Recombination | Zoology Optional Notes for UPSC PDF Download

Definition of Recombination

• The most important features of organisms are to adapt in the environment and to maintain their DNA sequence in the cells generation to generations with very little alterations. In long term survival of organisms depends on genetic variations, a key feature through which the organism can adapt to an environment which changes with time.
• This variability among the organisms occurs through the ability of DNA to undergo genetic rearrangements resulting in a little change in gene combination. Rearrangement of DNA occurs through genetic recombination.
• Thus, recombination is the process of formation of new recombinant chromosome by combining the genetic material from two organisms. The new recombinants show changes in phenotypic characters.
• Most of the eukaryotes show a complete sexual life cycle including meiosis, an important event that generates new allelic combinations by recombination. It is made possible through chromosomal exchange resulting from crossing over between the two homologous chromosomes containing identical gene sequences.
• Much work was done on eukaryotic genetics until 1945 that laid the foundation of classical genetics. The work on bacterial genetics was done between 1945 and 1965 that advanced the understanding of microbial genetics at molecular level.

Mechanism of Recombination

Basically, there are three theories viz., breakage and reunion, breakage and copying and complete copy choice that explain the mechanism of recombination (Fig.8.23).

Breakage and Reunion

Two homologous duplex of chromosome laying in paired form breaks between the gene loci a and b, and a⁺ and b⁺ (Fig. 8.23A). The broken segments rejoin crosswise and yield recombinants containing a and b⁺ segment, and a⁺ and b segment. This type of recombination does not require the synthesis of new DNA. This concept has been used to explain genetic recombination.

Breakage and Copying

One helix of paired homologous chromosome (ab and a⁺ b⁺) breaks between a and b (Fig. 8.23B). Segment b is replaced by a newly synthesized segment copied from b⁺ and attached to a section. Thus the recombinants contain and ab⁺ and a⁺ b⁺.

Complete Copy Choice:

• In, 1931, Belling proposed this theory for recombination of chromosome in higher animals. However, it has been questioned by several workers. Therefore, it has only historical importance.
• According to this theory a portion of one parental strand of homologous chromosome acts as template for the synthesis of a copy of its DNA molecule. The process of copying shifts to the other parental strand. Thus, the recombinants contain some genetic information of one parental strand and some of the other strand (Fig. 8.23 C).

Types of Recombination:

Many kinds of recombination occur in microorganisms.

These are classified basically into the following three groups:

• General recombination,
• Non-reciprocail recombination, and
• Site specific recombination.

(i) General Recombination:

• General recombination occurs only between the complementary strands of two homologous DNA molecules. Smith (1989) reviewed the homologous recombination in prokaryotes. General recombination in E. coli is guided by base pairing interactions between the complementary strands of two homologous DNA molecules.
• Double helix of two DNA molecules breaks and the two broken ends join to their opposite partners to reunite to form double helix. The site of exchange can occur anywhere in the homologous nucleotide sequence where a strand of one DNA molecule becomes base paired to the second strand to yield heteroduplex just between two double helices (Fig. 8.24).
• In the heteroduplex no nucleotide sequences are changed at the site of exchange due to cleavage and rejoining events. However, heteroduplex joints can have a small number of mismatched base pairs.
• General recombination is also known as homologous recombination as it requires homologous chromosomes. In bacteria and viruses general recombination is carried out by the products of rec genes such as RecA protein. The RecA protein is very important for DNA repair; therefore, it is recA dependent recombination.

Holliday Model for General Recombination

Holliday (1974) presented a model to show the general recombination (Fig. 8.25). According to this model recombination occurs in five steps such as strand breakage, strand pairing, strand invasion/assimilation, chiasma (crossing over) formation, breakage and reunion and mismatch repair.

(a) Strand breakage:

• General recombination occurs through crossing over by pairing between the complementary single strands of DNA duplex (a). Two homologous regions of DNA double helix undergo an exchange reaction.
• The homologous region contains a long sequence of complementary base pairing between a strand from one or two original double helices and a complementary strand from the other. However, it is unknown how the homologous region of DNA recognises each other.
• A list of recombination genes and their function have been given in Table 8.2. The RecBCD proteins of recBCD or recJ genes are required for recombination in E. coli. This protein enters the DNA from one end of double helix, and travels along the DNA at double helix, at the rate of about 300 nucleotides per second.
• It creates a loop of ssDNA along travelling DNA (b). It uses energy derived from hydrolysis of ATP molecules. A special recognition site (a) sequence of eight nucleotides scattered throughout E. coli chromosome (b) is nicked in the travelling loop of DNA formed by RecBCD protein.

Table 8.2 : Recombination (rec) genes and their function.

(b) Strand pairing:

The RecBCD proteins act as DNA helicase because these hydrolyse ATP and travel along DNA helix. Thus, the RecBCD proteins result in formation of single stranded whisker at the recognition site which is displaced from the helix (c). This initiates a base pairing interaction between the two complementary sequences of DNA double helix.

(c) Strand invasion/assimilation:

• A single strand (whisker) generated from one DNA double helix invades the another double helix (d). In E. coli recA gene produces RecA protein which is important for recombination between the chromosomes like single strand binding (SSB) proteins, The RecA protein binds firmly to single stranded DNA to form a nucleoprotein filament.
• Roca and Cox (1990) have reviewed the structure and function of RecA protein. RecA protein promotes rapid renaturation of complementary ssDNA hydrolyzing ATP in the process. RecA protein has several binding sites; therefore, it can bind a ssDNA and subsequently a dsDNA. RecA protein binds first to ssDNA, then search for homology between the donor strand and the recipient molecule.
• Due to the presence of these sites RecA protein catalyses a multistep reaction (called synapsis) between the homologous region of ssDNA and a DNA double helix. E. coli SSB protein helps the Rec protein to carry out these reactions. When a region of homology is identified by an initial base pairing between the complementary sequences, the crucial step in synapsis occurs.
• In vivo experiments have shown that several types of complexes are formed between a ssDNA covered with RecA protein and a dsDNA helix. First a non-base paired complex is formed which is converted into a three stranded structure (ssDNA, dsDNA and RecA protein) when a homologous region is found.
• This complex is unstable and spins out a DNA heteroduplex plus a displaced ssDNA from the original helix. Once the homologous regions are encountered and the ssDNA and dsDNA are complexed, a stable D-loop is formed (d).

(d) Branch migration:

• The next step is the assimilation of strand and nick ligation (e). The donor strand gradually displaces the recipient strand which is called branch migration. After formation of synapsis, the heteroduplex region is enlarged through protein-directed branch migration catalysed by RecA protein.
• RecA protein directed branch migration proceeds at a uniform rate in one direction due to addition of more RecA protein to one end of RecA protein filament on the ssDNA. Branch migration can take place at any point where two single strands with the sequence make attempts to pair with the same complementary strand.
• An unpaired region of the other single strand resulting in movement of branch point without changing the total number of DNA base pairs. Special DNA helicases that catalyse protein directed branch migration are involved in recombination. In contrast, the spontaneous branch migration proceeds in both the directions almost at the same rate. Therefore, it makes a little progress over a long distance.

(e) Chiasma or crossing over formation:

• Exchange of a single strand between two double helices is a different step in a general recombination event. After the initial cross strand exchange, further strand exchanges between the two closely opposed helices is thought to proceed rapidly. A nuclease cleaves and partly degrades the D-loop at some points.
• At this stage possibly different organisms follow different pathways. However, in most of the cases an important structure called cross-strand exchange (also called Holliday Juncture or chi form or chiasmas, is formed by the two participating DNA helices (g). A chi form of single stranded connections in the cross over region has also been observed under the electron microscope by Dressier and Potter (1982).
• The chi form of two homologous helices that initially paired and held together by mutual exchange of two of the four strands where one strand originates from each of the helices (g).
• The chi form has two important properties, (i) the point of exchange can migrate rapidly back and forth along the helices by a double branch migration, and (ii) it contains two pairs of strands, one pair of crossing strands and the other pair of non-crossing strands.

(f) Breakage and reunion:

• The chi structure can isomerize several rotations (h). This results in alteration of two original non-crossing strands into the crossing strands, and the crossing strands into the non-crossing strands. In order to regenerate two separate DNA helices, breakage and reunion in two crossing strands are required.
• If breakage and reunion occur before isomerization the two crossing strands would not occur. Therefore, isomerization is required for the breakage and reunion of two homologous DNA double helices resulting from general genetic recombination.
• Breakage and reunion occur either in the vertical or horizontal plane. If breakage occurs horizontally the recombinants would contain genotype ABlab with a little change in base sequences at the inner region (i).
• However, if breakage occurs vertically the recombinants would contain Ab/aB (J). The RurC protein and RecG protein expressed from ruvC and recG genes respectively are thought to be alternative endonucleases specific for Holliday structure.

(g) Mismatch Repair (Mismatch Proof Read­ing System):

• It is such a repair system which corrects mismatched base pairs of unpaired regions after recombination. This system recognises mis­matched function of DNA polymerase. The mecha­nism involves the excision of one of the other mismatched bases along with about 3,000 nucleotides. This RecFJO is involved in the repair of short mismatch either in the initial stage or at the end of recombination.
• The two proteins MutS and MutL are present in bacteria and eukaryotes. The MutS protein binds to mismatched base pair, whereas MutL scan the DNA for a nick (Fig. 8.26).
• When a nick is formed MutL triggers the degradation of the nicked strand all the way back through the mismatch, because the nicks are largely confined to the newly replicated strands in eukaryotes, replication errors are selectively removed. In bacteria the mechanism is the same except that an additional protein MutH nicks the un-methylated GATC sequences and begins the process.
  
• It has been demonstrated in yeast and bacteria that the same mismatch repair system which removes replication errors as in Fig. 8.26 also interrupts the genetic recombination events between imperfectly matched DNA sequences. It is known that homologous genes in two closely related bacteria (E. coli and S.typhimurium) generally will not recombine, even after having 80% identical nucleotide sequences.
• However, when mismatch repair system is inactivated by mutation, the frequency of such interspecies recombination increases by 100-fold. This mechanism protects the bacterial genome from sequence changes that would be caused by recombination with foreign DNA molecules entering in the cell.

(ii) Non-reciprocal Recombination (Gene Conversion):

• The fundamental law of genetics is that the two partners contribute the equal amount of genes to the offsprings. It means that the offsprings inherit the half complete set of genes from the male and half from the female. One diploid cell undergoes meiosis producing four haploid cells; therefore, the number of genes contributed by male gets halved and so the genes of female.
• In higher animals like man it is not possible to analyse these genes taking a single cell. However, in certain organisms such as fungi it is possible to recover and analyse all the four daughter cells produced from a single cell through meiosis.
• Occasionally, three copies of maternal allele and only one copy of paternal allele is formed by meiosis. This indicates that one of two copies of parental alleles has been altered to the maternal allele. This gene alteration is of non-reciprocal type and is called gene conversion. Gene conversion is thought to be an important event in the evolution of certain genes and occurs as a result of the mechanism of general recombination and DNA repair.
  
• Non-reciprocal general recombination is given in Fig. 8.27. Kobayashi (1992) has discussed the mechanism for gene conversion and homologous recombination.
• This process starts when a nick is made in one of the strands (a). From this point DNA polymerase synthesizes an extra copy of a strand and displaces the original copy as a single strand (b). This single strand starts pairing with the homologous region as in lower duplex of DNA molecule (b). The short unpaired strand produced in step (b) is degraded when the transfer of nucleotide sequence is completed. The results are observed (in the next cycle) when DNA replication has separated the two non-matching strands (c).

(iii) Site-Specific Recombination:

• Site specific recombination alters the relative position of nucleotide sequences in chromosome. The base pairing reaction depends on protein mediated recognition of the two DNA sequences that will combine. Very long homologous sequence is not required.
• Unlike general recombination, site specific recombination is guided by a recombination enzyme that recognises specific nucleotide sequences present on one of both recombining DNA molecules. Base pairing is not involved, however, if occurs the heteroduplex joint is only a few base pair long.
• It was first discovered in phage λ by which its genome moves into and out of the E. coli chromosome. After penetration phage encoded an enzyme, lambda integrase which catalyses the recombination process (Fig. 8.28). Lambda integrase binds to a specific attachment site of DNA sequence on each chromosome.
• It makes cuts and breaks a short homologous DNA sequences. The integrase switches the partner strands and rejoins them to form a heteroduplex joint of 7 bp long. The integrase resembles a DNA topoisomerase in rejoining the strands which have previously been broken.

Site specific recombination is of the following two types:

(a) Conservative site-specific recombina­tion:

Production of a very short heteroduplex by requiring some DNA sequence that is the same on the two DNA molecules is known as conservative site-specific recombination. The detail procedure is described in Fig. 8.28.

(b) Trans-positional site-specific recombi­nation:

• There is another type of recombination system known as trans-positional site-specific (TSS) recombination. The TSS recombination does not produce heteroduplex and requires no specific sequences on the largest DNA.
• There are several mobile DNA sequences including many viruses and transposable elements that encode integrates. The enzyme integrates by involving a mechanism different from phage λ insert its DNA into a chromosome. Each enzyme of integrates recog­nises a specific DNA sequence like phage λ.
• K. Mizuuchi (1992a) reviewed the mecha­nism of trans-positional recombination based on the studies of bacteriophage Mu and the other elements. The enzyme integrase was first purified from Mu. Similar to integrase of phage λ, the Mu integrase also carries out of its cutting and rejoining reactions without requirement of ATP. Also they do not require a specific DNA sequence in the target chromosome and do not form a joint of heteroduplex.
• Different steps of TSS recombinational events are shown in Fig. 8.29. The integrase makes a cut in one strand at each end of the viral DNA sequences, and exposes the 3′-OH group that protrudes out. Therefore, each of these 3′-OH ends directly invades a phosphodiester bond on opposite strands of a randomly selected site on a target chromosome.
• This facilitates to insert the viral DNA sequence into the target chromosome, leaving two short single stranded gaps on each side of recombinational DNA molecule.
• These gaps are filled in later on by DNA repair process (i.e. DNA polymerase) to complete the recombination process. This mechanism results in formation of short duplication (short repeats of about 3 to 12 nucleotide long) of the adjacent target DNA sequence. Formation of short repeats is the hall-marks of a TSS recombination.