12 - DNA Recombination

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Tookie
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281122
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12 - DNA Recombination
Updated:
2014-08-22 21:25:43
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dna recombination
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CMBM
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DNA Recombination
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  1. What are the four general types of recombination?
    Homologous recombination, transpositional recombination, conservative site-specific recombination, and illegitimate recombination; homologous recombination is the least drastic
  2. When does homologous recombination take place?
    Only takes place between two homologous DNA molecules since the process requires relatively large stretch of sequence similarity ( 50 bp) between the two DNA molecules undergoing recombination
  3. What is the importance of homologous recombination?
    Provides mechanisms for reassortment of genes, gene conversion, generation of new alleles, thereby bringing permanent change in the genome, a key step in evolution; it also repairs single and double strand breaks, repairs stalled replication forks and pairing of homologous chromosomes during meiotic cell division
  4. What are the key steps of homologous recombination?
    • 1. Introduction or formation of breaks in the DNA
    • 2. Strand invasion and formation of Holliday
    • junction
    • 3. Branch migration; movement of the Holliday junction along the DNA molecules
    • 4. Resolution; cleavage of Holliday junction
  5. What is a Holliday junction?
    Pairing of the strand from one DNA molecule with the strand from the other DNA molecule (forms a cross shaped region); requires identical or nearly identical nucleotide sequence for a stretch of ~100 bp
  6. What is the importance of branch migration?
    This will increase the heteroduplex region, important process for gene conversion and generation of new alleles (this occurs when neither the maternal or paternal DNA region is recreated but a totally new region is created)
  7. What are the two possible ways for a Holliday junction to be cleaved?
    Cleavage of the original invading strand (gives rise to non-recombinant, patch or non-crossover product) or the other strand (gives rise to recombinant, splice or crossover product)
  8. What are sources of DNA breaks in homologous recombination in prokaryotes?
    Ionizing radiation, damaging agents, DNA replication errors, and linear DNA that enters the bacterial cell during phage-mediated transduction or cell-cell conjugation
  9. What are sources of DNA breaks in homologous recombination in eukaryotes?
    • Mitotic: ionizing radiation, damaging agents, DNA replication errors, transposon movement and immunoglobulin synthesis
    • Meiotic: Spo11 is expressed during prophase of meiosis which induces double-strand breaks to initiate recombination
  10. What general proteins are required for recombination in E. coli?
    ssDNA-binding proteins, helicase, DNA polymerase, topoisomerases, and DNA ligase
  11. What specialized proteins which are required for recombination in E. coli?
    • RecBCD – generation of ssDNA with 3' overhangs and recruits
    • RecA; protects cells from foreign DNA which lack chi sites; MRX protein in eukaryotesRec A – forms a filamentous helical structure around 3’ overhang; important in stabilization of ssDNA, strand exchange; assembles Holliday junction (occurs when it finds a homologous sequence 50-100nt long)
    • RuvAB – branch migration; this functions as a chemomechanical motor to drive branch migration; this is an ATP dependent process
    • RuvC – makes the nicks for resolution; nicks two DNA strands with the same polarity
  12. What is Spo11?
    Recombination protein in Eukaryotes; introduces many strand breaks to assist in recombination between parent strands
  13. What does homologous recombination have to do with human disease?
    Decreased rate of homologous recombination can result in Down syndrome; errors during homologous recombination cause csome abnormatlities and these errors are primarily due to presence of repetitive DNA sequence; tandemly repeated DNA sequence and interspersed repetitive DNA sequence are hot spots for mediating disease-causing recombination errors
  14. What is transpositional recombination?
    Movement of certain sections of DNA to randomly different positions in the genome; in humans ~45% of the genome is transposon-related DNA sequence; also known as transposons
  15. What are the two classes of transposable elements?
    DNA transposons and RNA transposons (retrotransposons)
  16. What is the cut and paste mechanism of DNA transposons?
    The transposon is transcribed and translated; the transposase enzyme enters the nucleus and excises the transposon (sequence-specific endonuclease); a double strand break is formed at this site; transposase also cuts the target DNA (sequence independent); ligates the transposon at the new site
  17. What is the copy and paste mechanism of viral-like retrotransposons?
    The original copy of the retrotransposon is left intact at the original site, and a new copy is pasted at a new site in the genome; this is similar to retroviral infection; codes for integrase and RT
  18. How does Poly-A retrotransposition occur?
    • 1. First the transposon is transcribed synthesizing RNA-binding protein (ORF1) and another protein with endonuclease and RT activity (ORF2)
    • 2. The proteins bind the mRNA that it was
    • synthesized from and this complex moves into the nucleus
    • 3. Endonuclease breaks the DNA in a T-rich region and the PolyA of the transposon mRNA froms a basepair with a T in the DNA
    • 4. mRNA of the transposon is reverse transcribed into DNA using the 3’ end of the cut DNA as a primer and the mRNA as a template
    • 5. The transposon DNA is integrated into the csome DNA
  19. How to Poly-A retrotransposons give rise to processed pseudogenes?
    Pseudogenes are genomic DNA sequences similar to normal genes but non-functional; binding of the RNA-binding protein and endonuclease/RT protein of the retrotransposon to mRNA of cellular proteins can result in the formation of pseudo genes
  20. How are pseudogenes identified in csome DNA?
    Lack a promoter, introns, 5’ end; have DNA-encoded polyA region in the 3’ end; errors in the open reading frame
  21. What is the significance of transposons?
    There is little sequence selectivity in the choice of insertion sites which can result in disruption of gene function or gene regulation; causes ds breaks in DNA; increases chance of errors due to increase in repeptitive DNA sequences; common source of new mutations and gene rearrangements in many organisms; could be used as a vehicle in gene therapy
  22. What is conservative site-specific recombination?
    • Similar to transpositional recombination, but cutting and insertion sites are sequence-dependent; it is direction and the product of recombination depends on the relative direction of the two participating sites
  23. What is an example of insertion?
    Insertion of bacteriophage DNA into bacterial chromosome during lysogenic phase of bacteriophage infection
  24. What is an example of deletion?
    Excision of bacteriophage DNA during the lytic phase of bacteriophage infection
  25. What is an example of inversion?
    Switching between H1 and H2 flagellin by Salmonella to elude host immune system
  26. What is non-homologous end joining?
    This is the primary mechanis of ds-break repair in many eukaryotes; break ends are ligated directly without the need for a templet; only requires microhomologies with one or two base complementarity
  27. How does recombination play a role in generating antibody diversity?
    By joining two or three gene segments; or by joining two segements via NHEJ and the DNA sequence being changed at the junction of the two fragments

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