Apurinic/apyrimidinic endonucleases in repair of pyrimidine dimers and other lesions in DNA.

The characteristics of the nicks (single-strand breaks) introduced into damaged DNA by Escherichia coli endonucleases III, IV, and VI and by phage T4 UV endonuclease have been investigated with E. coli DNA polymerase I (DNA nucleotidyltransferase). Nicks introduced into depurinated DNA by endonuclease IV or VI provide good primer termini for the polymerase, whereas nicks introduced into depurinated DNA by endonuclease III or into irradiated DNA by T4 UV endonuclease do not. This result suggests that endonuclease IV nicks depurinated DNA on the 5' side of the apurinic site, as does endonuclease VI, whereas endonuclease III has a different incision mechanism. T4 UV endonuclease also possesses apurinic endonuclease activity that generates nicks in depurinated DNA with low priming activity for the polymerase. The priming activity of DNA nicked with endonuclease III or T4 UV endonuclease can be enhanced by an additional incubation with endonuclease VI and, to a lesser extent, by incubation with endonuclease IV. These results indicate that endonuclease III and T4 UV endonuclease (acting upon depurinated and irradiated DNA, respectively) generate nicks containing apurinic/apyrimidinic sites at their 3' termini and that such sites are not rapidly excised by the 3' leads to 5' activity of DNA polymerase I. However, endonuclease IV or VI apparently can remove such terminal apurinic/apyrimidinic sites as well as cleave on the 5' side of the unnicked sites. These results suggest roles for endonucleases III, IV, and VI in the repair of apurinic/apyrimidinic sites as well as pyrimidine dimer sites in DNA. Our results with T4 UV endonuclease suggest that the incision of irradiated DNA by T4 UV endonuclease involves both cleavage of the glycosylic bond at the 5' half of the pyrimidine dimer and cleavage of the phosphodiester bond originally linking the two nucleotides of the dimer. They also imply that the glycosylic bond is cleaved before the phosphodiester bond.     

X-ray structure of a dinucleoside monophosphate A2''p5''C that contains a 2''-5'' link found in (2''-5'')oligo(A)s induced by interferons: single-stranded helical conformation of 2''-5''-linked oligonucleotides.

In order to understand why DNA and RNA have the 3'-5' and not the 2'-5' link and to delineate the stereochemistry of the 2'-5' phosphodiester links, we crystallized and carried out a very accurate x-ray diffraction analysis of A2 p5'C, an analog of A2' p5'A. Contrary to numerous reports in the literature that conclude that the tendency for 2'-5' nucleotides to stack intramolecularly is stronger than for 3'-5' counterparts, we find hardly any intramolecular base stacking for this molecule but find an intramolecular "stacking" of the ribose oxygen-4' of cytidine on top of the adenine ring. Although A2' p5'C shows the standard conformational features usually found for 3'-5' nucleotides, the overall stereochemistry of 2'-5' nucleotides is quite different because the 2' link orients the backbone inwards to the bases unlike the 3' and 5' links that orient it away from the bases. With the conformational features found for A2' p5'C, it is possible to build a very compact right-handed single-stranded helix but not a double helix. Such a preference for single-stranded helices may be the reason for the absence of 2'-5' bonds in DNA and RNA even though the 2'-5' bonds are formed more readily then 3'-5' bonds.     

Human mitochondrial DNA: analysis of 7S DNA from the origin of replication.

Heat-treated samples of human mitochondrial DNA (mtDNA) exhibited a set of three low molecular weight DNA bands in addition to the major mtDNA band when electrophoresed in polyacrylamide gels. These DNA components were seen only after heat treatment or after relaxation of the mtDNA with a restriction endonuclease. The three components were single stranded and had sizes of 550, 585, and 629 nucleotides, close to the size (600 nucleotides) estimated from contour length measurements for the 7S DNA from the D loop of human mtDNA. Hybridization of the components with restriction endonuclease fragments of known position in the mtDNA confirmed this identification. Digestion of each 7S DNA component with the restriction endonuclease Hae III produced three fragments, two of which were identical in size among the components and the third of which varied. This third fragment, shown to be from the 5' end of each component, differed in length by approximately 35 nucleotides among the components. These results suggest that human 7S mtDNA synthesis is terminated at a distinct position and that it is either initiated at one of three possible sites in the same mtDNA or that the mtDNA population consists of three subpopulations, each differing from the others by the presence or absence of a nucleotide sequence immediately adjacent to the origin of replication.     

Physical linkage of the constant region genes for immunoglobulins lambda I and lambda III.

During differentiation from a stem cell to an antibody-secreting cell, the immunoglobulin genes within a B cell undergo a rearrangement that juxtaposes a variable region gene to a constant region gene. To analyze the genetic organization of an immunoglobulin gene family in nonrearranged, germ-line DNA, we have constructed a recombinant DNA library from randomly cleaved mouse kidney DNA fragments. From this library, we have isolated three overlapping recombinant clones containing the constant region gene for lambda I light chains (C lambda I). These clones spanned 24.9 kilobases of mouse DNA and contained no variable region sequences. Hybridization of these clones with lambda II cDNA demonstrated the presence of an additional constant region gene and a joining region 3.2 kilobases 5' of C lambda I. This gene was tentatively identified as C lambda III by the absence of an Ava I endonuclease site, which is present within C lambda II. The C lambda III amino acid sequence has recently been reported [Azuma, T., Steiner, L. A. & Eisen, H. N. (1981) Proc. Natl. Acad. Sci. USA 78, 569-573] and is very closely related to the C lambda II amino acid sequence.     

Abelson murine leukemia virus: molecular cloning of infectious integrated proviral DNA.

The integrated proviral genome of Abelson murine leukemia virus (A-MuLV) was cloned in lambda gtWES . lambda B bacteriophage after EcoRI endonuclease digestion and enrichment of proviral sequences by sequential RPC-5 column chromatography and agarose gel electrophoresis. Recombinant DNA clones containing a 7.8-kilobase-pair EcoRI insert were shown to have the entire integrated A-MuLV genome with both 5' and 3' ends flanked by mink cellular DNA sequences. This DNA fragment was shown to induce focus transformation upon transfection of NIH/3T3 mouse cells. Moreover, focus-forming virus could be rescued from transformed nonproducer cells upon superinfection with a type C helper virus. A polyprotein of molecular weight 120,000 (p120) containing murine leukemia virus gag gene determinants was invariably deteced by immunoprecipitation analysis of individual transformants induced by the 7.8-kilobase-pair DNA. Molecularly cloned integrated A-MuLV in its infectious form should be of use in elucidating the mechanisms involved in transformation by this virus.     

Recognition of local nucleotide conformation in contrast to sequence by a rRNA processing endonuclease.

RNase M5 of Bacillus subtilis cleaves twice in a double-helical region of a 179-nucleotide precursor of 5S rRNA to yield mature 5S rRNA (116 nucleotides) plus fragments (21 and 42 nucleotides) derived from both termini. Previous experiments had shown that the major recognition elements for the highly specific RNase M5 are in the mature domain of the precursor. However, one precursor residue, a G adjacent to the 5' cleavage site, significantly enhances the rate of its own cleavage as well as that of the 3' precursor fragment, so it must be an important component of the features recognized by the enzyme. This G residue is opposed in the helical substrate region to a C residue, which is at the 3' terminus of the mature domain, presenting the question of whether RNase M5 specifically contacts the cleavage site on the basis of nucleotide sequence (the G residue per se) or on the basis of more general aspects of helical conformation. We tested these alternatives by fabricating partially synthetic test substrates for RNase M5. Experiments were performed on 5' and 3' half-molecules derived from mature 5S rRNA. The 3'-terminal C was removed by periodate oxidation and beta elimination and replaced in a T4 RNA ligase condensation with each of the four mononucleoside bisphosphates. Artificial "precursor" segments containing each of the four nucleotides adjacent to the 5' cleavage site were added to the 5' terminus of the 5S rRNA half-molecule. We then annealed the modified half-molecules to yield test substrates containing all permutations of complementary in contrast to noncomplementary nucleotides at the cleavage site. The susceptibilities of these test substrates show that conformation, not sequence, is the important feature in the locale of the cleaved bonds.     

Related homing endonucleases I-BmoI and I-TevI use different strategies to cleave homologous recognition sites

A typical homing endonuclease initiates mobility of its group I intron by recognizing DNA both upstream and downstream of the intron insertion site of intronless alleles, preventing the endonuclease from binding and cleaving its own intron-containing allele. Here, we describe a GIY-YIG family homing endonuclease, I-BmoI, that possesses an unusual recognition sequence, encompassing 1 base pair upstream but 38 base pairs downstream of the intron insertion site. I-BmoI binds intron-containing and intronless substrates with equal affinity but can nevertheless discriminate between the two for cleavage. I-BmoI is encoded by a group I intron that interrupts the thymidylate synthase (TS) gene (thyA) of Bacillus mojavensis s87-18. This intron resembles one inserted 21 nucleotides further downstream in a homologous TS gene (td) of Escherichia coli phage T4. I-TevI, the T4 td intron-encoded GIY-YIG endonuclease, is very similar to I-BmoI, but each endonuclease gene is inserted within a different position of its respective intron. Remarkably, I-TevI and I-BmoI bind a homologous stretch of TS-encoding DNA and cleave their intronless substrates in very similar positions. Our results suggest that each endonuclease has independently evolved the ability to distinguish intron-containing from intronless alleles while maintaining the same conserved recognition sequence centered on DNA-encoding active site residues of TS.     

Inverted repeated DNA from Chinese hamster ovary cells studied with cloned DNA fragments.

Fragments from the DNA of Chinese hamster ovary cells produced by restriction endonuclease EcoRI were cloned in Charon 16A lambda bacteriophage and examined for the ability to hybridize in situ with 32P-labeled double-stranded regions from heterogeneous nuclear RNA (hnRNA). Of 235 clones tested, 87 (37%) contained sequences that hybridized with the double-stranded hnRNA. Nine of these were examined for the presence of inverted repeat DNA structures (ir-DNA) by electron microscopy. All nine contained at least two elements of ir-DNA. Analysis of heteroduplexes formed from the DNAs of the different clones as well as T1 fingerprint analysis of the double-stranded hnRNA hybridized to each of the nine clones suggest that there is detectable nucleotide sequence homology in the various ir-DNAs. There are ca 3 X 10(5) ir-DNA pairs in the haploid Chinese hamster ovary cell genome.     

Nucleosome phasing and micrococcal nuclease cleavage of African green monkey component alpha DNA.

The micrococcal nuclease cleavage of intact nuclear chromatin from African green monkey cells and of the completely deproteinized sequences was studied by using high-resolution analytical and DNA sequencing gels and secondary restriction enzyme analysis. When deproteinized component alpha DNA was used as substrate, not all phosphodiester bonds in the 172-base-pair repeat units were cleaved with equal frequency by the nuclease. A distinct preference for the cleavage of A-T rather than G-C bonds was observed; however, A + T-richness in itself did not confer susceptibility to cleavage by micrococcal nuclease. The results suggested that, in deproteinized DNA, nuclease cleavage at particular dinucleotides may be influenced more by the effect of adjacent sequences than by the composition of the dinucleotide. In contrast to complex cleavage patterns of the deproteinized component alpha DNA which arose because of multiple cleavage sites in the repeat unit, micrococcal nuclease cleaved component alpha nuclear chromatin at one site per nucleosome repeat, near position 126 in the nucleotide sequence. This simple chromatin cleavage pattern is consistent with the discrete nucleosomal structure of component alpha in chromatin and a direct phase relationship between the component alpha DNA sequence repeats and the nucleosome protein structural repeats.     

Structure of genes for human growth hormone and chorionic somatomammotropin.

A 2.6-kilobase (kb) EcoRI restriction endonuclease fragment containing human growth hormone (hGH; somatotropin) gene sequences and a 2.8-kb EcoRI fragment containing human chorionic somatomammotropin (hCS; choriomammotropin) gene sequences have been identified by hybridization to cloned cDNA. Human DNA was cleaved with EcoRI and fractionated by preparative agarose gel electrophoresis; DNA in the size range 2--3 kb was ligated to lambda gt WES.lambda B DNA and viable recombinant bacteriophage were recovered by in vitro packaging. After infection of Escherichia coli and screening of phage plaques, single isolates of hGH and hCS gene sequences were obtained. Restriction endonuclease mapping showed that the hGH gene contains three intervening sequences interrupting the coding sequence. Partial DNA sequence analysis of the hGH gene, obtained by the chain termination method, confirmed the location of the intervening sequences and the identity of the fragment.     

Mitochondrial DNA of chloramphenicol-resistant mouse cells contains a single nucleotide change in the region encoding the 3' end of the large ribosomal RNA.

The complete DNA sequence of the rRNA genes of mouse L cell mtDNA provides a basis for the examination of the nucleotide sequence of this region in a mutant mouse cell line that is resistant to chloramphenicol, a known inhibitor of mitochondrial protein synthesis. Resistance to chloramphenicol (CAPr) is conferred by a cytoplasmic determinant that is linked to mtDNA restriction endonuclease site polymorphisms. We have determined the sequence of a 212-nucleotide region of mtDNA from a CAPr mouse cell line that encodes a portion of the 1582-nucleotide large rRNA. This sequence is located 107-318 nucleotides from the 5' end of the heavy strand coding sequence, which corresponds to the 3' end of the rRNA. There is a single nucleotide difference in the large rRNA gene from CAPr cells, an A-to-G transition 243 nucleotides from the 5' end of the coding sequence. This single transition is located within a region of 10 nucleotides tht is otherwise completely homologous to human and yeast mitochondrial large rRNAs and Escherichia coli 23S rRNA and is positioned immediately adjacent to a single nucleotide transversion known to occur in a yeast CAPr mutant. This characterization of a mammalian mitochondrial mutant at the nucleotide level directly demonstrates that a mutant phenotype may result from a single mtDNA nucleotide change in an animal cell.     

Identification and molecular cloning of Moloney mouse sarcoma virus-specific sequences from uninfected mouse cells.

When uninfected mouse cell DNA is cleaved with restriction endonuclease EcoRI, a DNA fragment of 14.0 kilobases can be identified by hybridization to cloned DNA containing sarcoma specific sequences of Moloney mouse sarcoma virus (M-MSVsrc). The cellular DNA fragment contains the entire M-MSVsrc specific sequences. The 14.0-kilobase EcoRI DNA fragment was cloned in bacteriophage lambda. The sequence organization of a recombinant clone, lambda . MTX-1, was analyzed by restriction endonuclease mapping, nuclease S1 mapping, and electron microscopy. The results indicate that lambda . MTX-1 contains an uninterrupted stretch of 1.0 kilobase similar to that found in the M-MSV genome.     

Variable and constant parts of the immunoglobulin light chain gene of a mouse myeloma cell are 1250 nontranslated bases apart.

A DNA fragment carrying an immunoglobulin gene coding for both variable (V) and constant (C) regions of a mouse lambda light chain was enriched about 15-fold from a total endonuclease EcoRI digest of a plasmacytoma (HOPC 2020) DNA by preparative agarose gel electrophoresis. The DNA fraction was used for cloning of a lambda chain gene in the phage lambdagtWES vector. After screening [Benton, W. D. & Davis, R. W. (1977) Science 196, 180-182] of about 70,000 plaques, each arising from an independent transfection event, we isolated one clone (Ig 303) that contained both a Vlambda and a Clambda DNA sequence. Electron microscopy of R-loops formed between the cloned DNA and purified lambda chain mRNA (HOPC 2020) revealed that the Vlambda and Clambda DNA sequences are separated by a 1250-base DNA fragment.     

Relaxase (TraI) of IncP alpha plasmid RP4 catalyzes a site-specific cleaving-joining reaction of single-stranded DNA.

Conjugative DNA transfer of the self-transmissible broad-host-range plasmid RP4 is initiated by strand- and site-specific cleavage at the nick site (nic) of the transfer origin (oriT). Cleavage results in covalent attachment of the plasmid-encoded relaxase (TraI) to the 5'-terminal 2'-deoxycytidine residue at nic. We demonstrate that Tyr22 is the center of the catalytic site of TraI, mediating cleavage via formation of a phosphodiester between the DNA 5' phosphoryl and the aromatic hydroxyl group. The specificity of cleavage seen with form I oriT DNA was verified with short oligodeoxy-ribonucleotides embracing the nick region. The reaction requires TraI and Mg2+ but is independent of the relaxosome component TraJ. Cleavage produces one oligonucleotide fragment with a free 3' hydroxyl, the other part forms a covalent TraI-oligonucleotide adduct. Like nicking of form I oriT DNA, TraI-catalyzed oligonucleotide cleavage reaches an equilibrium when about 30% of the input TraI exists as a covalent protein-DNA complex. In the presence of two differently sized oligonucleotides, defined hybrid oligonucleotides are produced, demonstrating that TraI catalyzes recombination of two single strands at nic. This finding shows that TraI possesses cleaving-joining activity resembling that of a type I topoisomerase. Reactions are dependent on the sequence of the 3'-terminal 6 nucleotides adjacent to nic. Only certain base changes in a few positions are tolerated, whereas the sequence of the 5' terminal nucleotides apparently is irrelevant for recognition by TraI. The reactions described here further support the hypothesis that DNA transfer via conjugation involves a rolling circle-like mechanism which generates the immigrant single strand while DNA-bound TraI protein scans for the occurrence of a second cleavage site at the donor-recipient interface.     

Molecular cloning of the flavin-containing monooxygenase (form II) cDNA from adult human liver.

Complementary DNA (cDNA) clones encoding the adult human liver flavin-containing monooxygenase (FMO; dimethylaniline N-oxidase, EC 1.14.13.8) were isolated from lambda gt10 and lambda gt11 libraries. The cDNA libraries were screened with three synthetic 36-mer oligonucleotide probes derived from the nucleic acid sequence of the pig liver FMO cDNA. The deduced amino acid sequence for the adult human liver FMO was quite distinct from the pig liver FMO, and adult human liver FMO was designated form II (HLFMO II). The full-length cDNA sequence of HLFMO II [2119 base pairs (bp)] had an open reading frame of 1599 nucleotides, which encoded a 533-amino acid protein of Mr 59,179, a 5'-noncoding region of 136 nucleotides and a 3'-noncoding region of 369 nucleotides excluding the poly(A) tail. The deduced amino acid sequence of HLFMO II had 80% similarity with the rabbit liver FMO II but only a 52%, 55%, and 53% amino acid similarity with the rabbit liver (form I), the pig liver (form I), and fetal human liver (form I) FMOs, respectively. RNA analysis of adult human liver RNA showed that there was one HLFMO II mRNA species. Analysis of genomic DNA indicated that HLFMO II was the product of a single gene. These results indicated that the deduced amino acid sequence for HLFMO II contained highly conserved residues and suggested that FMO enzymes were closely related and, undoubtedly, derived from the same ancestral gene.     

Initiation sites for discontinuous DNA synthesis of bacteriophage T7

We have previously shown that the discontinuous replication of bacteriophage T7 DNA is primed by tetraribonucleotides (major component) or pentaribonucleotides. Both tetramers and pentamers start with pppA-C and are rich in A and C at the third and fourth nucleotides. In this study, the sites of transition from primer RNA to DNA in vivo have been located on a 340-nucleotide segment of the H strand of the T7 genome by 32P-labeling in vitro of the 5'-hydroxyl ends of DNA resulting from alkaline hydrolysis of RNA-linked T7 DNA fragments. Five strong transition sites were detected with a common sequence 5'-G-A-C-N1-N2-N3-N4-3', in which N1 was either C or A, N2 ws A, C, or G, and either N3 or N4 was the nucleotide for the switchover to DNA synthesis. We conclude that the complementary sequence 3'-C-T-G-G/T-N'2-(N'3)-5' in the template strand is the most frequently used signal for synthesis of primer RNA. Whereas primer-RNA synthesis starts at a precisely defined nucleotide, the transition to DNA synthesis varies within two nucleotides. Because the observed signal sequence would be present on a statistical basis once per 128 nucleotides, only about 10% of the existing signals are used for primer synthesis in each round of replication so that nascent fragments 1000-2000 long result. This provides an unexpected flexibility for RNA priming of DNA synthesis.