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DNA Recombination

  1. Shunichi Takeda,
  2. Mitsuyoshi Yamazoe

Published Online: 23 SEP 2005

DOI: 10.1038/npg.els.0005283

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How to Cite

Takeda, S. and Yamazoe, M. 2005. DNA Recombination. eLS.

Author Information

  1. Kyoto University, Kyoto, Japan

Publication History

  1. Published Online: 23 SEP 2005

Introduction

  1. Top of page
  2. Introduction
  3. DNA Recombination and DNA Repair
  4. Mechanism of Nonhomologous End-joining
  5. Mechanism of Homologous Recombination
  6. Regulation of the Two Different DSBR Pathways?
  7. Recombination at the Immunoglobulin Locus
  8. Medical Implications of DNA Recombination
  9. See also
  10. References
  11. Further Reading
  12. Web Links

Genetic recombination plays an important role in altering deoxyribonucleic acid (DNA) sequences to allow organisms to respond to a changing environment or in repairing damaged DNA caused exogenously or endogenously. Genetic recombination is classified into two major classes: general recombination and site-specific recombination. In general recombination, genetic exchange takes place between a pair of homologous DNA sequences, thus also referred to as homologous recombination (HR). In eukaryotes, it occurs during meiosis in germ cells to generate genetic diversity and at the G2 stage of the cell cycle in mitotic cells to repair damaged DNA. On the other hand, site-specific recombination is mediated by recombination enzymes that recognize short, specific nucleotide sequences present on one or both of the recombining DNA molecules. The most representative example in vertebrate cells is immunoglobulin gene rearrangement. In site-specific recombination, liberated DNA ends are joined by a mechanism called nonhomologous end-joining (NHEJ). Deficiency of HR or NHEJ leads to genome instability and sometimes causes chromosome translocation. There is a third class of recombination, called illegitimate recombination, but its mechanism is unclear. It includes the majority of translocation and random integration events that often occur after introduction of exogenous DNA into culture cells.

DNA Recombination and DNA Repair

  1. Top of page
  2. Introduction
  3. DNA Recombination and DNA Repair
  4. Mechanism of Nonhomologous End-joining
  5. Mechanism of Homologous Recombination
  6. Regulation of the Two Different DSBR Pathways?
  7. Recombination at the Immunoglobulin Locus
  8. Medical Implications of DNA Recombination
  9. See also
  10. References
  11. Further Reading
  12. Web Links

Of the many types of DNA damage, a double-strand break (DSB) is the most dangerous, because unrepaired DSBs eventually lead to cell death by activating a damage-checkpoint pathway. DSBs can be caused by numerous exogenous agents, including ionizing radiation and certain chemicals, or by endogenous sources such as free radicals generated during normal cellular metabolic reactions. DSBs can also arise during DNA replication: when a replication fork passes through a template that contains a single-strand break, the break will be converted into a DSB on one of the sister chromatids.

There are two distinct mechanisms of DSB repair (DSBR) in vertebrate cells: NHEJ and HR. These pathways are conserved between Saccharomyces cerevisiae and mammalian cells, although the relative importance in each differs considerably.

Mechanism of Nonhomologous End-joining

  1. Top of page
  2. Introduction
  3. DNA Recombination and DNA Repair
  4. Mechanism of Nonhomologous End-joining
  5. Mechanism of Homologous Recombination
  6. Regulation of the Two Different DSBR Pathways?
  7. Recombination at the Immunoglobulin Locus
  8. Medical Implications of DNA Recombination
  9. See also
  10. References
  11. Further Reading
  12. Web Links

NHEJ repairs DSBs by directly re-ligating DNA ends without the need for extensive homology between the DNA ends to be joined (Figure 1). The critical role of the NHEJ pathway in DSBR was first illustrated by the extreme sensitivity of NHEJ-deficient cells to ionizing radiation or genotoxic compounds. NHEJ is the major pathway of DSBR in mammalian cells (Lieber, 1999). This pathway is also required to process the DSB intermediates that are generated during V(D)J recombination.

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Figure 1. Double-strand break (DSB) repair by nonhomologous end-joining. Notched lines represent inaccurate genetic information by either insertion or deletion of nucleotides at the DSB. DNA-PKcs: DNA-protein kinase catalytic subunit; XRCC4: X-ray cross-complementing 4.

Two multiprotein complexes have been proven as factors in the NHEJ pathway. The first is the DNA-dependent protein kinase (DNA-PK). It is comprised of the Ku70/Ku80 heterodimer, which binds to DNA ends with a high affinity, and the DNA-PK catalytic subunit (DNA-PKcs), which is enzymatically activated by DSBs via the end-bound Ku complex. The second complex consists of DNA ligase IV and XRCC4 (X-ray cross-complementing 4), which catalyzes ligation during NHEJ. All of the NHEJ factors are evolutionally conserved from yeast to human, with the exception of DNA-PKcs, which is not found in yeast.

The first known DSBR-defective mouse mutant was the severe combined immunodeficiency (SCID) mouse, which carries a spontaneous mutation preventing the production of mature B and T cells, owing to a defect in V(D)J recombination. These mice not only have a defect in the development of their immune system, but also are hypersensitive to ionizing radiation. This phenotype is caused by a mutation in DNA-PKcs. The mutant mice develop lymphoid tumors of T-cell origin at high frequencies. Defects in Ku generally confer a similar but more severe phenotype than defects in DNA-PKcs. DNA-PK activity is undetectable in Ku-defective cell lines, indicating that DNA binding by the Ku70/80 heterodimer is essential for its activation.

As might be expected, cell lines that lack either the DNA ligase IV (LIG4, (ligase IV, DNA, ATP-dependent)) or XRCC4 (XRCC4; X-ray repair complementing defective repair in Chinese hamster cells 4) genes are sensitive to ionizing radiation. Knockout mice of either gene exhibit embryonic lethality, while Ku and DNA-PKcs-deficient mice are viable, suggesting that there may be an additional role for DNA ligase IV and XRCC4 during embryonic development. Mouse embryos deficient in either DNA ligase IV or XRCC4 undergo massive neuronal apoptosis and chromosomal aberrations. The severe phenotypes of mutants in any of the conserved NHEJ genes infer that normally dividing cells frequently sustain spontaneous DSBs, and NHEJ plays an essential role in maintaining genomic stability. It is unclear whether NHEJ has another role in the viability of neuronal cells during development.

Mechanism of Homologous Recombination

  1. Top of page
  2. Introduction
  3. DNA Recombination and DNA Repair
  4. Mechanism of Nonhomologous End-joining
  5. Mechanism of Homologous Recombination
  6. Regulation of the Two Different DSBR Pathways?
  7. Recombination at the Immunoglobulin Locus
  8. Medical Implications of DNA Recombination
  9. See also
  10. References
  11. Further Reading
  12. Web Links

HR can repair DSBs through interaction of a damaged DNA duplex with an intact homologous sequence, either on a sister chromatid or homologous chromosome (Figure 2). It has been believed that lower eukaryotes such as yeast preferentially use HR to repair DSBs, whereas vertebrates predominantly use NHEJ. However, several recent studies indicate that vertebrate cells also frequently use HR in DSBR, and this is an essential function for cell survival (Johnson and Jasin, 2001). HR may play a predominant role in repairing DNA damage generated endogenously during DNA replication, while NHEJ is a major player in repairing DSBs introduced exogenously such as by ionizing radiation.

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Figure 2. Double-strand break (DSB) repair by homologous recombination. Bold lines in (d)–(f) represent newly synthesized DNA regions. RPA: replication protein A.

HR in eukaryotes involves numerous proteins, many of which have been identified and extensively studied in S. cerevisiae (Haber, 2000; Sung et al., 2000). These proteins have been identified as members of the so-called Rad52 epistasis group, including Rad50, Rad51, Rad52, Rad54 and Mre11, and are evolutionally conserved from yeast to mammals. Rad51 is related to the Escherichia coli RecA protein, which promotes DNA strand-pairing and exchange reactions. The initial step of HR repair is mediated through the Rad50/Nbs1/Mre11 complex. Rad50 is an adenosine triphosphate (ATP)-dependent DNA-binding protein, and Mre11 has 3′–5′ double-stranded DNA exonuclease activity, which may process damaged DNA ends and expose short lengths of single-stranded DNA. The Nbs1-encoding gene (NBS1; Nijmegen breakage syndrome 1 (nibrin)) is mutated in patients with Nijmegen breakage syndrome. Nbs1 protein recruits the Rad50/Mre11 complex to DSB sites. The subsequently exposed single-stranded DNA is bound by RPA (replication protein A), which is ultimately replaced by Rad51, with the aid of Rad52. Rad51 protein, like RecA protein in E. coli, forms nucleoprotein filaments along the single-stranded DNA and plays a central role in HR. Mutation of Rad51 confers cellular lethality in vertebrates (Sonoda et al., 1998), while Rad51 in S. cerevisiae is dispensable for cellular viability. The Rad51 nucleoprotein filaments mediate the search for a homologous sequence in the template DNA and the formation of joint molecules between the damaged DNA and the template DNA. These reactions are stimulated by the Rad52 and Rad54 proteins. In the next step, DNA synthesis takes place to recover the degraded DNA strands, and finally the recombination intermediates are ligated and resolved.

Other relatives of Rad51 genes include Rad51B, Rad51C, Rad51D, Xrcc2 and Xrcc3. These possibly arose by gene duplication of Rad51 during evolution and are thus named Rad51 paralogs. Although Rad51 paralogs are not essential for cell viability, mutant cells of each paralog show indistinguishably similar phenotypes: impaired HR, spontaneous chromosomal aberrations and sensitivity to cross-linking reagents and ionizing radiation. Rad51 paralogs are thought to function as accessory proteins for Rad51 at various stages of HR.

Both the BRCA1 (breast cancer 1, early onset) and BRCA2 (breast cancer 2, early onset) genes were identified as tumor suppressor genes of hereditary breast and ovarian cancer syndromes. Both genes have been shown to be clearly involved in HR (Venkitaraman, 2002). Unlike the Rad52 epistasis group members, there are no obvious homologs in yeast. Loss of functional Brca1 or Brca2 results in spontaneous chromosomal aberrations and sensitivity to ionizing radiation and cross-linking chemicals. Brca1 is phosphorylated by the Atm (ataxia telangiectasia and Rad3 mutated) protein, a protein kinase activated by DSBs, and thus is thought to be one of the sensors of DNA damage in mammalian cells. Brca1 interacts with the Mre11/Rad50/Nbs1 complex and inhibits the nuclease activity of the complex under certain conditions. Brca2 has eight tandem copies of a repetitive sequence motif, named the BRC repeat. Six of the eight BRC repeats are highly conserved and bind to Rad51 in vitro. Therefore Brca2 may directly regulate the availability and activity of Rad51 in HR. Because both Brca1 and Brca2 are multifunctional proteins that have roles in cell cycle control and transcription, it is difficult to define the precise mechanism of their contribution to HR. Likewise, it is unclear why defects of BRCA1 and BRCA2 result in tumorigenesis predominantly in the mammary and ovarian tissues.

Regulation of the Two Different DSBR Pathways?

  1. Top of page
  2. Introduction
  3. DNA Recombination and DNA Repair
  4. Mechanism of Nonhomologous End-joining
  5. Mechanism of Homologous Recombination
  6. Regulation of the Two Different DSBR Pathways?
  7. Recombination at the Immunoglobulin Locus
  8. Medical Implications of DNA Recombination
  9. See also
  10. References
  11. Further Reading
  12. Web Links

At present there is no defined mechanism to explain how cells utilize two different pathways, NHEJ and HR, after induction of DNA damage. However, the relative predominance of the two pathways is likely to be dependent on the phase of the cell cycle. HR is apparent in late S to G2 phase, while NHEJ occurs throughout the cell cycle, including G0 phase. Cells with Ku70 deficiency show radiation sensitivity predominantly in G1 and early S phase, when HR activity is barely detected (Takata et al., 1998). The amount of Rad51 and Brca2, both of which are factors involved in HR, fluctuates during the cell cycle, the highest expression being in S phase, which is apparently consistent with predominance of the HR pathway in this cell cycle stage.

Spontaneous DSBs are likely to occur much more frequently and affect viability in a vertebrate cell than in a yeast cell, because a vertebrate's genome is hundreds of times larger than the yeast genome. HR repair is a process coupled with DNA replication and plays a more crucial role in repairing the spontaneous DSBs arising from DNA replication in vertebrate cells. On the other hand, NHEJ seems to be more directed to DNA repair of exogenously introduced DNA damage.

Recombination at the Immunoglobulin Locus

  1. Top of page
  2. Introduction
  3. DNA Recombination and DNA Repair
  4. Mechanism of Nonhomologous End-joining
  5. Mechanism of Homologous Recombination
  6. Regulation of the Two Different DSBR Pathways?
  7. Recombination at the Immunoglobulin Locus
  8. Medical Implications of DNA Recombination
  9. See also
  10. References
  11. Further Reading
  12. Web Links

Immunoglobulin gene rearrangement illustrates a typical DNA recombination event occurring in human lymphocytes. The rearrangement is carried out by both general and lymphocyte-specific mechanisms of DNA recombination, most of which have been extensively explored. These include V(D)J recombination, class switch recombination (CSR), somatic hypermutation and, in some species, gene conversion (Figure 3).

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Figure 3. Pathways involved in rearrangement of an immunoglobulin heavy chain locus. To simplify, only a fraction of genes are depicted as representatives of different segments: V: variable; D: diversity; and J: joining gene segments. Factors participating at each step are shown in blue. Gene conversion is also used in some species (see text). Note that both somatic hypermutation (SHM) and gene conversion can take place irrespective of a class switch recombination (CSR) event. Closed ovals represent switch regions located 5′ upstream of C genes. An asterisk represents a point mutation introduced into a VH region by somatic hypermutation. AID: activation-induced cytidine deaminase; CSR: class switch recombination; HR: homologous recombination; NHEJ: nonhomologous end-joining; SHM: somatic hypermutation.

V(D)J recombination

During human B cell development in the bone marrow, B cell precursors form intact exons for the variable region of antibody molecules by assembling variable (V), diversity (D) and joining (J) gene segments. For the V-region exon of the Ig heavy chain, V, D and J gene segments are joined, whereas the V-region exons of λ and κ light chains are composed of V and J segments. These gene rearrangements occur in an ordered fashion by a process called V(D)J recombination (Figure 4). First, a DH segment is rearranged to a JH segment; this is followed by a VH-to-DHJH joining.

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Figure 4. Mechanism of V(D)J recombination. An arrowhead in a rectangle represents a recombination signal sequence, consisting of a heptamer and a nonamer separated by 12 bp of spacer nucleotides. Factors participating in V(D)J recombination at each step are listed. Notched lines represent inserted extra nucleotides added at a junction between V and J segments. TdT: terminal deoxynucleoside transferase.

V(D)J recombination is initiated by a lymphocyte-specific endonuclease, consisting of the recombination activating gene (RAG1, RAG2) proteins (Rag1, Rag2), which induce DNA DSBs at specific recombination signal sequences (RSS) adjacent to the coding sequences of the V, D and J segments (Fugmann et al., 2000). Rag1 and Rag2 cleave precisely between the RSS and the coding sequences of the two rearranging gene segments in a concerted fashion, yielding two blunt RSS signal ends and two covalently sealed hairpin coding ends. The signal ends are precisely joined, releasing the intervening DNA between the rearranging gene segments as a DNA circle. The hairpin structures of the coding ends are opened by the recently identified Artemis and DNA-PKcs complex (Ma et al., 2002). During this reaction, nucleotides may be removed from the coding ends by exonucleolytic digestion, and nontemplated random sequences (N sequences) may be added by the lymphocyte-specific terminal deoxynucleoside transferase (TdT), further increasing the diversity of V-region genes. The process is completed by enzymes that are also involved in nonhomologous end-joining, including DNA-PK, DNA ligase IV and XRCC4, which join Rag-liberated V, D and J gene segments. Atm is recruited to DSBs accompanying V(D)J recombination simultaneously with Rag1 (Perkins et al., 2002). Although Atm is not necessary for V(D)J recombination, it may be required for surveillance of recombination intermediates. When V(D)J recombination is achieved successfully, Atm kinase activity may be attenuated. However, in the case of failed repair, Atm may remain active and could suppress potential deleterious translocations, possibly by inducing checkpoint responses.

Gene conversion and somatic hypermutation

When mature B cells are activated through binding of cognate antigen with their antigen receptors and interaction with T helper cells, they undergo fine tuning of the antibody response using mechanisms of somatic hypermutation (SHM) and/or gene conversion. Some species such as sheep, human and mouse exclusively use SHM for V-segment diversification, whereas others such as chicken, rabbit, cattle and pig rely predominantly on Ig gene conversion (Figure 3). For example, the avian genome has only one functional V gene segment, J gene segment and C gene segment. Upstream of the functional Vλ1 gene segment lie 25 Vλ pseudogenes, organized in either orientation. Only the Vλ1 gene segment recombines with the J–Cλ gene segment; however, sequences from the pseudogenes, between 10 and 120 bp in length, are substituted into the active Vλ1 region by gene conversion, resulting in considerable diversity of rearranged Vλ–J–Cλ gene segments. On the other hand, SHM is a mechanism that specifically introduces mutations into the V regions of Ig genes, giving rise to further variation of antibody. The process of SHM introduces mostly nucleotide substitutions into rearranged V genes, with minor events of deletions and duplications. Very recently it was shown that SHM is dependent on DNA polymerase ι (Faili et al., 2002) as well as DNA polymerase η (Zeng et al., 2001).

Class switch recombination

A fraction of B cells undergo CSR and thereby change the isotype of the expressed B cell receptor, resulting in altered effector functions of the antibody (Figure 5). Through class switching, the Cμ and Cδ genes that are originally expressed by a naive B cell are subsequently replaced by the Cγ, Cα or Cε genes. In humans, there are nine functional IgH constant-region genes (one Cμ, one Cδ, four Cγ, two Cα and one Cε), located downstream of the V, D and J gene cluster. Upstream of each CH gene, there are repetitive DNA sequences, known as the switch (S) regions. During CSR, DNA segments are removed between the switch μ region and one of the downstream S regions located immediately 5′ to each CH gene. CSR consists of three steps: (1) selection of a target S region; (2) cleavage of the targeted S and S regions by a putative class switch recombinase and (3) repair and ligation of the broken DNA ends at both S regions by the NHEJ repair system, including the DNA-PK complex. As a result, a new CH gene is placed downstream of the V-region exon.

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Figure 5. Mechanism of class switch recombination (CSR) at an immunoglobulin heavy chain locus. The organization of immunoglobulin heavy chain constant-region genes in humans is depicted. Factors participating in CSR at each step are listed on the right. CSR may take place successively, generating different isotypes. S region: switch region.

It has been demonstrated that the activation-induced cytidine deaminase (AID) gene is involved in the cleavage steps of CSR and SHM (Muramatsu et al., 2000). Also, AID is indispensable for gene conversion during remodeling of Ig loci in B cells (Arakawa et al., 2002). Expression of AID is restricted to stimulated B lymphocytes. Although it is not known whether AID itself cleaves DNA or regulates a putative DNA-cleaving enzyme, the predicted amino acid sequence of AID has homology to that of Apobec-1, a catalytic subunit of the RNA-editing complex for the apolipoprotein B mRNA precursor. Thus AID may edit an unknown pre-mRNA, converting it to mRNA for a nicking endonuclease specific to the recognition target for CSR, SHM and gene conversion.

Medical Implications of DNA Recombination

  1. Top of page
  2. Introduction
  3. DNA Recombination and DNA Repair
  4. Mechanism of Nonhomologous End-joining
  5. Mechanism of Homologous Recombination
  6. Regulation of the Two Different DSBR Pathways?
  7. Recombination at the Immunoglobulin Locus
  8. Medical Implications of DNA Recombination
  9. See also
  10. References
  11. Further Reading
  12. Web Links

Mutations in some of the genes involved in DNA recombination have been found to be responsible for hereditary diseases. Examples include LIG4, for Lig4 syndrome; MRE11A (MRE11 meiotic recombination 11 homolog A (S. cerevisiae)), for ataxia telangiectasia-like disorder; NBS1, for Nijmegen breakage syndrome; and BRCA1 and BRCA2, for early-onset breast–ovarian cancer. It is particularly intriguing that the diseases resulting from deficiency in the HR genes often cause cancer, probably because genome instability leads to a higher risk of malignant transformation. Exploration into the mechanism of homologous recombination is important for understanding the process of carcinogenesis.

Further studies into the mechanism of HR could also assist in the improvement of gene therapy. When plasmid DNA is introduced exogenously into cells, only a small number of plasmid molecules are integrated into chromosomal DNA, and this takes place in a random fashion (random integration). If the plasmid DNA has homologous sequence to a particular point on the chromosome, the plasmid DNA is integrated into a fixed region in the chromosome by HR (targeted integration). In vertebrate cells, the frequency of targeted integration is much lower than that of random integration. Therefore endeavor to increase the frequency of targeted integration could make it possible to correct deleterious mutation on the chromosomal sequence in safety.

References

  1. Top of page
  2. Introduction
  3. DNA Recombination and DNA Repair
  4. Mechanism of Nonhomologous End-joining
  5. Mechanism of Homologous Recombination
  6. Regulation of the Two Different DSBR Pathways?
  7. Recombination at the Immunoglobulin Locus
  8. Medical Implications of DNA Recombination
  9. See also
  10. References
  11. Further Reading
  12. Web Links

Further Reading

  1. Top of page
  2. Introduction
  3. DNA Recombination and DNA Repair
  4. Mechanism of Nonhomologous End-joining
  5. Mechanism of Homologous Recombination
  6. Regulation of the Two Different DSBR Pathways?
  7. Recombination at the Immunoglobulin Locus
  8. Medical Implications of DNA Recombination
  9. See also
  10. References
  11. Further Reading
  12. Web Links

Web Links

  1. Top of page
  2. Introduction
  3. DNA Recombination and DNA Repair
  4. Mechanism of Nonhomologous End-joining
  5. Mechanism of Homologous Recombination
  6. Regulation of the Two Different DSBR Pathways?
  7. Recombination at the Immunoglobulin Locus
  8. Medical Implications of DNA Recombination
  9. See also
  10. References
  11. Further Reading
  12. Web Links