27th October 2020
DNA-recombination-BioTrivia

What is DNA Recombination? (A Complete Guide)

DNA Recombination is the rearrangement of DNA segments by the involvement of DNA breakage, integration of DNA molecules, or by exchanging the parts of DNA. Breaking of DNA strands and the rejoining process in this recombination assist to produce genetic diversity.

The recombination process has performed both prokaryotes and eukaryotes. In prokaryotes, recombination undergoes through the conjugation, transformation, and transduction procedure. In eukaryotic cells, DNA recombination is seen during meiosis.

Types of DNA Recombination –

4 types of recombination have been found naturally.

  1. General or Homologous recombination
  2. Site-specific recombination
  3. Nonhomologous or Illegitimate recombination
  4. Replicative recombination

Mechanism of different types of Recombination–

1. General/Homologous recombination –

This type of recombination occurs in a very similar type of DNA sequence and relies on sequence complementarity. This type of recombination is seen in the meiosis process. As a result of crossing over, chiasmata forms, and this chiasmata portion help to unite the homologous chromosome during meiosis. In this process, only sequence exchange happens without any gain or loss of nucleotides.

This type of recombination can be explained by 3 models

i. The Holliday model

ii.Meselson Radding model

iii.Double strand break model

i. Holliday Model –

This model of homologous recombination was given by molecular biologist Robin Holliday in 1964 and also known as the heteroduplex model.

At the very first, nicks created by endonuclease at the identical positions of two homologous strands were found. Then one side of the broken end invaded to homologous strand. Invading strands are then attached covalently with homologous strand and Holliday Junction was generated finally by gap sealing with the help of ligase. Holliday junction can move which is termed as branch migration. As a sequel of branch migration, a heteroduplex is formed. The most important characteristic of this model is heteroduplex construction by interchanging the nucleotides segments between two homologous strands. As there was sequence matching between two strands so the heteroduplex’s stability initially is provided by the base pairing between the donor and recipient strand. Mismatch repair is fixed for the correction of heteroduplex. In the advanced stage, heteroduplex went for separation which resolves the intertwined DNA into two separate strands when cleavage occur either horizontally or vertically of chi (Crossover Hotspot Instigator) structure and gives two different results. Vertical cut of heteroduplex and reseal of it gave crossover products where interchanges of nucleotides were reciprocal. If a horizontal cut is introduced then it gives a non-crossover products in which gene conversion can take place.

Holliday Model for Homologous recombination
Fig – Holliday Model for Homologous recombination                         

ii. Meselson Radding model –

It is an extension of the Holliday model. Holliday model was failed to give a satisfactory clarification about the reason for two nicks’ introduction in two homologous strands in the same position. Meselson Radding model interpreted the reason regarding nick formation in two strands and how the double-stranded DNA interacted at the very initial stage to form heteroduplex.

In this model, the endonuclease cut was only in one strand of one DNA double helix. The free endpoint started invading the other duplex by chain synthesizing. D loop structure is created by the strand displacement. Then the unbroken displaced single strand was digested by endonuclease and nicks were formed at the equivalent position of two strands. This was the explanation of this model how two nicks are formed which was not clarified by the Holliday model. The next steps are the same as the Holliday model and the resulted product is also similar.

Meselson Radding Model
Fig – Meselson Radding Model. It gives the same product as the Holliday Model after the resolution of the Chi structure. The difference from the Holliday model is in the initial steps. After forming two nicks in two strands in the same position, the next steps are similar.

iii. Double-strand break model –

It has similarities to the previous model. Breakage of DNA takes place in a double strand of a DNA duplex.3’ overhang is created by exonuclease where one of the two overhangs invades to homologous region of the normal (donor) duplex. D loop is formed by the displacement of the normal strand. 3’ OH helps for DNA synthesis. A repair of a double-strand break can be done by DSBR (Double-Strand Break Repair) and SDSA (Single Displacement Strand Annealing) mechanism (1). DSBR model was established from the research on the transformation of Yeast which carries linear plasmid (2,3). The main feature of this model is the formation of a double Holliday junction. The existence of such a structure of meiotic homologous recombination was confirmed by gel analysis (4,5). Whenever an association of crossing over in DSBR was frequent then SDSA was introduced (6,7). The beginning steps of this process are similar to DSBR but capturing of the second end which gives DNA intermediate does not occur in SDSA. In DSBR if the crossover is not noticed then it is considered to occurring of SDSA(8).In single-strand annealing, double-strand break goes through processing for the single-stranded tail formation and anneal with each other. Repairing of SDSA always provides a non-crossover product.

Double-strand break repairing mechanism through homologous recombination.
Fig – Double-strand break repairing mechanism through homologous recombination.

2. Site-specific recombination –

This type of recombination occurs in specific short sized sequences. Sequence length remains within 12 to 24 bp. The transposition process is involved here which allows the transfer of DNA molecule for insertion into another part of DNA without any homology similarity. A special type of enzymatic involvement is necessary for site-specific recombination.

Example – In E.coli,λ (lambda) bacteriophage genome integration is an example of site-specific recombination. This recombination is taken place with the help of the attachment (att) site present on both E.coli and bacteriophage λ.

The attachment site in bacteria is attB, denoted as BOBʹ

“O” signifies the common sequence/core sequence where recombination occurs.

The attachment site in phage is attP, denoted as POPʹ

One tyrosine recombinase, λ integrase cut the DNA in an uneven manner because staggered end can easily integrate both genomes to each other. Another factor IHF (Integration host factor) is required to bind in the phage attachment site and provides λ integrase binding site in DNA. IHF is a double-stranded DNA binding protein-dependent on the sequence.

V(D)J recombination in immunoglobulin gene rearrangement and Cre-loxP recombination system is the example of this site-specific recombination.

Integration of lambda phage DNA in E.coli genome
Fig- Integration of lambda phage DNA in E.coli genome

3. Nonhomologous or Illegitimate recombination –

This type of recombination occurs where the homology of a DNA sequence is not matching to each other. Sometimes this recombination induces cancer by chromosomal translocation (9).

One common pathway where nonhomologous or illegitimate recombination performs is the nonhomologous end-joining (NHEJ) repair process (10).

The nonhomologous end-joining procedure does not require a template with the same homology. It works through insertion or deletion (also known as indel) of the DNA molecule during repairing. NHEJ can happen in any stage of the cell cycle but most common in G1 and G0 stage. NHEJ is very effective for emergency repairing for double stranded DNA but it is a highly error-prone mechanism. Broken extremities are ligated with losing one or more nucleotides from the ligated site.Ku70 and Ku80 heterodimer recognize the broken end and they make allowance for DNA-PKs (DNA dependent protein kinase to do its function. Kinase induced phosphorylation accommodates Artemis nuclease for end trimming so that ligation can be easily achieved. Complete repairing of dsDNA is done by DNA ligase IV with the assistance of XRCC4.

Nonhomologous end joining repairing mechanism by nonhomologous recombination
Fig – Nonhomologous end-joining repairing mechanism by nonhomologous recombination

4. Replicative Recombination –

This type of recombination forms a duplicate copy of DNA

Example – this recombination is mainly used by transposable elements

Replicative recombination

Chromosomal crossover is the process of recombinant chromosome formation by exchanging the genetic segment between non-sister chromatids of homologous chromosome pairs. Crossing over takes place during synapsis at 1st Prophase’s Pachytene stage of Meiosis. Synapsis/syndesis is the attachment of two homologous chromosomes at 1st Prophase of Meiosis.

Two genes on a chromosome may have a different percentages of chances of crossing over. Close genes have a lower percentage of probability to take part in crossing over whereas genes present far apart have high crossover chances.

Crossing over during Pachytene stage of Meiosis I
Fig- Crossing over during Pachytene stage of Meiosis I

Recombinant Frequency

As a result of crossing over the percentage found by recombinant progeny is recombinant frequency. When two genes are separated by crossing over the process they will be accounted for as recombined. Genes which are separated by crossing over shows high recombination frequency when frequency low it means they are not separated by crossing over.

The formula for recombination frequency = Formula-of-Recombination-Frequency

If 1% recombination present then it will be denoted as 1 CM (centimorgan) which is 1 map unit. This unit is useful for estimating the genetic distance along a chromosome.

If the recombinant frequency is 50% then it suggests that the genes are not present on the same chromosome or unlinked. Genes if hold this measure less than 50% will be determined as on the same chromosome or linked.50% recombination is expected independent assortment whereas 0% recombination considers complete linkage. Recombination frequency can’t be greater than 50% even in the case of multi-crossover.

The relationship of measured distance between genes and crossover frequency is helpful for constructing linkage maps from where relative positions of genes in chromosomes can be assessed.

Here the recombination frequency

Grey, Normal – 445    Recombination-Frequency

Grey, Vestigial – 49

Black, Normal – 51

Black, Vestigial -455

Factors affect the recombinant frequency –

  • Sex – In heterogametic species, female shows more recombination rate than male. Example – Drosophila.
  • Temperature –it varies in different species.
  • Distance between the gene
  • Chemical- Antinomycin D helps in recombination
  • Distance from centromere – Recombination rates are higher in the telomeric region than the centromeric part.

Gene Conversion

It is simply the duplicating of the genetic elements from the donor to the acceptor in a unidirectional way. It is a nonreciprocal transfer of genetic materials.

Different types of DNA sequences like palindromic repeats, direct repeats, minisatellite repeats are associated with gene conversion in human genes. In several cases, gene conversion can be an important cause for conducting human genetic disorders were either total or partial conversion of the functional gene occurs as a donor sequence is closely linked or in a highly homologous sequence.

Gene conversion can be allelic or nonallelic. In some genes, one allele can be replaced by another allele and in the case of nonallelic/ectopic one paralogous sequence of DNA changes another. Allelic conversion is observed in the meiosis process where the recombination is a homologous type between two heterozygotic sites (11).

Ectopic gene conversion happens during the repairing of double-strand DNA breakage. Repairing does not involve homologous chromosomes rather the broken duplex of sister chromatid is used here. Homologous sequences which are present at various gene loci are paralogous sequence. In ectopic gene conversion, the conversion takes place in paralogous sequences (12).

In B cells

In the early phase of cell development in Bone Marrow, regulated site-specific genetic recombination occurs at their immunoglobulin loci. This site-specific recombination is known as V(D)J recombination which shows genetic variation through the expression of IgM antigen receptor on the surface of B cell.

During the migration of mature B cells from Bone marrow to periphery secondary diversification occurs depending on antigen. As a consequence cytidine deaminase dependent class switching happens (13).

At the pro-B cell phase, IgM heavy chain is expressed by V(D)J recombination where firstly D to JH rearrangement is done after that VH to DJH rearrangement is completed. IgM heavy chain is displayed as a precursor B cell receptor on the surface of B cell after the association of light chain,b-proteins, CD79a with it. The signal given by pre-BCR helps for initiating VJ recombination of Igĸ or Igγ light chain (Herzog et al, 2009). Endonuclease RAG1&RAG2 makes cleavage at a specific recombination signal sequence (RSS). RAG complex introduces two hairpin-like extremities. DNA segment cutting shows blunt double-strand break at the extremities. These broken DNA segments are joined by nonhomologous end joining (NHEJ). As the NHEJ is an error-prone process, the joints formed by NHEJ can lose or gain certain nucleotides(14)

The junctional part of VDJ expresses CDR3 which plays a pivotal role in antigen-binding specificity.

Genetic Engineering

It is a process of manipulation of an organism’s gene with the collaboration of Biotechnology. This process is used for producing the desired genetic product. Recombinant DNA technology gives support for generating newly modified DNA. This new DNA is attached in a random way to the targeted portion of the gene.

Organisms which are the product of genetic engineering are known as genetically modified organism. Application of genetic engineering lies in a wide array including industrial, medicinal, research field, agriculture, etc. Different approaches to genetic engineering can be used as a potent health remedy against several genetic diseases by manufacturing the desired vaccine, hormones, and drugs under this process. Genetically modified foods provide strong economic support to a country although the health issues related to genetically modified food are a debatable topic.

In recombinant DNA technology, the main objective is to make the gene of interest or the numerous copy of the gene of interest. Different steps are involved in this process.

  1. Isolation and identification of the desired gene – Firstly need to open the cell to get the purified DNA (15). Different restriction enzymes are used for cutting the DNA so that genes of interest can be isolated (16). The identification of the desired gene is necessary. The genomic library, cDNA library provide the sequence of a known gene. If the sequence is known it chemical synthesis is possible. If the desired gene is found in very few amounts for cloning purposes, polymerase chain reaction (PCR) can be used for producing more copies of that gene.
  2. Joining with suitable vector – recombinant DNA formation by joining into a vector like a plasmid. Vector is a very short segment of DNA stretch taken from bacteria, the virus of the higher organism in which foreign DNA is inserted for numerous copies generating purpose (17).
  3. Recombinant DNA insertion in host cell- Different gene transfer strategies are used for delivering the recombinant DNA in the host like the chemical transfection method(by DEAE-dextran, by calcium phosphate, poly-glycol mediated), physical transfection method(microinjection, electroporation, particle bombardment, by liposome, DNA transfer via pollen, ultrasound-mediated), transformation and transduction(virus-mediated process).
  4. Recombinant cell selection – Before introducing in the targeted host, the gene of interest needs to be added with the promoter, terminator, and a selectable marker. Selectable markers help to determine whether the transformation has performed on not by differentiating them from the untransformed cells (18). Several methods are used for selection like –
  • By antibiotic resistance
  • By observing visible characters
  • By assessing biological activity
  • Culturing the selected cells in large scale
  • Blotting test.
  1. Cloning of gene of interest and its expression – By using the host cell’s enzymatic machinery, a recombinant vector will replicate and produce a large number of copies of the desired gene.
  2. Isolation and purification of the gene of interest – Affinity chromatography technique is used for this purpose.

An example of large quantities of products in cell culture is insulin hormone.

Gene targeting uses homologous recombination for modifying or manipulating a gene regarding interest. Genome editing is a technique that increases the frequency of gene targeting in an effective way. Artificially synthesized nucleases are used for creating breakage at the desired location in double stranded DNA. Nonhomologous end-joining and homologous recombination help to repair and the desired gene is formed. Gene editing is also used for creating some changes like a mutation in the target gene which can be able to knock out a gene (19).

Recombinational Repair

Recombinational repair eventuates after completion of replication if there is any damage in one strand of the double-strand DNA molecule. So it is also termed as a post-replication repair method.

  • If any damage such as pyrimidine dimer is formed in one strand during replication, this damage can bypass the replication process as it is used as a template. As an outcome, a gap is generated in the newly synthesized strand at the same position of damage in the parental strand. Filling of that gap is done by homologous single strand from normal undamaged DNA duplex. After exchanging the normal single strand, the acceptor duplex faces a damaged parental strand. On the other side, the parental strand of the donor duplex faces a gap. This gap is filled by generating a normal duplex by repair synthesis.
  • If the replication process is unable to bypass the damaged site then the daughter strand formation is halted. In this condition, the replication fork stops and for a short distance fork reverses to form a duplex between two daughter strands. Extension of this reversed incomplete portion is completed by DNA polymerase whereas the normal daughter strand act as a template. Then the replication fork would be able to move in forwarding direction by branch migration. So that replication is bypassed from the damage site and capable to continue.
Recombinational repair when replication fork collapses and restoration of a fork as well as a resume of the replication process.
Fig – Recombinational repair when replication fork collapses and restoration of a fork as well as a resume of the replication process.
  • If the parent strand contains a nick in the phosphodiester backbone in a single strand it can’t go through a repairing process if the replication fork is not passed at this point. When the replication fork approaches this nicked portion, the fork is degenerated and provides double-strand breakage on the newly synthesized strand. Homologous recombination repairs the broken part. RecBCD complex is involved here for processing 3ʹ overhang in a single strand. As a template donor duplex is acted for DNA synthesis, a D loop formation occurs when 3ʹ overhangs invade. Invasion of 3ʹ overhangs mediated by RecA protein yields Holliday junction. Separation or resolution of this junction assists in the recovery of the replication fork.

SOS Response

When alkylating agents or UV radiation creates DNA damage SOS response is initiated. RecA which is a LexA protease is activated and recognizes the damage site where LexA repressor is inhibited. RecA generates a filamentous arrangement around the DNA and facilitates annealing in single-stranded DNA. Rec A is an ATP-dependent protein (20). Activation of RecA favors the cleavage of LexA from the operator (20,21) SOS response accounts for high rates of repairing but error-prone.

LexA repressor represses the SOS genes when it attaches to SOS Box, a consensus 20bp sequence. Single-stranded DNA damage halts the movement of DNA polymerase; at that moment sos genes are expressed. RecA, LexA, UvrA, UvrB, UvrD genes are activated even in the presence of a weak signal. At first, it induces the Nucleotide Excision Repair (NER). If the NER would not be able to rectify the damage then the SOS response is fully generated. Then UmuC, UmuD and sulA are induced (20). Cell division is halted by sulA when it binds to FtsZ. With the help of UmuDC, mutational repair happens.

DNA Repair

Meiotic Recombination

Meiosis plays a key role in the formation of haploid gametes from diploid cells. Recombination and rearrangement in the homologous chromosomes at the time of meiosis confers evolutionary aspects as they constitute genetic variability. Mostly in meiotic division DNA replication occurs for a single time. Two sister chromatids are formed by duplicating chromosome and sister chromatids remain attached by cohesion.

Chromosomal segregation ensues in two phases where first in Anaphase-I and second in Anaphase-II after a short S phase. Homologous chromosomes recombine in Prophase-I and their association helps in bivalent formation at this stage. This homologous pair is separated in Meiosis-I. Reduction in ploidy during first division is dependent on bivalent formation. In most cases, meiotic recombination or crossing over occurs on bivalent. Crossing over in meiosis reciprocally exchanges the genetic material between two non-sister chromatids.

Achiasmy & Heterochiasmy

Dimorphism of recombination can happen on autosomes or on a sex chromosome. Where total lacking of autosomal recombination encounter in one sex this is known as achiasmy. It has been well identified in male Drosophila (22).

According to the Haldane-Huxley rule, when achiasmy happens in dioecious species, most of the time it is heterogametic sex (23,24). Haldane –Huxley rule has also been interpreted as a consequence of pleiotropic selection which speaks against recombination and supports evolutionary selection (in between X and Y or Z and W).

Sometimes business is observed in certain species where one sex (especially female) prefers higher rates of recombination. This pattern of differences varied in recombination rates is known as heterochiasmy. Heterochiasmy can be evolved when –

a. difference in epistasis in maternally and paternally derived genes,

b. if there is male-female gamete difference observed in haploid epistasis and

c. In diploid, a male-female difference in cis epistasis minus trans epistasis.

So many ideas were grown to find out the reason for heterochiasmy. These are –

  1. Mechanical explanation- Variation in internal environments like an ongoing physical and molecular process between males and females can be a reason for heteroplasmy (25).
  2. Neutral hypothesis- Burt et al, 1991 gave this theory when they were unable to favor the correlation between the scope of different sex selection and the enormity of heterchiasmy. They suggested that heterochiasmy can be neutral.
  3. Sexual selection, evolutionary explanation- Triver (1998) suggested that the phenomenon heterochiasmy can be an outcome of sexual selection.

Latest Research on this topic

  1. An approach for attacking cancer cells is the generating of specific monoclonal antibodies against the surface antigen which is decided to be introduced as an inserting vehicle of toxic substances only on the cancer cells. This is termed as recombinant immunotoxin as cancer therapeutic. It is a recombinant containing a portion of lethal toxin and Fv segment of antibodies. This type of protein expression is done in E.coli and goes through clinical trials for different types of cancer treatment.

Reference –Tapan K Bera.Recombinant immunotoxin as targeted therapeutic protein for Cancer therapy. International Conference on Genetic & Genetically modified organisms.August 12-13,2013, Doubletree by Hilton, Ralcigh, NC, USA.

Reference

  1. Patrick Sung Hannah Klein. Mechanism of homologous recombination: mediators and helicases take on regulatory functions. 2006 Oct; Nat Rev Mol Cell Biol.7(10):739-50.
  2. Orr-Weaver, T. L., Szostak, J. W. & Rothstein, R. J. Yeast transformation: a model system for the study of recombination. Proc. Natl Acad. Sci. USA 78, 6354–6358 (1981).
  3. Orr-Weaver, T. L. & Szostak, J. W. Yeast recombination: the association between double-strand gap repair and crossing-over. Proc. Natl Acad. Sci. USA 80, 4417–4421 (1983).
  4. Collins, I. & Newlon, C. S. Meiosis-specific formation of joint DNA molecules containing sequences from homologous chromosomes. Cell 76, 65–75 (1994). Presents the first physical demonstration of meiotic recombination DNA intermediates between homologous chromosomes.
  5. Schwacha, A. & Kleckner, N. Identification of joint molecules that form frequently between homologues but rarely between sister chromatids during yeast meiosis. Cell 76, 51–63 (1994). Presents evidence for DNA joint molecules between non-sister chromatids as recombination intermediates in meiosis.
  6. Ferguson, D. O. & Holloman, W. K. Recombinational repair of gaps in DNA is asymmetric in Ustilago maydis and can be explained by a migrating D-loop model. Proc. Natl Acad. Sci. USA 93, 5419–5424 (1996). Describes a recombination model, which is now known as the SDSA model, that does not entail crossover formation.
  7. Strathern, J. N. et al. Homothallic switching of yeast mating-type cassettes is initiated by a double-stranded cut in the MAT locus. Cell 31, 183–192 (1982). Discusses how yeast mating-type switching occurs by SDSA.
  8. Allers, T. & Lichten, M. Differential timing and control of noncrossover and crossover recombination during meiosis. Cell 106, 47–57 (2001).
  9. Zucman-Rossi J, Legoix P, Victor JM, Lopez B, Thomas G.Chromosome translocation based on illegitimate recombination in human tumors.1998. Proceeding of the National Academy of sciences of the United States of America.95(20):11786.
  10. Wilson TE.2006.Non-homologous End Joining: mechanisms, conversion & relationship ti illegitimate recombination in Aguilera A. Rothstein R (eds).Molecular Genetics of Recombination.Topics in Current Genetics. Berlin,Heidelberg, Springer.pp-487.
  11. Galtier N, Piganeu G,Mouchiroud D,Duret L.GC content evolution in mammalian genomes: the biased gene conversion hypothesis.2001.Genetics,159:907-911.
  12. Duret L,Galtier N.Biased gene conversion and evolution of mammalian genomic landscape.2009.Annu.Rev.Genom.Hum Genet.10:285-11.
  13. Maizels N,Immunoglobulin gene diversification.2005.Annu.Rev.Genet.39:23-46.
  14. G H Gauss ,M R Lieber.Mechanistic constraints on diversity in human V(D)J recombination. 1996 Jan; Mol Cell Biol.16(1):258-69.
  15. Nicholl, Desmond S.T.An introduction of genetic engineering.2008.Cambridge University Press.p-34.
  16. Alberts B, Johnson A, Lewis J et al.2002.”8” Isolating, cloning, and sequencing DNA (4th ed).New York, Garland Science.
  17. The process of genetic modification.www.fao.org. Retrieved 29 April 2017.
  18. Hohn B, Levy AA, Puchta H. Elimination of selection markers from transgenic plants.2001. Current Opinion in Biotechnology .12(2): 139-143.
  19. Ekker SC. Zinc finger-based knockout punches for Zebrafish genes.2008. Zebrafish. 5(2): 12-3.
  20. Maslowska KH, Makiela-Dzbcnska K, Fijalkowsa IJ. The SOS system: A complex and tightly regulated response to DNA damage. 2019. Environmental & Molecular Mutagenesis. 60(4): 368-384.
  21. Nelson DL, Cox MM. Lehninger Principles of Biochemistry.2004. 4th Edition, New York: W.H.Freeman and Company. p-1098.
  22. Lenormand, Thomas. The evolution of sex dimorphism in Recombination.2003.Genetics. 163(2):811-822.
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