Homologous Recombination at the Molecular level

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 _______ ________.

What causes recombination

The causes of recombination can also create _____ ______. Some chemicals do the same like ______

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

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

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 ________ _______

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.

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.

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 ______ _____

State one example of an indirect DSBs as a result of interference with the rep fork (there are two examples on the answer sheet)

In addition to repairing DSBs in chromosomal DNA, homologous recombination promotes ______ ______ in bacteria.

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.

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 ______ _______

5 key steps of homologous recombination as we understand it

Explain the alignment of two homologous DNA molecule (2)

Explain introduction of breaks in the DNA (1)

Explain strand invasion (3)

Explain the formation of a holliday junction (3)

Explain resolution of the holliday junction

The two homologous DNA molecules aligned at the start of homologous recombination, although nearly identical have different ______ of the same _____

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 _______ ______

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

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

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

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.

Resolution occurring in one of two ways gives rise to two distinct classes of DNA products

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 _______

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 ______.

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

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

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.

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.

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 ______ _______.

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

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 ______

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

RecBCD is composed of _____ subunits (name them) and has both DNA _____ and ______ activities.

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.

The activities of RecBCD are controlled by specific DNA sequence elements known as _____ _____. Why were they discovered?

The recB and RecD subunits are both ____ _______, meaning?

The RecB subunit contains a 3' to 5' ______ and also has a multifunctional ______ _______ that digests the DNA as it moves along.

RecD is a 5' to 3' ______, and RecC functions to recognize ____ ______

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 _______.

_____ 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.

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

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

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 _______

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.

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?

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

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

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.

The observation that recombination is stimulated specifically only on one side of the was initially puzzling. What do we know now?

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

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 ______.

_____ 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

What are the Eukaryotic counterparts to this: 

The ______ complex specifically recognizes holliday junctions and promotes branch migration

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 ______ _____

______ 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 ______ _______.

RuvB is similar to _______ _____ involved in DNA replication. The RuvB ATPase provides the ______ to drive the exchange of ______ ______ that moves the DNA branch.

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.

_____ cleaves specific DNA strands at the holliday junction to finish recombination

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 _____& _____