to virtually all of the infinite number of foreign agents. Ability rests in diversity of receptors (B cells, antibody, immonoglobulin) on surface of WBC. Once a certain receptor is deemed useful, it can be activated and replicated to help the body clear the specific pathogen. In the spleen and lymph nodes, lymphocytes that have proven useful are saved for future use (immunologic memory), while the rest are iliminated.

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V(D)J Recombination

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Diversity of antibodies is derived through V(D)J recombination. Occurs in DNA in developing atibodies. Multiple V, D, and J coding segments are distributed at a specific locus on the chromosome. On each of the segments is then assembled a code for the complete variable domain of the mature receptor. This variable domain provides specificity for different foreign agents. Over 100 million different receptors are possible although we don't have this many at any one time.

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How V(D)J Recombination Works

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Step 1: A lymphocytic initiation step (clevage). Reactivating Genes 1 and 2 (RAGI and RAGII) found only in developing B/T cells, recognize a pair of recombination signals and introduce DBS at border between signal and coding regions.
Step 2: A resolution step (joining). Uses regular cellular DBS repair mechanisms to join the ends of coding segment DNA. Signal ends are also joined to create signal junction products (this circular DNA product is usually lost in next replication). If there is a loss (from mutation?) of DBS repair mechanisms, immunodeficiency is common due to inability to complete V(D)J recombination.

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Severe Combined Immunodeficiency (scid)

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Failure in cleavage step (RAGS) of V(D)J recombination results in complete deficiency in both B and T cells. Patients with rs-scid (radio-sensitive scid) have a general cellular sensitivity to DBSs. Often have a defect in gene required for end joining.

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Lymphoid Malignancy

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Leukemia, Lymphoma, Thymoma are all lymphoid malignancies. Often due to translocatons with breakpoints near in antibody T cell receptor loci near where DBS intermediates in V(D)J recombinations occur.