Genetic exchange works constantly to blend and rearrange chromosomes, most obviously during meiosis, when homologous chromosomes pair before the first nuclear division. During this pairing, genetic exchange between the chromosomes occurs. This exchange, classically termed crossing over, is one of the results of homologous recombination. DNA Recombination involves the physical exchange of DNA sequences between the chromosomes.
The frequency of crossing over between two genes on the same chromosome depends on the physical distance between these genes, with long distances giving the highest frequencies of exchange. In fact, genetic maps derived from early measurements of crossing-over frequencies gave the first real information about chromosome structure by revealing that genes are arranged in a fixed, linear order. Sometimes, however, gene order does change: For example, movable DNA segments called transposons occasionally “jump” around chromosomes and promote DNA rearrangements, thus altering chromosomal organization.
Elegant early experiments using heavy isotopes of atoms incorporated into DNA provided the first molecular view of the process of homologous recombination. This is the same approach used by Matthew Meselson and Franklin W. Stahl to show that DNA replicates in a semiconservative manner. In their experiments, Meselson and Stahl showed that the products of replication contain one old and one newly synthesized DNA strand. In contrast, this same experimental approach revealed that the recombination process under investigation involved the direct breakage and rejoining of DNA molecules.
HOMOLOGOUS DNA RECOMBINATION
Key steps of homologous recombination present in any models include the following :-
- Alignment of two homologous DNA molecules :- Homologous are DNA sequences that are identical or nearly identical for a region of at least 100 bp or so. Despite this high degree of similarity, DNA molecules can have small regions of sequence difference and may, for example, carry different sequence variants, known as alleles, of the same gene.
- Introduction of breaks in the DNA :- Once the breaks are formed, the ends at the breaks are further processed to generate regions of single-stranded DNA.
- Strand invasion :- Initial short regions of base pairing are formed between the two recombining DNA molecules. This pairing occurs when a singlestranded region of DNA originating from one parental molecule pairs with its complementary strand in the homologous duplex DNA molecule. This event is called strand invasion. As a result of the strand invasion process, regions of new duplex DNA are generated; this DNA, which often contains some mismatched base pairs, is called heteroduplex DNA.
- Formation of the Holliday junction :- After strand invasion, the two DNA molecules become connected by crossing DNA strands to form a structure that is called a Holliday junction. This 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 process is called branch migration.
- Resolution of the Holliday junction :- The process to regenerate DNA molecules and therefore finish genetic exchange is called resolution. Resolution can be achieved in one of two ways, either by cleavage of the Holliday junction or (in eukaryotes) by a process of “dissolution.” In the first, cutting the DNA strands within the Holliday junction regenerates two separate duplexes.
PROTEINS INVOLVE IN HOMOLOGOUS DNA RECOMBINATION
The RecBCD enzyme processes broken DNA molecules to generate these regions of ssDNA. RecBCD also helps load the RecA strand-exchange protein onto these ssDNA ends.
- RecBCD is composed of three subunits (the products of the recB, recC, and recD genes) and has both DNA helicase and nuclease activities.
- The complex binds to DNA molecules at the site of a DSB and tracks along DNA using the energy of ATP hydrolysis. As a result of its action, the DNA is unwound, with or without the accompanying nucleolytic destruction of one or both of the DNA strands.
- The activities of RecBCD are controlled by specific DNA sequence elements known as Chi sites (for “crossover hot spot instigator”). Chi sites were discovered because they stimulate the frequency of homologous recombination.
- RecB subunit contains a 30 -to-50 helicase and has also a multifunctional nuclease domain that digests theDNA as it moves along.
- RecD is a 50 -to-30 helicase, andRecC functions to recognizeChi sites.
- Upon encountering the Chi sequence, the complex pauses for a few seconds, then continues at about one-half the initial rates.
- RecC signals recD to stop unwinding DNA.
- RecD then signals rec B to cut the DNA
- RecB creates the nick at chi
- Unwinding continues and produces 3 –OH single strand tail with chi near the terminus.
- The recA protein is then actively loaded onto the 3 prime tail by recBCD.
- The RecA protein coated DNA filament that is able to the intact double helix and set up the D loop.
- After the strand invasion step of recombination is complete, the two recombining DNA molecules are connected by a DNA branch known as a Holliday junction.
- Movement of the site of this branch requires exchange of DNA base pairs between the two homologous DNA duplexes. Cells encode proteins that greatly stimulate the rate of branch migration.
- RuvA recognizes and binds to Holliday junctions and recruits the RuvB protein to this site.
- RuvB is a hexameric ATPase, similar to the hexameric helicases involved in DNA replication.
- Resolution by RuvC occurs when RuvC recognizes the Holliday junction—likely in a complex with RuvA and RuvB—and specifically nicks two of the homologous DNA strands that have the same polarity.
- This cleavage results in DNA ends that terminate with 50 -phosphates and 30 -OH groups that can be directly joined by DNA ligase.
- Depending on which pair of strands is cleaved by RuvC, the resulting ligated recombination products will be of either the “splice” (crossover) or “patch” (noncrossover) type.
DOUBLE STRAND BREAK MODEL
Double strand break model is similar in many ways to the model discuss previously. But initiation is by double strand break in one of the two DNA is resected from the breaking point by an exonuclease that leaves 3-OH overhangs. One of the overhang invades the homologous region in the donor duplex. This is called single strand invasion.
The formation of heteroduplex DNA generates a D loop, in which one strand of the donor duplex is displaced. Becuase the invading strand is with 3 termini, it serves as primer for new DNA synthesis. Elongation from this DNA end using the complementary strands in homologous duplex as a template serves to regenerate the region of the DNA that were destroy during the processing of the strand at the break site. The two holiday junctions found in recombination intermediate generation by this model move by branch migration and ultimately are resolve to finish recombination.
Meiosis involves two rounds of nuclear division, resulting in a reduction of the DNA content from the normal content of diploid cells (2N) to the content present in gametes (1N). . During S phase, these chromosomes are replicated to give a total DNA content of 4N. The products of replication—that is, the sister chromatids—stay together. Then, in preparation for the first nuclear division, these duplicated homologous chromosomes must pair and align at the center of the cell. It is this pairing of homologs that requires homologous recombination. These events are carefully timed. Recombination must be complete before the first nuclear division to allow the homologs to properly align and then separate. During this process, sister chromatids remain paired.
Then, in the second nuclear division, it is the sister chromatids that separate. The products of this division are the four gametes, each with one copy of each chromosome (i.e., the 1N DNA content). In the absence of recombination, chromosomes often fail to align properly for the first meiotic division, and, as a result, there is a high incidence of chromosome loss. This improper segregation of chromosomes, called nondisjunction, leads to a large number of gametes without the correct chromosome complement. Gametes with either too few or too many chromosomes cannot develop properly once fertilized; thus, a failure in homologous recombination is often reflected in poor fertility. The homologous recombination events that occur during meiosis are called meiotic recombination.
Meiotic recombination also frequently gives rise to crossing over between genes on the two homologous parental chromosomes. An important consequence is that the alleles present on the parental DNA molecules are reassorted for the next generation.
Initial models for the mechanism of homologous recombination were formulated largely to explain the genetic consequences of the process. Now that the basic steps involved in recombination are understood, it is useful to review how the process of homologous recombination alters DNA molecules and thereby generates specific genetic changes.
IMAGES AND CONTENT ARE TAKEN FROM
- Molecular biology of the genes (seventh edition) by Watson, Baker, Bell, Gann, Levine, Losick
- Life sciences fundamental and practices sixth edition, pathfinder publication
:- Article Written By Zahra Madraswala