Homologous Recombination at the Molecular level

  1. All DNA is _______ DNA. Genetic exchange works constantly to blend and rearrange chromosomes pair before the first nuclear division. During this pairing, ______ exchange between chromosomes occurs. This exchange, classically termed ______ ______, is one of the results of _______ ________.
    • recombinant
    • genetic exchange
    • cross over
    • homologous recombination
  2. What causes recombination
    ribose ionization & DSB
  3. The causes of recombination can also create _____ ______. Some chemicals do the same like ______
    • free radicals
    • bleomycin
  4. Recombination involves the ______ exchange of DNA sequences between the chromosomes. The frequency of crossing over between two genes on the same chromosome depends on the ______ ______ between the two genes, with _____ ______ giving the highest frequencies of exchange
    • physical 
    • physical distance 
    • long distances
  5. Genetic maps derived from early measurements of crossing over frequencies gave the first real info about chromosome structure by revealing that genes are arranged in a _____ and _____ order
    • fixed 
    • linear
  6. Occassionally, gene order does change, for example, movable DNA segments called _______ can jump around chromosomes and promote DNA ________, thus altering chromosomal organization. The recombination mechanisms responsible for ________ and other genome rearrangements, are distinct from those of ________ _______
    • transposons
    • rearrangements
    • transposition
    • homologous recombination
  7. Double stranded breaks in DNA arise often and if these breaks are not repaired, the consequence to the cell is disastrous. For example, a single DSB in the E. coli chromosome is _____ to a cell that lacks the ability to ______ it.
    • lethal
    • repair
  8. The major mechanism used to repair DSBs in most cells is _______ _______. Some cell types also use a simpler mechanism such as _________ _____ _____ to heal their chromosomes.
    • homologous recombination
    • nonhomologous end joining (NHEJ)
  9. In bacteria, the major biological role of homologous recombination is to ______ ______. These broken DNA ends arise by several means. _______ _______ and other ______ agents can directly break both strands of the DNA backbone. Many types of DNA damage also indirectly give rise to DSBs by interfering with the progress of a ______ _____
    • repair DSBs 
    • Ionizing radiation 
    • damaging 
    • replication fork
  10. State one example of an indirect DSBs as a result of interference with the rep fork (there are two examples on the answer sheet)
    • an unrepaired nick in one DNA strand will lead to collapse of a passing rep fork. 
    • a lesion in DNA that makes a strand unable to serve as a template will stop a rep fork 
    • These types of stalled fork can be processed by several different pathways (fork regression or nuclease digestion) that give rise to a DNA end with a DSB
    • The broken ends then initiate recombination with a homologous DNA molecule to heal the break.
  11. In addition to repairing DSBs in chromosomal DNA, homologous recombination promotes ______ ______ in bacteria.
    genetic exchange
  12. In euk cells, homologous recombination is critical for repairing _____ _____ and _______ _____ ______. This roleof chromosome repair and rep restart is the principal function of homologous recombination in most ______ cells in complex organisms as well as in vegetatively growing single cell euks.
    • DNA breaks 
    • collapsed rep forks 
    • somatic
  13. There are other times when recomb. for genetic exchange and chromosome maintenance is specially needed. Recombination is essential to the process of chromosome ______ during ______. In this case, as cell enter ______, they produce a specific protein to introduce DSBs into the DNA and thereby initiate the _______ pathway. Thus, although they arise from many different sources, the appearance of DSBs in DNA is a key early event in ______ _______
    • pairing 
    • meiosis
    • meiosis 
    • recombination pathway
    • homologous recombination
  14. 5 key steps of homologous recombination as we understand it
    • alignment of two homologous DNA molecules
    • introduction of breaks in the DNA 
    • strand invasion 
    • formation of the holliday junction
    • resolution of the holliday unction
  15. Explain the alignment of two homologous DNA molecule (2)
    • By homologous, we mean that the DNA sequences are identical or nearly identical for a region of at least 100bp 
    • Despite this high degree of similarity, DNA molecules can have small regions of sequence difference and may, for example, carry different sequence variants nown as alleles, of the same gene
  16. Explain introduction of breaks in the DNA (1)
    Once breaks are formed, the ends at the breaks are further processed to generate regions of single-stranded DNA
  17. Explain strand invasion (3)
    • Initial short regions, base pairings are formed between the two recombining DNA molecules.
    • This pairing occurs when a single stranded region of DNA originating from one parental molecule pairs with its complementary strand in the homologous duplex DNA molecule (this is called strand invasion)
    • As a result, of the strand invasion process, regions of new duplex DNA are generated. **It often has mismatched base pairs called heteroduplex DNA
  18. Explain the formation of a holliday junction (3)
    • After strand invasion, the two DNA molecules become connected by crossing DNA strands to form a structure that is called a Holliday junction.
    • The junction can move along the DNA by the repeated melting and formation of base pairs
    • Each time the junction moves, base pairs are broken in the parental DNA molecules while identical base pairs are formed in the recombination intermediate (this is called branch migration)
  19. Explain resolution of the holliday junction
    • The process to regenerate DNA molecules and therefore finish genetic exchange is called resolution.
    • It can be achieved in one of two ways, either by cleavage of the holliday junction or (in euk cells) by process of dissolution 
    • In the first, cutting the DNA astrands within the Holliday junction regenerates two separate duplexes
    • In the second, resolution is achieved by dissolution, a sort of convergence/collapse mechanism
  20. The two homologous DNA molecules aligned at the start of homologous recombination, although nearly identical have different ______ of the same _____
    • alleles 
    • gene
  21. Recombination is initiated by the presence of a _____ in one of the DNA molecules. DNA strands near the _____ site can then be peeled away from their complementary strands, freeing these strands to invade and ultimately _____ _____ with the _______ ______
    • DSB
    • break
    • base pair 
    • homologous duplex
  22. Strand invasion is the _____ step in homologous recombination, because it is this invasion and then _______ of complementary strands between the two ________ duplexes that establishes the stable pairing between these molecules
    • central 
    • pairing
    • homologous
  23. Strand invasion also initiates the ______ of DNA strands between the two ______ DNAs. The enzymes that catalyze strand invasion are called _____ ______ proteins because they promote this critical reaction
    • exchange
    • parental 
    • strand exchange proteins
  24. Strand invasion generates a ______ _______ that can then move along the DAN by branch migration. This migration increases the ______ of the DNA exchanged. If the two DNA molecules are not identical but for example, carry a few small sequence differences, as is often true between two alleles of the same gene, _____ ______ through these regions of sequence difference generates DNA duplexes carrying one or a few sequence mismatches. Repairing them in a _______ DNA can have important genetic consequences
    • holliday junction
    • length 
    • branch migration 
    • heteroduplex DNA
  25. Finishing recombination usually requires resolution of the ______ ______ by cutting the DNA strands near the site of the cross; this reaction separates the two _______ DNA molecules and thus completes the ______ exchange.
    • holliday junction 
    • recombining 
    • genetic exchange
  26. Resolution occurring in one of two ways gives rise to two distinct classes of DNA products
    • crossover product 
    • noncrossover products
  27. Homologous recombination is often initiated by DSBs in DNA. A common model describing this type of genetic exchange is the _____ _____ ______ ______ pathway. This pathway starts with the introduction of a _____ in one of two homologous duplex DNA moleucels. The other DNA duplex remains ______. The asymmetric initial ______ of the two DNA molecules in the ____ repair model necessitates that later stages in the recombination process are also _______
    • double strand break repair pathway 
    • DSB
    • intact
    • breakage
    • DSB
    • asymmetric
  28. After introduction of the DSB, a DNA ______ enzyme sequentially degrades the broken DNA molecule to generate regions of ______. This processing creates ______ ______ extensions, known as ______ _____, on the broken DNA molecules; and they terminate at ____ ends. In some cases both strands at a DSB are processed whereas in other cases, only the 5' terminating end is ______.
    • cleaving
    • ssDNA 
    • single strand 
    • ssDNA tails
    • 3'
    • degraded
  29. The ssDNA tails generated by this process then _____ the unbroken homologous DNA duplex. The ______ strand base pairs with its ________ strand in the other DNA molecule. Because the ______ strands end with 3' termini, they can serve as _______ for new DNA synthesis. _______ from these DNA ends, using the complementary strand in the homologous duplex as a ______, serves to ________ the regions of DNA that were destroyed during the processing of the strands at the _____ site
    • invade
    • invading 
    • complementary 
    • invading 
    • primers 
    • Elongation
    • templates
    • regenerate 
    • break
  30. If the two original DNA duplexes were not identical in sequence near the site of the break, sequence information could be lost during ________ by the ____ _____ pathway. Sequence info lost from the DNA molecule as a result of DNA processing is replaced by the sequence present on the other ______ as a result of DNA _______. This nonreciprocal step in DSB repair sometimes leaves a genetic trace, giving rise to a ______ ______ event
    • recombination 
    • DSB repair 
    • duplex 
    • synthesis
    • gene conversion
  31. The two holliday junctions found in the ________ intermediates generated by this model move by ______ _______ and ultimately are resolved to finish recomb. The strands that are _____ during resolution of these holliday junctions determine whether or not the product DNA molecules will contain ______ genes in the regions flanking the site of recomb.
    • recomb. 
    • branch migration 
    • cleaved 
    • reassorted
  32. Organisms from all branches of life encode enzymes that catalyze the chemical steps of recombination. In some cases, members of homologous ______ families provide the same function in all organisms. In contrast, other recombination steps are catalyzed by different classes of ______ in different organisms but withe same general outcome.
    • protein
    • protein
  33. Our most detailed understanding of the mechansim of recombination comes from studies of ______ and its phage. It promotes recombination via a major DSB repair pathway known as the ______ _______.
    • E. coli
    • RecBCD pathway
  34. Despite the fact that recombination via the RecBCD pathway requires a _____ on one of the two recombining DNA molecules, in _______, no specific protein  has been found that performs this task. Name 3 ways the break can occur
    • DSB
    • bacteria/prok
    • DNA damage, error in DNA repair or collapse of the rep fork.
  35. DNA molecules with ssDNA extensions or tails are the preferred _______ for initiating strand exchange between regions of ______ sequence. The ______ enzyme processs broken DNA molecules to generate these regions of ______
    • substrate 
    • homologous 
    • RecBCD
    • ssDNA
  36. RecBCD also helps load the _____ ____ exchange protein onto these ______ ends. In addition, the multiple enzymatic activities of RecBCD provide a means for cells to determine whether to ______ with or ______ DNA molecules that enter a cell
    • RecA strand exchange protein
    • ssDNA
    • recombine 
    • destroy
  37. RecBCD is composed of _____ subunits (name them) and has both DNA _____ and ______ activities.
    • three
    • recB, recC, and recD genes
    • DNA helicase 
    • nuclease
  38. The complex binds to DNA molecules at the site of a DSB and tracks along DNA using the energy of ____ _______. As a result of its action, the DNA is _______, with or without the accompanying _______ destruction of one or both of the DNA strands.
    • ATP hydrolysis
    • unwound
    • nucleolytic
  39. The activities of RecBCD are controlled by specific DNA sequence elements known as _____ _____. Why were they discovered?
    • chi sites
    • because they stimulate the frequency of homologous recomb.
  40. The recB and RecD subunits are both ____ _______, meaning?
    • DNA helicases
    • They use ATP hydrolysis to melt and unwind DNA bse pairs
  41. The RecB subunit contains a 3' to 5' ______ and also has a multifunctional ______ _______ that digests the DNA as it moves along.
    • helicase 
    • nuclease domain
  42. RecD is a 5' to 3' ______, and RecC functions to recognize ____ ______
    • helicase 
    • chi sites
  43. RecB and RecD helicase "motors" move ________ along opposite strands of the DNA duplex and at _______ speeds. Together they are capable of driving the ______ complex along the DNA at rates of 1000bp per second. ____ _____ within DNA act as a sort of molecular throttle to regulate the activities of the helicases and therefore the speed of DNA _______.
    • independently 
    • different 
    • RecBCD
    • Chi sites 
    • translocation
  44. _____ subunit runs faster than _____ and therefor leads the complex. As _____ tries to keep up, a loop of ssDNA from the ___ end bulges out ahead of the complex.
    • RecD
    • RecB
    • RecB
    • 3'
  45. Upon encountering the ____ ______, the complex pauses for a few seconds, then continues at about one-half the initial rate. During the pause, three events occur to change the activity of the complex. Name them
    chi sites

    • First, the looped out ssDNA is pulled or reeled back in by the RecB subunit, and RecB becomes the primary motor now leading the complex
    • Second, a possible conformational change occurs that results in uncoupling of the RecD subunit
    • Third, the nuclease activity of the RecBCD complex is altered. As RecBCD moves into the sequence beyond the Chi site, the nuclease no longer cleaves the DNA strand with 3' to 5' polarity
  46. The opposite DNA strand is cleaved more frequently than it was before the _____ ____ was encountered. As a result of this change in activity, the DNA duplex now has a ___ ____ _____ extension terminating with the chi sequence at the ____ end. This structure is ideal for assembly of ____ and _____ of strand exchange
    • chi site
    • 3' single strand
    • 3'
    • RecA 
    • initiation
  47. The structure of the RecBCD complex bound to DNA provides further insight into how this _____ subunit machine functions and how its activity changes upon encountering a chi site. The protein complex has an overall _______ shape, with duplex DNA entering the protein from the top point of the ____ of the _______
    • three subunit 
    • triangular 
    • top of the triangle
  48. Once the DNA has entered the top of the triangular structure, it encounters a pin structure protruding from the _____ subunit that _____ the DNA duplex and guides the _____ _______ ______ of DNA to the two motors within the enzyme. The _____ subunit channels the 3' strand to the _____ motor and the 5' strand to the _____ motor.
    • RecC 
    • splits 
    • two individual strands 
    • RecC 
    • RecB motor
    • RecD motor
  49. In this manner, RecC, which is not itself a helicase, contributes to the overall efficiency of the ______ activity of the complex. The organization of the channels within the enzyme causes the ____ DNA tail to be fed along a groove that emerges at the at the _____ active site on the _____ subunit. What happens as a result?
    • helicase 
    • 3' 
    • nuclease 
    • RecB

    As a result, before the enzyme complex encounters a CHi site, this strand is efficiently and processively degraded
  50. The 5' DNA tail also moves past the nuclease active site upon leaving the _____ motor, but it is digested _____ ______ than the the 3' tail, because it must compete with the more favorably ______ 3' strand. However, upon encountering a _____ _____, the situation changes. _____ recognizes and binds tightly to this DNA site, and once this 3' end is bound, it is prevents from entering the _______. What two things does this binding accomplish
    • RecD motor
    • less frequently 
    • positioned
    • chi site
    • RecC  
    • nuclease
    • prevents further digestion of the 3' tail and promotes digestion of the 5' tail by removing the competitor
  51. The ssDNA tail generated by RecBCD must be coated by the _____ protein for recombination to occur. However, cells also contain _____ that can bind to this DNA. To ensure that _____, rather than _____ binds these ssDNA tails, _______ interacts directly with RecA and promotes its assembly. This loading activity involves a direct _____-_____ interaction between the nuclease domain of the ______ subunit and the _____ protein and serves to load RecA on the DNA with the ____ tail
    • RecA
    • SSBs
    • RecA 
    • SSBs
    • RecBCD
    • protein-protein
    • RecB subunit
    • RecA 
    • 3'
  52. Chi sites increase the frequency of ________ about _____ fold. This stimulation is most pronounced directly ______ to the chi site. Although elevated recombination frequencies are observed for about 10kb distal to the chi site, they _____ _____ gradually over this distance.
    • recombination
    • 10 
    • adjacent 
    • drop off
  53. The observation that recombination is stimulated specifically only on one side of the was initially puzzling. What do we know now?
    The DNA between the DSB (entry point of RecBCD) and the chi site is cut into small pieces by the enzyme and is thereoree not available for recombination. In constrast, DNA sequences met by RecBCD after its encounter with Chi are preserved in a recombinogenic, single strand form and are specifically loaded with RecA
  54. The ability of Chi sites to control the nuclease activity of RecBCD also helps _____ _____ protect themselves from foreign DNA that may enter via ______ _______ or _______. The 8 nucleotide chi site (GCTGGTGG) is highly _________ in the E.coli genome
    • bacteria cells 
    • phage infection or conjugation
    • overrepresented
  55. DNA form the bacteriophage or from another species (in which E. coli Chi sites are not overrepresented) will lack frequent _____ _____. RecBCD action on this DNA will lead to its extensive ________, rather than activation for ______.
    • chi sites
    • degradation 
    • recombination
  56. _____ is the central protein in homologous recombination. It assembles on ______ and promotes ______ ______. _____ is the founding member of a family of enzymes called strand exchange proteins
    • RecA 
    • ssDNA
    • strand invasion
    • RecA
  57. What are the Eukaryotic counterparts to this: Image Upload 1
    Image Upload 2
  58. The ______ complex specifically recognizes holliday junctions and promotes branch migration
  59. After the strand invasion step of recombination is complete, the two recombining DNA molecules are connected by a DNA branch known as a ________ _______. Movement of the site of this branch requires exchange of DNA base pairs between the two ________ ________. Cells encode proteins that greatly stimulate the rate of ______ _____
    • holliday junction
    • homologous duplexes 
    • branch migration
  60. ______ protein is a holliday junction-specific DNA-proteins binding protein that recognizes the structure of the DNA _______, regardless of its specific DNA ______. ______ recognizes and binds to holliday junctions and recruits the ______ protein to this site. It is a ______ _______.
    • RuvA 
    • junction 
    • sequence
    • RuvA 
    • RuvB 
    • Hexameric ATPase
  61. RuvB is similar to _______ _____ involved in DNA replication. The RuvB ATPase provides the ______ to drive the exchange of ______ ______ that moves the DNA branch.
    • hexameric helicases
    • energy 
    • base pairs
  62. The energy from the RuvB is needed to move the branch rapidly in _____ direction. Structural models for _____ complexes at a ______ _______ show a _______ of RuVA together with two ________ of RuvB work together to power this DNA exchange process.
    • one 
    • RuvAB 
    • holliday junction
    • tetramer
    • hexamer
  63. _____ cleaves specific DNA strands at the holliday junction to finish recombination
  64. Completion of recombination requires that the ______ _____ between the two recombining DNA molecules be resolved. In bacteria, the major ______ that accomplishes this RuvC. Evidence suggests it works in concert with both _____& _____
    • holliday junction
    • endonuclease 
    • RuvA & RuvB
Card Set
Homologous Recombination at the Molecular level
Ch 11