The analysis of genes in evolutionarily distant but morphologically similar species is of major importance to unravel the changes in genomes over millions of years, which led to gene silencing and functional diversification. We report the analysis of Wnt8a gene expression in the medakafish and provide a detailed comparison to other vertebrates. In all teleosts analyzed there are two paralogous Wnt8a copies. These show largely overlapping expression in the early developing zebrafish embryo, an evolutionarily distant relative of medaka. In contrast to zebrafish, we find that both maternal and zygotic expression of particularly one Wnt8a paralog has diverged in medaka. While Wnt8a1 expression is mostly conserved at early embryonic stages, the expression of Wnt8a2 differs markedly. In addition, both genes are distinctly expressed during organogenesis unlike the zebrafish homologs, which may hint at the emergence of functional diversification of Wnt8a ligands during evolution.
Secondary immunoglobulin (Ig) gene diversification relies on activation-induced cytidine deaminase (AID) to create U:G mismatches that are subsequently fixed by mutagenic repair pathways. AID activity is focused to Ig loci by cis-regulatory DNA sequences named targeting elements. Here we show that in contrast to prevailing thought in the field, the targeting elements in the chicken IGL locus are distinct from classical transcriptional enhancers. These mutational enhancer elements (MEEs) are required over and above transcription to recruit AID-mediated mutagenesis to Ig loci. We identified a small 222 bp fragment in the chicken IGL locus that enhances mutagenesis without boosting transcription, and this sequence represents a key component of a MEE. Lastly, MEEs are evolutionarily conserved amongst birds, both in sequence and function, and contain several highly conserved sequence modules that are likely involved in recruiting trans-acting targeting factors. We propose that MEEs represent a novel class of cis-regulatory elements whose function is to control genomic integrity.
Activation-induced cytidine deaminase (AID) is a key enzyme for antibody-mediated immune responses. Antibodies are encoded by the immunoglobulin genes and AID acts as a transcription-dependent DNA mutator on these genes to improve antibody affinity and effector functions. An emerging theme in field is that many transcribed genes are potential targets of AID, presenting an obvious danger to genomic integrity. Thus there are mechanisms in place to ensure that mutagenic outcomes of AID activity are specifically restricted to the immunoglobulin loci. Cis-regulatory targeting elements mediate this effect and their mode of action is likely a combination of immunoglobulin gene specific activation of AID and a perversion of faithful DNA repair towards error-prone outcomes.
The Recombination Activating Genes 1 and 2 (Rag1 and Rag2) encode the key enzyme that is required for the generation of the highly diversified antigen receptor repertoire central to adaptive immunity. The longstanding model proposed that this gene pair was acquired by horizontal gene transfer to explain its abrupt appearance in the vertebrate lineage. The analyses of the enormous amount of sequence data created by many genome sequencing projects now provides the basis for a more refined model as to how this unique gene pair evolved from a selfish DNA transposon into a sophisticated DNA recombinase essential for immunity.
RAG1; RAG2; transposase; evolution; adaptive immunity
V(D)J recombination is a somatic gene rearrangement process that assembles antigen receptor genes from individual segments during lymphocyte development. The access of the RAG1/RAG2 recombinase to these gene segments is regulated at the level of chromatin modifications, in particular histone tail modifications. Trimethylation of lysine 4 in histone H3 (H3K4me3) correlates with actively recombining gene elements, and this mark is recognized and interpreted by the plant homeodomain (PHD) of RAG2. Here we report that the PHD domain of the only known invertebrate homolog of RAG2, the SpRAG2L protein of the purple sea urchin (Strongylocentrotus purpuratus) also binds to methylated histones, but with a unique preference for H3K4me2. While the cognate substrate for the sea urchin RAG1L/RAG2L complex remains elusive, the affinity for histone tails and the nuclear localization of ectopically expressed SpRAG2L strongly support the model that this enzyme complex exerts its activity on DNA in the context of chromatin.
V(D)J recombination; SpRAG1L; SpRAG2L; RAG2; PHD domain; Strongylocentrotus purpuratus; H3K4 methylation; chromatin
Diversity of T cell receptor (TCR) genes is primarily generated by nucleotide insertions upon rearrangement from their germ line-encoded V, D and J segments. Nucleotide insertions at V-D and D-J junctions are random, but some small subsets of these insertions are exceptional, in that one to three base pairs inversely repeat the sequence of the germline DNA. These short complementary palindromic sequences are called P nucleotides. We apply the ImmunoSeq deep-sequencing assay to the third complementarity determining region (CDR3) of the β chain of T cell receptors, and use the resulting data to study P nucleotides in the repertoire of naïve and memory CD8+ and CD4+ T cells. We estimate P nucleotide distributions in a cross section of healthy adults and different T cell subtypes. We show that P nucleotide frequency in all T cell subtypes ranges from 1% to 2%, and that the distribution is highly biased with respect to the coding end of the gene segment. Classification of observed palindromic sequences into P nucleotides using a maximum conditional probability model shows that single base P nucleotides are very rare in VDJ recombination; P nucleotides are primarily two bases long. To explore the role of P nucleotides in thymic selection, we compare P nucleotides in productive and non-productive sequences of CD8+ naïve T cells. The naïve CD8+ T cell clones with P nucleotides are more highly expanded.
The programmed degradation of the RAG2 enzyme upon entry to S phase restricts V(D)J recombination to the G0–G1 phase of the cell cycle. In this issue of Immunity Zhang et al. (2011) show that this is critical to prevent lymphoma formation.
Activation induced deaminase (AID) mediates class switch recombination and somatic hypermutation of immunoglobulin (Ig) genes in germinal centre B cells. In order to regulate its specific activity and as a means to keep off-target mutations low, several mechanisms have evolved, including binding to specific cofactors, phosphorylation and destabilization of nuclear AID protein. Although ubiquitination at lysine residues of AID is recognized as an essential step in initiating degradation of nuclear AID, any functional relevance of lysine modifications has remained elusive.
Here, we report functional implications of lysine modifications of the human AID protein by generating a panel of lysine to arginine mutants of AID and assessment of their catalytic class switch activity. We found that only mutation of Lys22 to Arg resulted in a significant reduction of class switching to IgG1 in transfected primary mouse B cells. This decrease in activity was neither reflected in reduced hypermutation of Ig genes in AID-mutant transfected DT40 B cell lines nor recapitulated in bacterial deamination assays, pointing to involvement of post-translational modification of Lys22 for AID activity in B cells.
Our results imply that lysine modification may represent a novel level of AID regulation and that Lys22 is important for effective AID activity.
Protein evolution includes the birth and death of structural motifs. For example, a zinc finger or a salt bridge may be present in some, but not all, members of a protein family. We propose that such transitions are manifest in sequence phylogenies as concerted shifts in substitution rates of amino acids that are neighbors in a representative structure. First, we identified rate shifts in a quartet from the Fpg/Nei family of base excision repair enzymes using a method developed by Xun Gu and coworkers. We found the shifts to be spatially correlated, more precisely, associated with a flexible loop involved in bacterial Fpg substrate specificity. Consistent with our result, sequences and structures provide convincing evidence that this loop plays a very different role in other family members. Second, then, we developed a method for identifying latent protein structural characters (LSC) given a set of homologous sequences based on Gu's method and proximity in a high-resolution structure. Third, we identified LSC and assigned states of LSC to clades within the Fpg/Nei family of base excision repair enzymes. We describe seven LSC; an accompanying Proteopedia page (http://proteopedia.org/wiki/index.php/Fpg_Nei_Protein_Family) describes these in greater detail and facilitates 3D viewing. The LSC we found provided a surprisingly complete picture of the interaction of the protein with the DNA capturing familiar examples, such as a Zn finger, as well as more subtle interactions. Their preponderance is consistent with an important role as phylogenetic characters. Phylogenetic inference based on LSC provided convincing evidence of independent losses of Zn fingers. Structural motifs may serve as important phylogenetic characters and modeling transitions involving structural motifs may provide a much deeper understanding of protein evolution.
Non-productive antigen receptor genes with frame shifts generated during the assembly of these genes are found in many mature lymphocytes. Transcripts from these genes have premature termination codons (PTCs) and could encode truncated proteins if they are not either inactivated or destroyed by nonsense-mediated decay (NMD). In mammalian cells, NMD can be activated by pathways that rely on the presence of an intron downstream of the PTC; however, NMD can also be activated by pathways that do not rely on these downstream introns, and pathways independent of NMD can inactivate PTC-containing transcripts. Here, through the generation and analysis of mice with gene-targeted modifications of the endogenous T cell receptor beta (Tcrb) locus, we demonstrate that in T cells in vivo, optimal clearance of PTC-containing Tcrb transcripts depends on the presence of an intron downstream of the PTC.
Somatic hypermutation (SHM) of immunoglobulin genes is initiated by activation-induced cytidine deaminase (AID) in activated B cells. This process is strictly dependent on transcription. Hence, cis-acting transcriptional control elements have been proposed to target SHM to immunoglobulin loci. The Mus musculus Igκ locus is regulated by the intronic enhancer (iE/MAR) and the 3′ enhancer (3′E), and multiple studies using transgenic and knock-out approaches in mice and cell lines have reported somewhat contradictory results about the function of these enhancers in AID-mediated sequence diversification. Here we show that the M. musculus iE/MAR and 3′E elements are active solely as transcriptional enhancer when placed in the context of the IGL locus in Gallus gallus DT40 cells, but they are very inefficient in targeting AID-mediated mutation events to this locus. This suggests that either key components of the cis-regulatory targeting elements reside outside the murine Igκ transcriptional enhancer sequences, or that the targeting of AID activity to Ig loci occurs by largely species-specific mechanisms.
Adaptive immunity is mediated through numerous genetic and cellular processes that generate favourable somatic variants of antigen-binding receptors under evolutionary selection pressure by pathogens and other factors. Advances in our understanding of immunity in mammals and other model organisms are revealing the underlying basis and complexity of this remarkable system. Although the evolution of adaptive immunity has been considered to occur by acquisition of novel molecular capabilities, an increasing amount of information from new model systems suggest that co-option and redirection of preexisting systems are the major source of innovation. We combine evidence from a wide range of organisms to obtain an integrated view of the origins and patterns of divergence in adaptive immunity.
The discovery and characterization of activation-induced cytidine deaminase (AID) 10 years ago provided the basis for a mechanistic understanding of secondary antibody diversification and the subsequent generation and maintenance of cellular memory in B lymphocytes, which signified a major advance in the field of B cell immunology. Here we celebrate and review the triumphs in the mission to understand the mechanisms through which AID influences antibody diversification, as well as the implications of AID function on human physiology. We also take time to point out important ongoing controversies and outstanding questions in the field and highlight key experiments and techniques that hold the potential to elucidate the remaining mysteries surrounding this vital protein.
Metazoan immunity is mainly associated with specialized cells that are directly involved with the immune response. Nevertheless, both in vertebrates and invertebrates other organs might respond to immune activation and participate either directly or indirectly in the ongoing immune process. However, most of what is known about invertebrate immunity has been restricted to immune effector cells and little information is available on the immune responses of other tissues or organs. We now focus on the immune reactions of the intestinal tissue of an echinoderm. Our study employs a non-conventional model, the echinoderm Holothuria glaberrima, to identify intestinal molecules expressed after an immune challenge presented by an intra-coelomic injection of lipopolysaccharides (LPS). The expression profiles of intestinal genes expressed differentially between LPS-injected animals and control sea water-injected animals were determined using a custom-made Agilent microarray with 7209 sea cucumber intestinal ESTs. Fifty (50) unique sequences were found to be differentially expressed in the intestine of LPS-treated sea cucumbers. Seven (7) of these sequences represented homologues of known proteins, while the remaining (43) had no significant similarity with any protein, EST or RNA database. The known sequences corresponded to cytoskeletal proteins (Actin and alpha-actinin), metabolic enzymes (GAPDH, Ahcy and Gnmt), metal ion transport/metabolism (major yolk protein) and defense/recognition (fibrinogen-like protein). The expression pattern of 11 genes was validated using semi-quantitative RT-PCR. Nine of these corroborated the microarray results and the remaining two showed a similar trend but without statistical significance. Our results show some of the molecular events by which the holothurian intestine responds to an immune challenge and provide important information to the study of the evolution of the immune response.
The Cre-loxP system has been used to enable tissue specific activation, inactivation and mutation of many genes in vivo and has thereby greatly facilitated the genetic dissection of several cellular and developmental processes. In such studies, Cre-reporter strains, which carry a Cre-activated marker gene, are frequently utilized to validate the expression profile of Cre transgenes, to act as a surrogate marker for excision of a second allele, and to irreversibly label cells for lineage tracing experiments.
We have studied three commonly used Cre-reporter strains, Z/AP, Z/EG and R26R-EYFP and have demonstrated that although each reporter can be reliably activated by Cre during early development, exposure to Cre in adult hematopoietic cells results in a much lower frequency of marker-positive cells in the Z/AP or Z/EG strains than in the R26R-EYFP strain. In marker negative cells derived from the Z/AP and Z/EG strains, the transgenic promoter is methylated and Cre-mediated recombination of the locus is inhibited.
These results show that the efficiency of Cre-mediated recombination is not only dependent on the genomic context of a given loxP-flanked sequence, but also on stochastic epigenetic mechanisms underlying transgene variegation. Furthermore, our data highlights the potential shortcomings of utilizing the Z/AP and Z/EG reporters as surrogate markers of excision or in lineage tracing experiments.
Somatic hypermutation (SHM) and gene conversion (GCV) are closely related processes that increase the diversity the primary immunoglobulin repertoire. In both processes the activation-induced cytidine deaminase (AID) converts cytosine residues to uracils within the DNA of the immunoglobulin (Ig) genes in a transcription-dependent manner, and subsequent error-prone repair processes lead to changes in the antigen recognition site of the encoded receptors. This activity is specifically recruited to the Ig loci by unknown mechanisms. Our analyses of the chicken B-cell line DT40, and derivatives thereof, now revealed that even the closest neighbors of the Ig light chain (IgL) gene are protected from AID activity, albeit being transcribed and thus acting as potential targets of AID. Our findings are in support of a model in which cis-acting DNA boundary elements restrict AID activity to the IgL locus and guard the genome in the vicinity of the IgL gene from deleterious mutations.
Somatic Hypermutation; Gene Conversion; Immunoglobulin; DT40
Embryos of most fish develop externally and are exposed to an aquatic environment full of potential pathogens, whereas they have little or only limited ability to mount an efficient and protective response. How fish embryos survive pathogenic attacks remains poorly defined. Here we demonstrate that the maternal immunization of female zebrafish with formalin-killed Aeromonas hydrophila causes a significant increase in C3 and Bf contents in the mother, a corresponding rise in the offspring, and induces a remarkable increase in the hemolytic activities in both the mother and offspring. In addition, the embryos derived from the immunized mother are significantly more tolerant to A. hydrophila challenge than those from the unimmunized fish, and blocking C3 and Bf activities by injection of the antibodies against C3 and Bf into the embryos render them more susceptible to A. hydrophila. These results clearly show that the protection of zebrafish embryos against A. hydrophila can be achieved by the maternally-transferred immunity of the complement system operating via the alternative pathway. This appears to be the first report providing in vivo evidences for the protective role of the alternative complement components in the early embryos of zebrafish, paving the way for insights into the in vivo function of other maternally-transferred factors in fish.
Conditional gene targeting using the Cre-loxp system is a well established technique in numerous in vitro and in vivo systems. Ligand regulated forms of Cre have been increasingly used in these applications in order to gain temporal and spatial control over conditional targeting. The tamoxifen-regulated Cre variant mer-Cre-mer (mCrem) is widely utilized because of its reputation for tight regulation in the absence of its tamoxifen ligand. In the DT40 chicken B cell line, we generated an mCrem-based reversible switch for conditional regulation of a transgene, and in contrast with previous work, observed significant constitutive activity of mCrem. This prompted us to use our system for analysis of the parameters governing tamoxifen-regulated mCrem recombination of a genomic target. We find that robust mCrem expression correlates with a high level of tamoxifen-independent Cre activity, while clones expressing mCrem at the limit of western blot detection exhibit extremely tight regulation. We also observe time and dose-dependent effects on mCrem activity which suggest limitations on the use of conditional targeting approaches for applications which require tight temporal coordination of Cre action within a cell population.
Due to essentially maternal inheritance and a bottleneck effect during early oogenesis, newly arising mitochondrial DNA (mtDNA) mutations segregate rapidly in metazoan female germlines. Consequently, heteroplasmy (i.e. the mixture of mtDNA genotypes within an organism) is generally resolved to homoplasmy within a few generations. Here, we report an exceptional transpecific heteroplasmy (predicting an alanine/valine alloacceptor tRNA change) that has been stably inherited in oniscid crustaceans for at least thirty million years. Our results suggest that this heteroplasmy is stably transmitted across generations because it occurs within mitochondria and therefore escapes the mtDNA bottleneck that usually erases heteroplasmy. Consistently, at least two oniscid species possess an atypical trimeric mitochondrial genome, which provides an adequate substrate for the emergence of a constitutive intra-mitochondrial heteroplasmy. Persistence of a mitochondrial polymorphism on such a deep evolutionary timescale suggests that balancing selection may be shaping mitochondrial sequence evolution in oniscid crustaceans.
DNA damage such as double-stranded DNA breaks (DSBs) has been reported to stimulate mitochondrial biogenesis. However, the underlying mechanism is poorly understood. The major player in response to DSBs is ATM (ataxia telangiectasia mutated). Upon sensing DSBs, ATM is activated through autophosphorylation and phosphorylates a number of substrates for DNA repair, cell cycle regulation and apoptosis. ATM has been reported to phosphorylate the α subunit of AMP-activated protein kinase (AMPK), which senses AMP/ATP ratio in cells, and can be activated by upstream kinases. Here we provide evidence for a novel role of ATM in mitochondrial biogenesis through AMPK activation in response to etoposide-induced DNA damage.
Three pairs of human ATM+ and ATM- cells were employed. Cells treated with etoposide exhibited an ATM-dependent increase in mitochondrial mass as measured by 10-N-Nonyl-Acridine Orange and MitoTracker Green FM staining, as well as an increase in mitochondrial DNA content. In addition, the expression of several known mitochondrial biogenesis regulators such as the major mitochondrial transcription factor NRF-1, PGC-1α and TFAM was also elevated in response to etoposide treatment as monitored by RT-PCR. Three pieces of evidence suggest that etoposide-induced mitochondrial biogenesis is due to ATM-dependent activation of AMPK. First, etoposide induced ATM-dependent phosphorylation of AMPK α subunit at Thr172, indicative of AMPK activation. Second, inhibition of AMPK blocked etoposide-induced mitochondrial biogenesis. Third, activation of AMPK by AICAR (an AMP analogue) stimulated mitochondrial biogenesis in an ATM-dependent manner, suggesting that ATM may be an upstream kinase of AMPK in the mitochondrial biogenesis pathway.
These results suggest that activation of ATM by etoposide can lead to mitochondrial biogenesis through AMPK activation. We propose that ATM-dependent mitochondrial biogenesis may play a role in DNA damage response and ROS regulation, and that defect in ATM-dependent mitochondrial biogenesis could contribute to the manifestations of A-T disease.
The beyond 12/23 (B12/23) rule ensures inclusion of a Dβ gene segment in the assembled T-cell receptor (TCR) β variable region exon and is manifest by a failure of direct Vβ-to-Jβ gene segment joining. The restriction is enforced during the DNA cleavage step of V(D)J recombination by the recombination-activating gene 1 and 2 (RAG1/2) proteins and the recombination signal sequences (RSSs) flanking the TCRβ gene segments. Nothing is known about the step(s) at which DNA cleavage is defective or how TCRβ locus sequences contribute to these defects. To address this, we examined the steps of DNA cleavage by the RAG proteins using TCRβ locus V, D, and J RSS oligonucleotide substrates. The results demonstrate that the B12/23 rule is enforced through slow nicking of Jβ substrates and to some extent through poor synapsis of Vβ and Jβ substrates. Nicking is controlled largely by the coding flank and, unexpectedly, the RSS spacer, while synapsis is controlled primarily by the RSS nonamer. The results demonstrate that different Jβ substrates are crippled at different steps of cleavage by distinct combinations of defects in the various DNA elements and strongly suggest that the DNA nicking step of V(D)J recombination can be rate limiting in vivo.
It is thought that gene conversion (GCV) and somatic hypermutation (SHM) of immunoglobulin (Ig) genes occur in two steps: the generation of uracils in DNA by activation-induced cytidine deaminase, followed by their subsequent repair by various DNA repair pathways to generate sequence-diversified products. It is not known how either of the two steps is targeted specifically to Ig loci. Because of the tight link between transcription and SHM, we have investigated the role of endogenous Ig light chain (IgL) transcriptional control elements in GCV/SHM in the chicken B cell line DT40. Promoter substitution experiments led to identification of a strong RNA polymerase II promoter incapable of supporting efficient GCV/SHM. This surprising finding indicates that high levels of transcription are not sufficient for robust GCV/SHM in Ig loci. Deletion of the IgL enhancer in a context in which high-level transcription was not compromised showed that the enhancer is not necessary for GCV/SHM. Our results indicate that cis-acting elements are important for Ig gene diversification, and we propose that targeting specificity is achieved through the combined action of several Ig locus elements that include the promoter.
During lymphocyte development, V(D)J recombination assembles antigen receptor genes from component V, D, and J gene segments. These gene segments are flanked by a recombination signal sequence (RSS), which serves as the binding site for the recombination machinery. The murine Jβ2.6 gene segment is a recombinationally inactive pseudogene, but examination of its RSS reveals no obvious reason for its failure to recombine. Mutagenesis of the Jβ2.6 RSS demonstrates that the sequences of the heptamer, nonamer, and spacer are all important. Strikingly, changes solely in the spacer sequence can result in dramatic differences in the level of recombination. The subsequent analysis of a library of more than 4,000 spacer variants revealed that spacer residues of particular functional importance are correlated with their degree of conservation. Biochemical assays indicate distinct cooperation between the spacer and heptamer/nonamer along each step of the reaction pathway. The results suggest that the spacer serves not only to ensure the appropriate distance between the heptamer and nonamer but also regulates RSS activity by providing additional RAG:RSS interaction surfaces. We conclude that while RSSs are defined by a “digital” requirement for absolutely conserved nucleotides, the quality of RSS function is determined in an “analog” manner by numerous complex interactions between the RAG proteins and the less-well conserved nucleotides in the heptamer, the nonamer, and, importantly, the spacer. Those modulatory effects are accurately predicted by a new computational algorithm for “RSS information content.” The interplay between such binary and multiplicative modes of interactions provides a general model for analyzing protein–DNA interactions in various biological systems.
Spacers not only ensure that the distance between the nonamer and heptamer is correct but they also regulate recombination activity by providing protein-binding sites along the DNA sequences that affect recombination
The recent development of High Throughput Sequencing technologies has enabled an individual’s TCR repertoire to be efficiently analysed at the nucleotide level. However, with unique clonotypes ranging in the tens of millions per individual, this approach gives a surfeit of information that is difficult to analyse and interpret in a biological context and gives little information about TCR structural diversity. Using publicly available TCR CDR3 sequence data, we analysed TCR repertoires by converting the encoded CDR3 amino acid sequences into Kidera Factors, a set of orthogonal physico-chemical properties that reflect protein structure. This approach enabled the TCR repertoire from different individuals to be distinguished and demonstrated the close similarity of the repertoire in different samples from the same individual.
Because cells are constantly subjected to DNA damaging insults, DNA repair pathways are critical for genome integrity . DNA damage recognition protein complexes (DRCs) recognize DNA damage and initiate DNA repair. The DNA-Damage Binding protein 2 (DDB2) complex is a DRC that initiates nucleotide excision repair (NER) of DNA damage caused by ultraviolet light (UV) –. Using a purified DDB2 DRC, we created a probe (“DDB2 proteo-probe”) that hybridizes to nuclei of cells irradiated with UV and not to cells exposed to other genotoxins. The DDB2 proteo-probe recognized UV-irradiated DNA in classical laboratory assays, including cyto- and histo-chemistry, flow cytometry, and slot-blotting. When immobilized, the proteo-probe also bound soluble UV-irradiated DNA in ELISA-like and DNA pull-down assays. In vitro, the DDB2 proteo-probe preferentially bound 6-4-photoproducts [(6-4)PPs] rather than cyclobutane pyrimidine dimers (CPDs). We followed UV-damage repair by cyto-chemistry in cells fixed at different time after UV irradiation, using either the DDB2 proteo-probe or antibodies against CPDs, or (6-4)PPs. The signals obtained with the DDB2 proteo-probe and with the antibody against (6-4)PPs decreased in a nearly identical manner. Since (6-4)PPs are repaired only by nucleotide excision repair (NER), our results strongly suggest the DDB2 proteo-probe hybridizes to DNA containing (6-4)PPs and allows monitoring of their removal during NER. We discuss the general use of purified DRCs as probes, in lieu of antibodies, to recognize and monitor DNA damage and repair.