CasPEDIA Database: a functional classification system for class 2 CRISPR-Cas enzymes.

CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.

Characterization of a thermostable Cas13 enzyme for one-pot detection of SARS-CoV-2.

Type VI CRISPR-Cas systems have been repurposed for various applications such as gene knockdown, viral interference, and diagnostics. However, the identification and characterization of thermophilic orthologs will expand and unlock the potential of diverse biotechnological applications. Herein, we identified and characterized a thermostable ortholog of the Cas13a family from the thermophilic organism Thermoclostridium caenicola (TccCas13a). We show that TccCas13a has a close phylogenetic relation to the HheCas13a ortholog from the thermophilic bacterium Herbinix hemicellulosilytica and shares several properties such as thermostability and inability to process its own pre-CRISPR RNA. We demonstrate that TccCas13a possesses robust cis and trans activities at a broad temperature range of 37 to 70 °C, compared with HheCas13a, which has a more limited range and lower activity. We harnessed TccCas13a thermostability to develop a sensitive, robust, rapid, and one-pot assay, named OPTIMA-dx, for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. OPTIMA-dx exhibits no cross-reactivity with other viruses and a limit of detection of 10 copies/µL when using a synthetic SARS-CoV-2 genome. We used OPTIMA-dx for SARS-CoV-2 detection in clinical samples, and our assay showed 95% sensitivity and 100% specificity compared with qRT-PCR. Furthermore, we demonstrated that OPTIMA-dx is suitable for multiplexed detection and is compatible with the quick extraction protocol. OPTIMA-dx exhibits critical features that enable its use at point of care (POC). Therefore, we developed a mobile phone application to facilitate OPTIMA-dx data collection and sharing of patient sample results. This work demonstrates the power of CRISPR-Cas13 thermostable enzymes in enabling key applications in one-pot POC diagnostics and potentially in transcriptome engineering, editing, and therapies.

Characterization of Cas12a nucleases reveals diverse PAM profiles between closely-related orthologs.

CRISPR-Cas systems comprise diverse adaptive immune systems in prokaryotes whose RNA-directed nucleases have been co-opted for various technologies. Recent efforts have focused on expanding the number of known CRISPR-Cas subtypes to identify nucleases with novel properties. However, the functional diversity of nucleases within each subtype remains poorly explored. Here, we used cell-free transcription-translation systems and human cells to characterize six Cas12a single-effector nucleases from the V-A subtype, including nucleases sharing high sequence identity. While these nucleases readily utilized each other's guide RNAs, they exhibited distinct PAM profiles and apparent targeting activities that did not track based on phylogeny. In particular, two Cas12a nucleases encoded by Prevotella ihumii (PiCas12a) and Prevotella disiens (PdCas12a) shared over 95% amino-acid identity yet recognized distinct PAM profiles, with PiCas12a but not PdCas12a accommodating multiple G's in PAM positions -2 through -4 and T in position -1. Mutational analyses transitioning PiCas12a to PdCas12a resulted in PAM profiles distinct from either nuclease, allowing more flexible editing in human cells. Cas12a nucleases therefore can exhibit widely varying properties between otherwise related orthologs, suggesting selective pressure to diversify PAM recognition and supporting expansion of the CRISPR toolbox through ortholog mining and PAM engineering.

Programmed DNA destruction by miniature CRISPR-Cas14 enzymes.

CRISPR-Cas systems provide microbes with adaptive immunity to infectious nucleic acids and are widely employed as genome editing tools. These tools use RNA-guided Cas proteins whose large size (950 to 1400 amino acids) has been considered essential to their specific DNA- or RNA-targeting activities. Here we present a set of CRISPR-Cas systems from uncultivated archaea that contain Cas14, a family of exceptionally compact RNA-guided nucleases (400 to 700 amino acids). Despite their small size, Cas14 proteins are capable of targeted single-stranded DNA (ssDNA) cleavage without restrictive sequence requirements. Moreover, target recognition by Cas14 triggers nonspecific cutting of ssDNA molecules, an activity that enables high-fidelity single-nucleotide polymorphism genotyping (Cas14-DETECTR). Metagenomic data show that multiple CRISPR-Cas14 systems evolved independently and suggest a potential evolutionary origin of single-effector CRISPR-based adaptive immunity.

Single-strand DNA processing: phylogenomics and sequence diversity of a superfamily of potential prokaryotic HuH endonucleases.

BACKGROUND: Some mobile genetic elements target the lagging strand template during DNA replication. Bacterial examples are insertion sequences IS608 and ISDra2 (IS200/IS605 family members). They use obligatory single-stranded circular DNA intermediates for excision and insertion and encode a transposase, TnpAIS200, which recognizes subterminal secondary structures at the insertion sequence ends. Similar secondary structures, Repeated Extragenic Palindromes (REP), are present in many bacterial genomes. TnpAIS200-related proteins, TnpAREP, have been identified and could be responsible for REP sequence proliferation. These proteins share a conserved HuH/Tyrosine core domain responsible for catalysis and are involved in processes of ssDNA cleavage and ligation. Our goal is to characterize the diversity of these proteins collectively referred as the TnpAY1 family. RESULTS: A genome-wide analysis of sequences similar to TnpAIS200 and TnpAREP in prokaryotes revealed a large number of family members with a wide taxonomic distribution. These can be arranged into three distinct classes and 12 subclasses based on sequence similarity. One subclass includes sequences similar to TnpAIS200. Proteins from other subclasses are not associated with typical insertion sequence features. These are characterized by specific additional domains possibly involved in protein/DNA or protein/protein interactions. Their genes are found in more than 25% of species analyzed. They exhibit a patchy taxonomic distribution consistent with dissemination by horizontal gene transfers followed by loss. The tnpAREP genes of five subclasses are flanked by typical REP sequences in a REPtron-like arrangement. Four distinct REP types were characterized with a subclass specific distribution. Other subclasses are not associated with REP sequences but have a large conserved domain located in C-terminal end of their sequence. This unexpected diversity suggests that, while most likely involved in processing single-strand DNA, proteins from different subfamilies may play a number of different roles. CONCLUSIONS: We established a detailed classification of TnpAY1 proteins, consolidated by the analysis of the conserved core domains and the characterization of additional domains. The data obtained illustrate the unexpected diversity of the TnpAY1 family and provide a strong framework for future evolutionary and functional studies. By their potential function in ssDNA editing, they may confer adaptive responses to host cell physiology and metabolism.

Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems.

The CRISPR-Cas-derived RNA-guided Cas9 endonuclease is the key element of an emerging promising technology for genome engineering in a broad range of cells and organisms. The DNA-targeting mechanism of the type II CRISPR-Cas system involves maturation of tracrRNA:crRNA duplex (dual-RNA), which directs Cas9 to cleave invading DNA in a sequence-specific manner, dependent on the presence of a Protospacer Adjacent Motif (PAM) on the target. We show that evolution of dual-RNA and Cas9 in bacteria produced remarkable sequence diversity. We selected eight representatives of phylogenetically defined type II CRISPR-Cas groups to analyze possible coevolution of Cas9 and dual-RNA. We demonstrate that these two components are interchangeable only between closely related type II systems when the PAM sequence is adjusted to the investigated Cas9 protein. Comparison of the taxonomy of bacterial species that harbor type II CRISPR-Cas systems with the Cas9 phylogeny corroborates horizontal transfer of the CRISPR-Cas loci. The reported collection of dual-RNA:Cas9 with associated PAMs expands the possibilities for multiplex genome editing and could provide means to improve the specificity of the RNA-programmable Cas9 tool.

Activity, specificity and structure of I-Bth0305I: a representative of a new homing endonuclease family.

Novel family of putative homing endonuclease genes was recently discovered during analyses of metagenomic and genomic sequence data. One such protein is encoded within a group I intron that resides in the recA gene of the Bacillus thuringiensis 03058-36 bacteriophage. Named I-Bth0305I, the endonuclease cleaves a DNA target in the uninterrupted recA gene at a position immediately adjacent to the intron insertion site. The enzyme displays a multidomain, homodimeric architecture and footprints a DNA region of ~60 bp. Its highest specificity corresponds to a 14-bp pseudopalindromic sequence that is directly centered across the DNA cleavage site. Unlike many homing endonucleases, the specificity profile of the enzyme is evenly distributed across much of its target site, such that few single base pair substitutions cause a significant decrease in cleavage activity. A crystal structure of its C-terminal domain confirms a nuclease fold that is homologous to very short patch repair (Vsr) endonucleases. The domain architecture and DNA recognition profile displayed by I-Bth0305I, which is the prototype of a homing lineage that we term the 'EDxHD' family, are distinct from previously characterized homing endonucleases.

Neisseria gonorrhoeae FA1090 carries genes encoding two classes of Vsr endonucleases.

A very short patch repair system prevents mutations resulting from deamination of 5-methylcytosine to thymine. The Vsr endonuclease is the key enzyme of this system, providing sequence specificity. We identified two genes encoding Vsr endonucleases V.NgoAXIII and V.NgoAXIV from Neisseria gonorrhoeae FA1090 based on DNA sequence similarity to genes encoding Vsr endonucleases from other bacteria. After expression of the gonococcal genes in Escherichia coli, the proteins were biochemically characterized and the endonucleolytic activities and specificities of V.NgoAXIII and V.NgoAXIV were determined. V.NgoAXIII was found to be multispecific and to recognize T:G mismatches in every nucleotide context tested, whereas V.NgoAXIV recognized T:G mismatches in the following sequences: GTGG, CTGG, GTGC, ATGC, and CTGC. Alanine mutagenesis of conserved residues showed that Asp50 and His68 of V.NgoAXIII and Asp51 and His69 of V.NgoAXIV are essential for hydrolytic activity. Glu25, His64, and Asp97 of V.NgoAXIV and Glu24, Asp63, and Asp97 of V.NgoAXIII are important but not crucial for the activity of V.NgoAXIII and V.NgoAXIV. However, Glu24 and Asp63 are also important for the specificity of V.NgoAXIII. On the basis of our results concerning features of Vsr endonucleases expressed by N. gonorrhoeae FA1090, we postulate that at least two types of Vsr endonucleases can be distinguished.

Unconventional GIY-YIG homing endonuclease encoded in group I introns in closely related strains of the Bacillus cereus group.

Several group I introns have been previously found in strains of the Bacillus cereus group at three different insertion sites in the nrdE gene of the essential nrdIEF operon coding for ribonucleotide reductase. Here, we identify an uncharacterized group IA intron in the nrdF gene in 12 strains of the B. cereus group and show that the pre-mRNA is efficiently spliced. The Bacillus thuringiensis ssp. pakistani nrdF intron encodes a homing endonuclease, denoted I-BthII, with an unconventional GIY-(X)8-YIG motif that cleaves an intronless nrdF gene 7 nt upstream of the intron insertion site, producing 2-nt 3' extensions. We also found four additional occurrences of two of the previously reported group I introns in the nrdE gene of 25 sequenced B. thuringiensis and one B. cereus strains, and one non-annotated group I intron at a fourth nrdE insertion site in the B. thuringiensis ssp. Al Hakam sequenced genome. Two strains contain introns in both the nrdE and the nrdF genes. Phylogenetic studies of the nrdIEF operon from 39 strains of the B. cereus group suggest several events of horizontal gene transfer for two of the introns found in this operon.

Thermodynamics of DNA target site recognition by homing endonucleases.

The thermodynamic profiles of target site recognition have been surveyed for homing endonucleases from various structural families. Similar to DNA-binding proteins that recognize shorter target sites, homing endonucleases display a narrow range of binding free energies and affinities, mediated by structural interactions that balance the magnitude of enthalpic and entropic forces. While the balance of DeltaH and TDeltaS are not strongly correlated with the overall extent of DNA bending, unfavorable DeltaH(binding) is associated with unstacking of individual base steps in the target site. The effects of deleterious basepair substitutions in the optimal target sites of two LAGLIDADG homing endonucleases, and the subsequent effect of redesigning one of those endonucleases to accommodate that DNA sequence change, were also measured. The substitution of base-specific hydrogen bonds in a wild-type endonuclease/DNA complex with hydrophobic van der Waals contacts in a redesigned complex reduced the ability to discriminate between sites, due to nonspecific DeltaS(binding).

The structure of I-CeuI homing endonuclease: Evolving asymmetric DNA recognition from a symmetric protein scaffold.

Homing endonucleases are highly specific catalysts of DNA strand breaks, leading to the transfer of mobile intervening sequences containing the endonuclease ORF. We have determined the structure and DNA recognition behavior of I-CeuI, a homodimeric LAGLIDADG endonuclease from Chlamydomonas eugametos. This symmetric endonuclease displays unique structural elaborations on its core enzyme fold, and it preferentially cleaves a highly asymmetric target site. This latter property represents an early step, prior to gene fusion, in the generation of asymmetric DNA binding platforms from homodimeric ancestors. The divergence of the sequence, structure, and target recognition behavior of homing endonucleases, as illustrated by this study, leads to the invasion of novel genomic sites by mobile introns during evolution.

DNase II is a member of the phospholipase D superfamily.

MOTIVATION: DNase II is an endodeoxyribonuclease involved in apoptosis and essential for the mammalian development. Despite the understanding of biochemical properties of this enzyme, its structure and relationships to other protein families remain unknown. RESULTS: Using protein fold-recognition we found that DNase II exhibits a catalytic domain common to the phospholipase D superfamily. Our model explains the available experimental data and provides the first structural platform for sequence-function analyses of this important nuclease.

DmGEN, a novel RAD2 family endo-exonuclease from Drosophila melanogaster.

A novel endo-exonuclease, DmGEN (Drosophila Melanogaster XPG-like endonuclease), was identified in D.melanogaster. DmGEN is composed of five exons and four introns, and the open reading frame encodes a predicted product of 726 amino acid residues with a molecular weight of 82.5 kDa and a pI of 5.36. The gene locus on Drosophila polytene chromosomes was detected at 64C9 on the left arm of chromosome 3 as a single site. The encoded protein showed a relatively high degree of sequence homology with the RAD2 nucleases, especially XPG. Although the XPG-N- and XPG-I-domains are highly conserved in sequence, locations of the domains are similar to those of FEN-1 and EXO-1, and the molecular weight of the protein is close to that of EXO-1. In vitro, DmGEN showed endonuclease and 3'-5' exonuclease activities with both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), but the endonuclease action with dsDNA was quite specific: 5'-3' exonuclease activity was found to occur with nicked DNA, while dsDNA was endonucleolytically cut at 3-4 bp from the 5' end. Homologs are widely found in mammals and higher plants. The data suggest that DmGEN belongs to a new class of RAD2 nuclease.

Mismatch cleavage by single-strand specific nucleases.

We have investigated the ability of single-strand specific (sss) nucleases from different sources to cleave single base pair mismatches in heteroduplex DNA templates used for mutation and single-nucleotide polymorphism analysis. The TILLING (Targeting Induced Local Lesions IN Genomes) mismatch cleavage protocol was used with the LI-COR gel detection system to assay cleavage of amplified heteroduplexes derived from a variety of induced mutations and naturally occurring polymorphisms. We found that purified nucleases derived from celery (CEL I), mung bean sprouts and Aspergillus (S1) were able to specifically cleave nearly all single base pair mismatches tested. Optimal nicking of heteroduplexes for mismatch detection was achieved using higher pH, temperature and divalent cation conditions than are routinely used for digestion of single-stranded DNA. Surprisingly, crude plant extracts performed as well as the highly purified preparations for this application. These observations suggest that diverse members of the S1 family of sss nucleases act similarly in cleaving non-specifically at bulges in heteroduplexes, and single-base mismatches are the least accessible because they present the smallest single-stranded region for enzyme binding. We conclude that a variety of sss nucleases and extracts can be effectively used for high-throughput mutation and polymorphism discovery.

SURVEY AND SUMMARY: holliday junction resolvases and related nucleases: identification of new families, phyletic distribution and evolutionary trajectories.

Holliday junction resolvases (HJRs) are key enzymes of DNA recombination. A detailed computer analysis of the structural and evolutionary relationships of HJRs and related nucleases suggests that the HJR function has evolved independently from at least four distinct structural folds, namely RNase H, endonuclease, endonuclease VII-colicin E and RusA. The endonuclease fold, whose structural prototypes are the phage lambda exonuclease, the very short patch repair nuclease (Vsr) and type II restriction enzymes, is shown to encompass by far a greater diversity of nucleases than previously suspected. This fold unifies archaeal HJRs, repair nucleases such as RecB and Vsr, restriction enzymes and a variety of predicted nucleases whose specific activities remain to be determined. Within the RNase H fold a new family of predicted HJRs, which is nearly ubiquitous in bacteria, was discovered, in addition to the previously characterized RuvC family. The proteins of this family, typified by Escherichia coli YqgF, are likely to function as an alternative to RuvC in most bacteria, but could be the principal HJRs in low-GC Gram-positive bacteria and AQUIFEX: Endonuclease VII of phage T4 is shown to serve as a structural template for many nucleases, including MCR:A and other type II restriction enzymes. Together with colicin E7, endonuclease VII defines a distinct metal-dependent nuclease fold. As a result of this analysis, the principal HJRs are now known or confidently predicted for all bacteria and archaea whose genomes have been completely sequenced, with many species encoding multiple potential HJRs. Horizontal gene transfer, lineage-specific gene loss and gene family expansion, and non-orthologous gene displacement seem to have been major forces in the evolution of HJRs and related nucleases. A remarkable case of displacement is seen in the Lyme disease spirochete Borrelia burgdorferi, which does not possess any of the typical HJRs, but instead encodes, in its chromosome and each of the linear plasmids, members of the lambda exonuclease family predicted to function as HJRs. The diversity of HJRs and related nucleases in bacteria and archaea contrasts with their near absence in eukaryotes. The few detected eukaryotic representatives of the endonuclease fold and the RNase H fold have probably been acquired from bacteria via horizontal gene transfer. The identity of the principal HJR(s) involved in recombination in eukaryotes remains uncertain; this function could be performed by topoisomerase IB or by a novel, so far undetected, class of enzymes. Likely HJRs and related nucleases were identified in the genomes of numerous bacterial and eukaryotic DNA viruses. Gene flow between viral and cellular genomes has probably played a major role in the evolution of this class of enzymes. This analysis resulted in the prediction of numerous previously unnoticed nucleases, some of which are likely to be new restriction enzymes.

Crystal structure of NaeI-an evolutionary bridge between DNA endonuclease and topoisomerase.

NAE:I is transformed from DNA endonuclease to DNA topoisomerase and recombinase by a single amino acid substitution. The crystal structure of NAE:I was solved at 2.3 A resolution and shows that NAE:I is a dimeric molecule with two domains per monomer. Each domain contains one potential DNA recognition motif corresponding to either endonuclease or topoisomerase activity. The N-terminal domain core folds like the other type II restriction endonucleases as well as lambda-exonuclease and the DNA repair enzymes MutH and Vsr, implying a common evolutionary origin and catalytic mechanism. The C-terminal domain contains a catabolite activator protein (CAP) motif present in many DNA-binding proteins, including the type IA and type II topoisomerases. Thus, the NAE:I structure implies that DNA processing enzymes evolved from a few common ancestors. NAE:I may be an evolutionary bridge between endonuclease and DNA processing enzymes.

A novel 3-methyladenine DNA glycosylase from Helicobacter pylori defines a new class within the endonuclease III family of base excision repair glycosylases.

The cloning, purification, and characterization of MagIII, a 3-methyladenine DNA glycosylase from Helicobacter pylori, is presented in this paper. Sequence analysis of the genome of this pathogen failed to identify open reading frames potentially coding for proteins with a 3-methyladenine DNA glycosylase activity. The putative product of the HP602 open reading frame, reported as an endonuclease III, shares extensive amino acid sequence homology with some bacterial members of this family and has the canonic active site helix-hairpin-helix-GPD motif. Surprisingly, this predicted H. pylori endonuclease III encodes a 25,220-Da protein able to release 3-methyladenine, but not oxidized bases, from modified DNA. MagIII has no abasic site lyase activity and displays the substrate specificity of the 3-methyladenine-DNA glycosylase type I of Escherichia coli (Tag) because it is not able to recognize 7-methylguanine or hypoxanthine as substrates. The expression of the magIII open reading frame in null 3-methyladenine glycosylase E. coli (tag alkA) restores to this mutant partial resistance to alkylating agents. MagIII-deficient H. pylori cells show an alkylation-sensitive phenotype. H. pylori wild type cells exposed to alkylating agents present an adaptive response by inducing the expression of magIII. MagIII is thus a novel bacterial member of the endonuclease III family, which displays biochemical properties not described for any of the members of this group until now.

A new member of the endonuclease III family of DNA repair enzymes that removes methylated purines from DNA.

DNA is constantly exposed to endogenous andexogenous alkylating agents that can modify its bases,resulting in mutagenesis in the absence of DNA repair [1,2]. Alkylation damage is removed by the action of DNA glycosylases, which initiate the base excision repair pathway and protect the sequence information of the genome [3-5]. We have identified a new class of methylpurine DNA glycosylase, designated MpgII, that is a member of the endonuclease III family of DNA repair enzymes. We expressed and purified MpgII from Thermotoga maritima and found that the enzyme releases both 7-methylguanine and 3-methyladenine from DNA. We cloned the MpgII genes from T. maritima and from Aquifex aeolicus and found that both genes could restore methylmethanesulfonate (MMS) resistance to Escherichia coli alkA tagA double mutants, which are deficient in the repair of alkylated bases. Analogous genes are found in other Bacteria and Archaea and appear to be the only genes coding for methylpurine DNA glycosylase activity in these organisms. MpgII is the fifth member of the endonuclease III family of DNA repair enzymes, suggesting that the endonuclease III protein scaffold has been modified during evolution to recognize and repair a variety of DNA damage.

Purification, characterization, and amino acid sequencing of DNase gamma from rat spleen.

An endonuclease named DNase gamma was purified to apparent homogeneity from rat splenocyte nuclei and its properties were characterized. We also determined the NH2-terminal and partial amino acid sequences of the proteolytic internal peptides. The molecular mass of gamma DNase was 33,000 daltons as determined by SDS-polyacrylamide gel electrophoresis. A native molecular mass of 30,000 was estimated by gel filtration. Purified DNase gamma is active in the presence of both Ca2+ and Mg2+ or Mn2+ alone and inhibited by Co2+, Ni2+, Cu2+, and especially Zn2+. Maximal activity was achieved at pH 7.2 in Mops-NaOH buffer. The sequence data on the NH2-terminal and seven internal peptides obtained by sequential digestions with Achromobacter protease I and endoproteinase Asp-N revealed that DNase gamma is a novel endonuclease that shows sequence homology with DNase I.

A new class of uracil-DNA glycosylases related to human thymine-DNA glycosylase.

Mispairs in DNA of guanine with uracil and thymine can arise as a result of deamination of cytosine and 5-methylcytosine, respectively. In humans such mispairs are removed by thymine-DNA glycosylase (TDG). By deleting the carboxy and amino termini of this enzyme we have identified a core region capable of processing G/U but not G/T mispairs. We have further identified two bacterial proteins with strong sequence homology to this core and shown that the homologue from Escherichia coli (dsUDG) can remove uracil from G/U mispairs. This enzyme is likely to act as a back-up to the highly efficient and abundant enzyme uracil-DNA glycosylase (UDG) which is found in most organisms. Pupating insects have been reported to lack UDG activity, but we have identified an enzyme similar to dsUDG in cell lines from three different insect species. These data imply the existence of a family of double-strand-specific uracil-DNA glycosylases which, although they are subservient to UDG in most organisms, may constitute the first line of defence against the mutagenic effects of cytosine deamination in insects.