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Restriction endonucleases naturally target DNA duplexes. Systematic screening has identified a small minority of these enzymes that can also cleave RNA/DNA heteroduplexes and that may therefore be useful as tools for RNA biochemistry. We have chosen AvaII (G↓GWCC, where W stands for A or T) as a representative of this group of restriction endonucleases for detailed characterization. Here, we report crystal structures of AvaII alone, in specific complex with partially cleaved dsDNA, and in scanning complex with an RNA/DNA hybrid. The specific complex reveals a novel form of semi-specific dsDNA readout by a hexa-coordinated metal cation, most likely Ca2+ or Mg2+. Substitutions of residues anchoring this non-catalytic metal ion severely impair DNA binding and cleavage. The dsDNA in the AvaII complex is in the A-like form. This creates space for 2'-OH groups to be accommodated without intra-nucleic acid steric conflicts. PD-(D/E)XK restriction endonucleases of known structure that bind their dsDNA targets in the A-like form cluster into structurally similar groups. Most such enzymes, including some not previously studied in this respect, cleave RNA/DNA heteroduplexes. We conclude that A-form dsDNA binding is a good predictor for RNA/DNA cleavage activity., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In Escherichia coli, errors in newly-replicated DNA, such as the incorporation of a nucleotide with a mis-paired base or an accidental insertion or deletion of nucleotides, are corrected by a methyl-directed mismatch repair (MMR) pathway. While the enzymology of MMR has long been established, many fundamental aspects of its mechanisms remain elusive, such as the structures, compositions, and orientations of complexes of MutS, MutL, and MutH as they initiate repair. Using atomic force microscopy, we--for the first time--record the structures and locations of individual complexes of MutS, MutL and MutH bound to DNA molecules during the initial stages of mismatch repair. This technique reveals a number of striking and unexpected structures, such as the growth and disassembly of large multimeric complexes at mismatched sites, complexes of MutS and MutL anchoring latent MutH onto hemi-methylated d(GATC) sites or bound themselves at nicks in the DNA, and complexes directly bridging mismatched and hemi-methylated d(GATC) sites by looping the DNA. The observations from these single-molecule studies provide new opportunities to resolve some of the long-standing controversies in the field and underscore the dynamic heterogeneity and versatility of MutSLH complexes in the repair process., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Uniform bimetallic nanowires, tunable in size, have been grown on artificial DNA templates via a two-step metallization process. Alkyne-modified cytosines were incorporated into 900-base-pair polymerase-chain-reaction fragments. The alkyne modifications serve as addressable metal-binding sites after conversion to a sugar triazole derivative via click chemistry. Reaction of the Tollens reagent with these sugar-coated DNA duplexes generates Ag0 metallization centers around the sugar modification sites of the DNA. After a subsequent enhancement step using gold, nanowires < or = 10 nm in diameter with a homogeneous surface profile were obtained. Furthermore, the advantage of this two-step procedure lies in the high selectivity of the process, due to the exact spatial control of modified DNA base incorporation and hence the confinement of metallization centers at addressable sites. Besides experiments on a membrane as a proof for the selectivity of the method, atomic force microscopy (AFM) studies of the wires produced on Si-SiO2 surfaces are discussed. Furthermore, we demonstrate time-dependent metallization experiments, monitored by AFM.

Mutation detection and mismatch repair investigations based on heteroduplex formation require a linear DNA structure. DNA branching, described previously under physiological conditions, has been analysed in the heteroduplex formation process. Symmetrical chi-structures were detected after heteroduplex formation by gel electrophoresis and electron microscopy. Buffer composition, DNA concentration and duplex end-sequences influence DNA branching. Duplexes with homologous central regions but non-complementary ends do not form hybrid heteroduplexes or hybrid Holliday junctions. Our results explain the requirements for efficient heteroduplex formation, which were previously determined empirically: special solution composition, optimal DNA concentration and GC clamps. This provides the theoretical background for further optimisation of the procedure.

The disease-associated expansion of (CTG)*(CAG) repeats is likely to involve slipped-strand DNAs. There are two types of slipped DNAs (S-DNAs): slipped homoduplex S-DNAs are formed between two strands having the same number of repeats; and heteroduplex slipped intermediates (SI-DNAs) are formed between two strands having different numbers of repeats. We present the first characterization of S-DNAs formed by disease-relevant lengths of (CTG)*(CAG) repeats which contained all predicted components including slipped-out repeats and slip-out junctions, where two arms of the three-way junction were composed of complementary paired repeats. In S-DNAs multiple short slip-outs of CTG or CAG repeats occurred throughout the repeat tract. Strikingly, in SI-DNAs most of the excess repeats slipped-out at preferred locations along the fully base-paired Watson-Crick duplex, forming defined three-way slip-out junctions. Unexpectedly, slipped-out CAG and slipped-out CTG repeats were predominantly in the random-coil and hairpin conformations, respectively. Both the junctions and the slip-outs could be recognized by DNA metabolizing proteins: only the strand with the excess repeats was hypersensitive to cleavage by the junction-specific T7 endonuclease I, while slipped-out CAG was preferentially bound by single-strand binding protein. An excellent correlation was observed for the size of the slip-outs in S-DNAs and SI-DNAs with the size of the tract length changes observed in quiescent and proliferating tissues of affected patients-suggesting that S-DNAs and SI-DNAs are mutagenic intermediates in those tissues, occurring during error-prone DNA metabolism and replication fork errors.

The MkaH protein from the archaeon Methanopyrus kandleri, an unusual assembly of two histone-fold domains in a single polypeptide chain, demonstrates high structural similarity to eukaryal histones. We studied the DNA binding and self-association properties of MkaH by means of the electrophoretic mobility shift assay (EMSA), electron microscopy (EM), chemical cross-linking, and analytical gel filtration. EMSA showed an increased mobility of linear DNA complexed with MkaH protein with a maximum at a protein-DNA weight ratio (R(w)) of approximately 3; the mobility decreased at higher protein concentration. EM of the complexes formed at Rw or=9) thickened compact nucleoprotein structures were observed; no individual loops were seen within the complexes. Gel filtration chromatography and chemical fixation indicated that in the absence of DNA the dominant form of the MkaH in solution, unlike other archaeal histones, is a stable dimer (pseudo-tetramer of the histone-fold domain) apparently resembling the eukaryal (H3-H4)(2) tetramer. Similarly, dimers are the dominant form of the protein interacting with DNA. The properties of MkaH supporting the assignment of its intermediate position between other archaeal and eukaryal histones are discussed.

Previously, we demonstrated the interaction of homologous linear duplexes with formation of four-way DNA structures on the model of five PCR products. We propose that homologous duplex interaction is initiated by the nucleation of several dissociated base pairs of the complementary ends of two fragments with Holliday junction formation, in which cross point migration occurs via spooling of DNA strands from one duplex to the other one, finally resulting in complete resolution into new or previously existing duplexes. To confirm that DNA-DNA interaction involves formation of four-way DNA structures with strand exchange at the cross point, we have demonstrated the strand exchange process between identical duplexes using homologous fragments, harboring either biotin label or (32)P-label. Incubation of the mixture resulted in the addition of (32)P-label to biotin-labeled fragments, and the intensity of (32)P-labeling of biotinylated fragments was dependent upon the incubation duration. DNA-DNA interaction is not based on surface-dependent denaturing, as Triton X-100 does not decrease the formation of complexes between DNA duplexes. The equilibrium concentration of Holliday junctions depends on the sequences of the fragment ends and the incubation temperature. The free energy of Holliday junction formation by the fragments with GC and AT ends differed by 0.6 kcal/mol. Electron microscopic analysis demonstrated that the majority of Holliday junctions harbor the cross point within a 300 base pair region of the fragment ends. This insight into the mechanism of homologous duplex interaction extends our understanding of different DNA rearrangements. Understanding of DNA-DNA interaction is of practical use for better interpretation and optimization of PCR-based analyses.

DNA heteroduplexes as models for slipped strand DNA have been analyzed by polyacrylamide gel migration and atomic force microscopy (AFM). All heteroduplexes containing one hairpin or loop have reduced electrophoretic mobilities compared with that expected for their molecular weights. The retarded gel mobility correlates with the formation of a sharp kink detected by AFM. Increasing the hairpin length from 7 bp to 50 bp results in a monotonous decrease in gel mobility of heteroduplexes. This secondary retardation effect appears to depend only on the hairpin size since the AFM data show no dependence of the kink angle on the hairpin length. Heteroduplex isomers with a loop or hairpin in opposite strands migrate with distinct mobilities. Analysis of gel migration of heteroduplexes with altered hairpin orientations as well as of truncated heteroduplexes indicates that the difference in mobility is due to an inherent curvature in one of the long arms. This is confirmed by the end-to-end distance measurements from AFM images. In addition, significant variation of the end-to-end distances is consistent with a dynamic structure of heteroduplexes at the three-way junction. Double heteroduplexes containing one hairpin in each of the complementary strands also separate in a gel as two isomers. Their appearance in AFM showed a complicated pattern of flat representations of the three-dimensional structure and may indicate a certain degree of interaction between complementary parts of the hairpins that are several helical turns apart., (Copyright 1999 Academic Press.)

Replication factor C (RF-C) is a eukaryotic heteropentameric protein required for DNA replication and repair processes by loading proliferating cell nuclear antigen (PCNA) onto DNA in an ATP-dependent manner. Prior to loading PCNA, RF-C binds to DNA. This binding is thought to be restricted to a specific DNA structure, namely to a primer/template junction. Using the electron microscope we have examined the affinity of human heteropentameric RF-C and the DNA-binding region within the large subunit of RF-C from Drosophila melanogaster (dRF-Cp140) to heteroduplex DNA. The electron microscopic data indicate that both human heteropentameric RF-C and the DNA-binding region within dRF-Cp140 are sequestered by single-stranded DNA. No preferential affinity for the 3' or 5' transition points from single- to double-stranded DNA was evident.

Interaction of Escherichia coli MutS and MutL with heteroduplex DNA has been visualized by electron microscopy. In a reaction dependent on ATP hydrolysis, complexes between a MutS dimer and a DNA heteroduplex are converted to protein-stabilized, alpha-shaped loop structures with the mismatch in most cases located within the DNA loop. Loop formation depends on ATP hydrolysis and loop size increases linearly with time at a rate of 370 base pairs/min in phosphate buffer and about 10,000 base pairs/min in the HEPES buffer used for repair assay. These observations suggest a translocation mechanism in which a MutS dimer bound to a mismatch subsequently leaves this site by ATP-dependent tracking or unidimensional movement that is in most cases bidirectional from the mispair. In view of the bidirectional capability of the methyl-directed pathway, this reaction may play a role in determination of heteroduplex orientation. The rate of MutS-mediated DNA loop growth is enhanced by MutL, and when both proteins are present, both are found at the base of alpha-loop structures, and both can remain associated with excision intermediates produced in later stages of the reaction.

Molecular modelling studies resulted in the design of a variety of non-nucleotidic covalent linkers to bridge the 3'-end of the (+)-strand and the 5'-end of the (-)-strand in DNA duplexes. Three of these linkers were synthesized and used to prepare singly cross-linked duplexes d(GTGGAATTC)-linker-d(GAATTCCAC). Linker I is an assembly of a propylene-, a phosphate- and a second propylene-group and is thought to mimic the backbone of two nucleotides. Linkers II and III consist of five and six ethyleneglycol units, respectively. The melting temperatures of the cross-linked duplexes are 65 degrees C for I and 73 degrees C for II and III, as compared with 36 degrees C for the corresponding non-linked nonadeoxynucleotide duplex. The three cross-linked duplexes were structurally characterized by nuclear magnetic resonance spectroscopy. The 1H and 31P resonance assignments in the DNA stem were obtained using standard methods. For the resonance assignment of the linker protons, two-dimensional 1H-31P heteronuclear COSY and two-quantum-experiments were used. Distance geometry calculations with NOE-derived distance constraints were performed and the resulting structures were energy-minimized. In duplex I, the nucleotides flanking the propylene-phosphate-propylene-linker do not form a Watson-Crick base pair, whereas in duplexes II and III the entire DNA stem is in a B-type double helix conformation.

During genetic recombination a heteroduplex joint is formed between two homologous DNA molecules. The heteroduplex joint plays an important role in recombination since it accommodates sequence heterogeneities (mismatches, insertions or deletions) that lead to genetic variation. Two Escherichia coli proteins, RuvA and RuvB, promote the formation of heteroduplex DNA by catalysing the branch migration of crossovers, or Holliday junctions, which link recombining chromosomes. We show that RuvA and RuvB can promote branch migration through 1800 bp of heterologous DNA, in a reaction facilitated by the presence of E.coli single-stranded DNA binding (SSB) protein. Reaction intermediates, containing unpaired heteroduplex regions bound by SSB, were directly visualized by electron microscopy. In the absence of SSB, or when SSB was replaced by a single-strand binding protein from bacteriophage T4 (gene 32 protein), only limited heterologous branch migration was observed. These results show that the RuvAB proteins, which are induced as part of the SOS response to DNA damage, allow genetic recombination and the recombinational repair of DNA to occur in the presence of extensive lengths of heterology.

The RuvA and RuvB proteins of Escherichia coli, which are induced in response to DNA damage, are important in the formation of heteroduplex DNA during genetic recombination and related recombinational repair processes. In vitro studies show that RuvA binds Holiday junctions and acts as a specificity factor that targets the RuvB ATPase, a hexameric ring protein, to the junction. Together, RuvA and RuvB promote branch migration, an ATP-dependent reaction that increases the length of the heteroduplex DNA. Electron microscopic visualization of RuvAB now provides a new insight into the mechanism of this process. We observe the formation of a tripartite protein complex in which RuvA binds the crossover and is sandwiched between two hexameric rings of RuvB. The Holliday junction within this complex adopts a square-planar structure. We propose a molecular model for branch migration, a unique feature of which is the role played by the two oppositely oriented RuvB ring motors.

Using electron microscopy and indirect end-labeling methods, we have examined excision tracts produced by the Escherichia coli methyl-directed mismatch repair system on a closed circular G-T heteroduplex that contains a single d(GATC) site. Despite differing polarities of the unmodified strand in the two hemimethylated derivatives of the heteroduplex, that portion of the unmethylated strand spanning the shorter path between the d(GATC) site and mismatch is targeted for excision in both cases. Mismatch-provoked excision occurring on both hemimethylated DNAs requires DNA helicase II, but exonuclease requirements for the reaction depend on heteroduplex orientation. When the d(GATC) sequence on the unmodified strand resides 3' to the mismatch as viewed along the shorter path, excision requires exonuclease I. Excision occurring on the alternate hemimethylated heteroduplex depends on the 5'--> 3' hydrolytic activity of exonuclease VII. Coupled with the previous demonstration that repair initiates via the mismatch-provoked, MutHLS-dependent incision of the unmethylated strand at a d(GATC) sequence (Au, K.G., Welsh, K., and Modrich, P. (1992) J. Biol. Chem. 267, 12142-12148), these findings indicate an excision mechanism in which helicase II displacement renders the incised strand sensitive to the appropriate single-strand exonuclease. Our data imply that hydrolysis commences at the d(GATC) site, proceeds to a point beyond the mismatch, and terminates at a number of discrete sites within a 100-nucleotide region just beyond this site. The extent of excision is therefore controlled by one or more components of the repair system.

Electron microscopic examination of nucleic acids requires the use of special spreading techniques. The classical method was developed by Kleinschmidt and Zahn in 1959. Modifications of this method increased sensitivity to allow detection of a total amount of about 1 x 10(-3) micrograms of single-stranded DNA and 2 x 10(-5) micrograms of double-stranded DNA. Here we describe two rapid and simple procedures increasing sensitivity by 1-2 orders of magnitude to visualize at least 1 x 10(-5) micrograms of single- and/or double-stranded DNA.

RecA protein promotes a substantial DNA strand exchange reaction in the presence of adenosine 5'-O-3-(thio)triphosphate (ATP gamma S) (Menetski et al., 1990), calling into question the role of ATP hydrolysis in this reaction. We demonstrate here that the ATP gamma S-mediated process is restricted to homologous strand exchange reactions involving three strands. In four-strand exchanges between a gapped duplex circle and a second linear duplex, joint molecules are formed in the gap but are not extended into the four-strand region when ATP gamma S is present. This result provides evidence that one function of ATP hydrolysis in the recA system is to facilitate reciprocal DNA strand exchange involving four strands. Implications with respect to the role of four-stranded pairing intermediates and the mechanistic relationship between three- and four-strand exchange reactions are discussed.

The plasmids pON5300 and pON5304, nonconjugative variants of the plasmid R1drd-19Km, were analyzed by electron microscopy. It was found by heteroduplex mapping that a 1.4 kb DNA segment was inserted into EcoRI E fragment of both plasmids, where some tra-genes and oriT are localized. Although this DNA segment was mapped to the same region its orientation was different in each of the two plasmids. The inserted DNA segment was identified as an IS10R sequence on the basis of analysis of self-annealed molecules of pON5304 and their cleavage with EcoRV restriction enzyme. These methods enable us not only to map IS10R sequences on 87 kb pON5300 and 65 kb pON5304 molecules, respectively, but also to define their orientation.

The rat submandibular gland-specific GRP-Ca gene (encoding C-terminal variant A of the glutamine/glutamic acid-rich protein) has been cloned from a male Wistar/Furth genomic library. The complete sequence, including 2.0 kb of 5' flanking and 0.5 kb of 3' flanking DNA has been determined. Electron microscopic heteroduplex analysis and sequence analysis established that transcripts coding for GRP-Ca and GRP-Cb are encoded by separate genes. The GRP-Ca gene is approx. 4.5 kb in size and is comprised of four exons and three introns. Comparison of this gene with several rodent and human salivary proline-rich protein-encoding genes (PRP) indicates that GRP-Ca shares this exon-intron structure with the rat SMR-2 gene, the hamster H29 gene, and the human PRP genes. In addition, a 28-bp element found in the proximal promoter region of GRP-Ca was found to be highly conserved among the superfamily of PRP genes.

RecA protein promotes an unexpectedly efficient DNA strand exchange between circular single-stranded DNA and duplex DNAs containing short (50-400-base pair) heterologous sequences at the 5' (initiating) end. The major mechanism by which this topological barrier is bypassed involves DNA strand breakage. Breakage is both strand and position specific, occurring almost exclusively in the displaced (+) strand of the duplex within a 15-base pair region of the heterology/homology junction. Breakage also requires recA protein, ATP hydrolysis, and homologous sequences 3' to the heterology. Although the location of the breaks and the observed requirements clearly indicate a major role for recA protein in this phenomenon, the molecular mechanism is not yet clear. The breakage may reflect a DNA structure and/or some form of structural stress within the DNA during recA protein-mediated DNA pairing which either exposes the DNA at this precise position to the action of a contaminating nuclease or induces a direct mechanical break. We also find that when heterology is located at the 3' end of the linear duplex, strand exchange is halted (without DNA breakage) about 500 base pairs from the homology/heterology junction.

We have characterized three temperate bacteriophages of pneumococcus (HB-3, HB-623, and HB-746). Although all the phages belong to the same family, the polypeptide composition of the virions and the DNA restriction endonuclease analysis of their DNAs revealed differences among the three phages. The genomes of these bacteriophages have been isolated as DNA-protein complexes. The protein is specifically associated with the two 5' termini of the DNA as shown by experiments carried out with exonucleases. The protein bound to the DNA in the three phages studied, iodinated in vitro with 125I, has a molecular weight of 23,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment of the complexes with chaotropic agents suggested that the protein is covalently bound to the 5' termini of the DNA. Comparative pulsed-field gel electrophoresis analysis and Southern hybridization of the SmaI restriction fragments of DNAs from one lysogenic bacteria and its parental strain revealed that the prophage genome was integrated in the host chromosome.

The genomes of two new human papillomavirus (HPV) types, named HPV54 and HPV55, were cloned from penile lesions of 2 patients. HPV54 was isolated from a verrucous carcinoma (Buschke-Löwenstein tumour) together with full-length HPV6 genomes and HPV6 DNA molecules with a deletion of about 0.3 kb located in the non-coding region. HPV55 was isolated from a condyloma acuminatum. No cross-hybridization was observed between HPV54 DNA and the DNAs of the known cutaneous and genital HPVs by blot hybridization experiments performed under stringent conditions. In contrast, significant cross-hybridization was detected between HPV55 DNA and the DNA of HPV13, associated with benign oral lesions, and, to a lesser extent, with the DNAs of HPV6, 11, and 44, associated with benign genital proliferative lesions. The DNA sequence homology between HPV55 and HPV6, 11, and 13 was estimated at 12%, 12%, and 20%, respectively, by hybridization in liquid phase at saturation, followed by nuclease S1 analysis. The physical maps of HPV54 and 55 were aligned with the genetic maps of HPV16 and 11, respectively, by heteroduplex mapping and partial DNA sequencing. HPV54 is thus only weakly related to the known HPVs, while HPV55 represents an additional HPV6-related HPV type. HPV54 and HPV55 are uncommon genital HPV types since, in a survey of a large series of specimens of benign, pre-malignant or malignant anogenital and orolaryngeal tumours, HPV54 was not detected, and HPV55 was found in another case of condyloma acuminatum.

The analysis of heteroduplexes between EcoRI digested DNA derived from IncM plasmids in the transfer-off and in the transfer-on state revealed an internal loop of about 1 kbp on a distinct large EcoRI fragment. The presence of an invertible element (Min) is suggested which enables the formation of conjugative pili at 30 degrees C, but switches off the pilus formation at 37 degrees C incubation temperature.

Eleven pro-1 homologous clones from human nasopharyngeal carcinoma cell line CNE2 were studied with respect to their ability to transfer sensitivity to tumor promoter-induced neoplastic transformation (P+ activity), their molecular structure, and their homology to both mouse pro-1 and each other. Restriction mapping and Southern analysis of CNE2 pro-1 homologous clones revealed three structural classes, all of which showed P+ activity. The approximate limits of mouse pro-1 hybridizing sequences within CNE2 clones from each structural class were identified, and some of these minimum homologous sequences were tested for P+ activity by transfection into P- cells. For all classes, a strong association between P+ activity and pro-1 homology was observed. This finding implies that, for CNE2 clones to be P+-active, structural homology to mouse pro-1 is required. The minimum P+-active sequence thus far identified was a 2.6-kbp EcoRI-SstI fragment. One clone was inactive, even though it was indistinguishable from active clones of the same class by restriction mapping, Southern analysis, and electron microscope examination of heteroduplexes formed by an active and an inactive clone. This raises the possibility that discrete changes, involving a few nucleotides rather than gross rearrangement, may determine the P+ activation of these pro-1-homologous sequences.

Natural recombinant genomes between several, phenotypically distinct forms of phages kappa and theta were isolated and characterized by DNA restriction fragment mapping and electron microscopic heteroduplex analysis. The phenotypes of the recombinants were correlated with the physical maps of the genomes, and several genetic functions were therefore defined and mapped. All genes necessary for the assembly of infectious virus particles map in a contiguous tract of DNA comprising about 20 kb, or nearly one third of the genome length. No DNA homology occurs within these domains of the two genomes, so that homologous recombination does not take place here and phenotypic mixing of the phages is eo ipso excluded. Other regions of heterology contain regulatory genes responsible for the lytic or temperature character of the phages, and for exclusion of phage kappa by 9.

The genes encoding procyclin, the major glycoprotein expressed on the surface of procyclic forms of Trypanosoma brucei, comprise a multigene family. It has previously been demonstrated that procyclin genes in cloned trypanosome strains from Kenya and Uganda show restriction fragment polymorphisms. A detailed study of the Kenyan strain 227 has revealed that procyclin genes are arranged in tandem at 3 distinct loci (Pro A, B and C) and that the polymorphism is due to the duplication of 1.3 kb in the Pro A locus, which has generated an additional procyclin gene. Northern blot analysis has shown that at least 2 loci are transcribed and that a minimum of 3 procyclin genes are expressed within a cloned line. The transcription of procyclin genes is resistant to 1 mg ml-1 alpha-amanitin, whereas that of the 5' flanking gene in the Pro A locus is sensitive. This observation suggests that the two genes form part of separate transcription units with a promoter between them.

Heteroduplexes that contain single-stranded loops are repaired very efficiently in mammalian cells. The strand that does not contain the loop is used as the template strand for repair nearly twice as often as the looped strand. In this study we tested the influence of nearby nicks on the choice of template strand. We find that strand selection in repair of heteroduplexes with single-stranded loops is influenced by the presence of a nick located 71 or 125 base pairs from the loop, but only to a minor degree. Thus the loop itself is a stronger signal for repair than is a nearby nick. On the other hand, if a break is introduced into the single strand that forms the loop, the looped strand is marked for excision and rarely, if ever, is used as the template for repair.

Previously isolated human DNA clones containing asparagine transfer RNA (tRNAAsn) genes have been used to determine the genomic organization of this multigene family in man. One clone also contained a gene for U1 RNA, and so the organization of the two multigene families could be directly compared. The majority, and perhaps all, of the human tRNAAsn genes map to the same chromosome bands as do the U1 RNA true genes and class I pseudogenes located on the short and long arms, respectively, of chromosome 1. These two gene clusters were independently isolated using a somatic-cell hybrid minipanel, and use of repeat-unit DNA polymorphisms showed that one tRNA gene clone maps to the short-arm gene cluster and the other to the long-arm gene cluster. Electron microscopy of heteroduplexes between these two clones showed duplex formation along the proposed region of overlap between them, indicating that the short- and long-arm gene clusters are structurally related. I suggest that the split into two distinct loci was facilitated by a pericentric chromosome inversion. This would have had the effect of positioning the genes currently on the long arm adjacent to the centromeric heterochromatin, perhaps resulting in a "position effect" on transcription of these genes. Restriction fragments of different sizes were found that were common to a majority of repeat units, depending on the restriction enzyme being used. Pulsed-field electrophoresis revealed that fragments of molecular weight of 180 kb were common to each unit (or multiples of units). These fragments also contained U1 RNA gene sequences. I therefore propose that these two gene families are closely linked on repeat units (or multiples of units) of 180 kb in size, which are probably organized in tandem arrays.