Human nucleotide excision nuclease removes thymine dimers from DNA by incising the 22nd phosphodiester bond 5'' and the 6th phosphodiester bond 3'' to the photodimer.

By using a human cell-free system capable of nucleotide excision repair, a synthetic substrate consisting of a plasmid containing four thymidine dimers at unique locations, and deoxyribonucleoside 5'-[alpha-thio]triphosphates for repair synthesis, we obtained DNA fragments containing repair patches with phosphorothioate linkages. Based on the resistance of these linkages to digestion by exonuclease III and their sensitivity to cleavage by I2, we were able to delineate the borders of the repair patch to single-nucleotide resolution and found an asymmetric patch with sharp boundaries. That the repair patch was produced by filling in a gap generated by an excision nuclease and not by nick-translation was confirmed by the finding that the thymidine dimer was released in a 27- to 29-nucleotide oligomer.     

Structural origins of the exonuclease resistance of a zwitterionic RNA

Nuclease resistance and RNA affinity are key criteria in the search for optimal antisense nucleic acid modifications, but the origins of the various levels of resistance to nuclease degradation conferred by chemical modification of DNA and RNA are currently not understood. The 2'-O-aminopropyl (AP)-RNA modification displays the highest nuclease resistance among all phosphodiester-based analogues and its RNA binding affinity surpasses that of phosphorothioate DNA by 1 degrees C per modified residue. We found that oligodeoxynucleotides containing AP-RNA residues at their 3' ends competitively inhibit the degradation of single-stranded DNA by the Escherichia coli Klenow fragment (KF) 3'-5' exonuclease and snake venom phosphodiesterase. To shed light on the origins of nuclease resistance brought about by the AP modification, we determined the crystal structure of an A-form DNA duplex with AP-RNA modifications at 1.6-A resolution. In addition, the crystal structures of complexes between short DNA fragments carrying AP-RNA modifications and wild-type KF were determined at resolutions between 2.2 and 3.0 A and compared with the structure of the complex between oligo(dT) and the D355A/E357A KF mutant. The structural models suggest that interference of the positively charged 2'-O-substituent with the metal ion binding site B of the exonuclease allows AP-RNA to effectively slow down degradation.     

A general method for maximizing the expression of a cloned gene.

We present a method, utilizing a combination of restriction endonuclease cleavage and digestion with Escherichia coli exonuclease III and Aspergillus orizae nuclease S1, that allows us to position a restriction fragment bearing the promoter of the lacZ gene of E. coli at virtually any distance in front of any cloned gene. In particular, we have used this method to examine the effect on protein production of gene-promoter separation for the cro gene of phage lambda and to produce plasmids that, upon transformation into appropriate E. coli hosts, direct the synthesis of up to 190,000 cro protein monomers per cell.     

Polarity of heteroduplex formation promoted by Escherichia coli recA protein.

When recA protein pairs circular single strands with linear duplex DNA, the circular strand displaces its homolog from only one end of the duplex molecule and rapidly creates heteroduplex joints that are thousands of base pairs long [DasGupta, C., Shibata, T., Cunningham, R. P. & Radding, C. M. (1980) Cell 22, 437-446]. To examine this apparently polar reaction, we prepared chimeric duplex fragments of DNA that had M13 nucleotide sequences at one end and G4 sequences at the other. Circular single strands homologous to M13 DNA paired with a chimeric fragment when M13 sequences were located at the 3' end of the complementary strand but did not pair when the M13 sequences were located at the 5' end. Likewise circular single-stranded G4 DNA paired with chimeric fragments only when G4 sequences were located at the 3' end of the complementary strand. To confirm these observations, we prepared fd DNA labeled only at the 5' or 3' end of the plus strand, and we examined the susceptibility of these labeled ends to digestion by exonucleases when joint molecules were formed. Eighty percent of the 5' label in joint molecules became sensitive to exonuclease VII. Displacement of that 5' end by recA protein was concerted because it did not occur in the absence of single-stranded DNA or in the presence of heterologous single strands. By contrast, only a small fraction of the 3' label became sensitive to exonuclease VII or exonuclease I. These observations show that recA protein forms heteroduplex joints in a concerted and polarized way.     

Uptake of homologous single-stranded fragments by superhelical DNA: a possible mechanism for initiation of genetic recombination.

Superhelical [3-H]DNA (replicative form I, RFI) of bacteriophage phiX174 slowly but spontaneously took up 32-P-labeled homologous single-stranded fragments at 4 degrees. Uptake was accelerated by heating to 75 degrees. RFI did not take up single-stranded fragments derived from DNA of Escherichia coli or from separated strands of phage lambda. Uptake was inhibited by low concentrations of ethidium bromide. Relaxed circular phiX174 DNA did not take up homologous fragments. Per molecule of RFI, the complexes contained as much as 90 nucleotide residues of homologous fragment. The 32-P-lebeled fragments were largely resistant to digestion by exonuclease I, and were not displaced by heating complexes at 60 degrees for 1 min in 16 mM or 100 mM NaCl. Under comparable conditions of temperature and salt all of the fragments were displaced from complexes in which at least one phosphodiester bond was cleaved by pancreatic DNase, but a significant fraction of the fragments was retained in complexes that were relaxed by digestion with S1 nuclease. These observations are interpreted to mean that S1 nuclease digested the plus (viral) strand of the recipient RF at the site of uptake in some instances. Transfection of E. coli by heterozygous complexes produced recombinant progeny, thereby showing that genetic information can be transferred from the fragment of plus strand to progeny plus strands. We propose that both uptake of a third strand by superhelical DNA and the action of nucleases on the resulting complex may simulate early steps in genetic recombination.     

Exonuclease III and endonuclease IV remove 3'' blocks from DNA synthesis primers in H2O2-damaged Escherichia coli.

Escherichia coli deficient in exonuclease III (xth gene mutants) are known to be hypersensitive to hydrogen peroxide. We now show that such mutants accumulate many more DNA single-strand breaks than do wild-type bacteria upon exposure to H2O2. DNA isolated from H2O2-treated xth- cells contains strand breaks that do not efficiently support synthesis by E. coli DNA polymerase I, indicating the presence of blocking groups at the DNA 3' termini. Purified E. coli exonuclease III activates this blocked DNA to allow substantial synthesis by polymerase I in vitro. Another E. coli enzyme, endonuclease IV, also activates primers for DNA polymerase. Exonuclease III accounts for greater than 95% of the total activity in E. coli crude extracts for removal of 3'-terminal phosphoglycolaldehyde esters from model DNA substrates. Purified exonuclease III and endonuclease IV can each efficiently remove 3'-terminal phosphoglycolaldehyde in vitro. An important physiological function for exonuclease III is thus the activation of blocked 3' ends for DNA repair synthesis. Endonuclease IV can also initiate the repair of ruptured 3'-deoxyribose in DNA.     

The Length of the Terminal Repetition in Adenovirus-2 DNA

Adenovirus-2 DNA was end-labeled by partial digestion with Escherichia coli exonuclease III and resynthesis with the DNA polymerase from avian myeloblastosis virus and α-³²P-labeled deoxyribonucleoside triphosphates. This end-labeled DNA was cleaved with several specific endonucleases and the terminal fragments were characterized by gel electrophoresis and pyrimidine tract analysis. Two endonucleases gave identical fragments from both ends, presumably from cleavage within the inverted terminal repetition, while all other endonucleases gave dissimilar fragments from the two ends. From the sizes of these fragments it is estimated that the inverted terminal repetition is between 100 and 140 nucleotide pairs long.     

Adenovirus-2 DNA Contains an Inverted Terminal Repetition

Denaturation and renaturation of the adenovirus-2 chromosome (a duplex rod) generates single-stranded circles of unit length. These circles can be opened into linear DNA molecules by digestion with exonuclease III, indicating that hydrogen bonding between the two ends of an adenovirus strand is responsible for maintaining the rod in a circular state.The formation of adenovirus single-stranded circles, and their sensitivity to exonuclease III, indicate that the mature adenovirus-2 DNA molecule contains an inverted terminal repetition. That is, the base sequence at one end of the molecule is inverted and appears again at the other end of the molecule. This is the first example of such a structure, and its function is unknown.     

Spliced adenovirus-associated virus RNA.

We describe the structure of cytoplasmic RNA species transcribed from the DNA of adenovirus-associated virus, a defective parvovirus. The RNA was hybridized with minus strand template DNA and visualized in the electron microscope. Alternatively, the DNA.RNA duplex molecules were digested with nuclease S1 or Escherichia coli exonuclease VII and analyzed by agarose gel electrophoresis. A set of RNA species was observed with 5' terminal at map positions 5, 13, 19, or 39 and a 3' terminus and poly(A) tail at position 96 (one map unit is equivalent to 1% of genome length). Most of these RNAs are spliced and lack sequences approximately between positions 40 and 49. Some RNA preparations also contained unspliced molecules with 5' and 3' terminal at positions similar to those in the spliced RNA.     

Strand specificity of DNA unwinding by RecBCD enzyme.

RecBCD enzyme (exonuclease V) of Escherichia coli unwinds DNA, frequently forming asymmetric structures with two single-stranded tails of unequal length abutting a single-stranded loop at the junction with double-stranded DNA. Their lengths are consistent with the longer tail being one strand of the duplex and the loop plus the shorter tail being the other strand. The strand polarity of the unwinding was determined by labeling the 3' or 5' ends of duplex DNA with biotinylated nucleotides, reacting the DNA with RecBCD enzyme, and distinguishing the labeled ends, in the electron microscope, by their binding to streptavidin-gold complex. The shorter tail was formed from the DNA strand with its 3' terminus at the duplex end where RecBCD enzyme entered. We conclude that RecBCD enzyme unwinds DNA by forming a loop on the strand with a 3' end at the entry point. This result is concordant with a previously proposed model of recombination, which we discuss.     

MECHANISM OF DNA CHAIN GROWTH, IV. DIRECTION OF SYNTHESIS OF T4 SHORT DNA CHAINS AS REVEALED BY EXONUCLEOLYTIC DEGRADATION

T4 nascent short chains labeled at their growing ends with H(3)-thymidine and uniformly with C(14)-thymidine were prepared, separated into complementary strands, and degraded by E. coli exonuclease I in the 3' to 5' direction or by B. subtilis nuclease in the 5' to 3' direction. The kinetics of release of H(3) and C(14) labels by both enzymes was consistent with the conclusion that the H(3) label is at the 3' end of the nascent short chains of both strands and that the short chains are products of discontinuous synthesis in the 5' to 3' direction along the two template strands.     

Enhanced resolution of DNA restriction fragments: a procedure by two-dimensional electrophoresis and double-labeling.

A probe-free method was developed to detect DNA rearrangement in bacteria based on the electrophoretic separation of twice-digested restriction fragments of genomic DNA into a two-dimensional (2-D) pattern. The first restriction enzyme digestion was done in solution, followed by electrophoresis of the restriction fragments in one dimension. A second restriction enzyme digestion was carried out in situ in the gel, followed by electrophoresis in a second dimension perpendicular to the first electrophoresis. The 2-D pattern provides for the resolution of 300-400 spots, which are defined and indexed by an "x,y" coordinate system with size markers. This approach has greatly increased the resolution power over conventional one-dimensional (1-D) electrophoresis. To study DNA rearrangement, a 2-D pattern from a test strain was compared with the 2-D pattern from a reference strain. After the first digestion, genomic DNA fragments from the test strain were labeled with 35S, while those from the reference strain were labeled with 35P. This was done to utilize the difference in the energy emission of 35S and 32P isotopes for autoradiography when two x-ray films were exposed simultaneously on top of the gel after the 2-D electrophoresis. The irradiation from the decay of 35S exposed only the lower film, whereas the irradiation from the decay of 32P exposed both the lower and upper films. Different DNA fragments existed in the test DNA compared with the reference DNA can be identified unambiguously by the differential two 2-D patterns produced on two films upon exposure to the 35S and 32P fragments in the same gel. An appropriate photographic procedure further simplified the process, allowing only the difference in DNA fragments between these two patterns to be shown in the map. We have utilized the difference map obtained from Escherichia coli strains HB101 and HB101 (lambda) genomic DNA to show the incorporation of one copy of phage lambda DNA without the use of a lambda DNA probe. This is the same test system that was used previously.     

Mechanism of Action of Ribonuclease H Isolated from Avian Myeloblastosis Virus and Escherichia coli

Purified preparations of RNA-dependent DNA polymerase isolated from avain myeloblastosis virus contain RNase H activity. Labeled ribohomopolymers are degraded in the presence of their complementary deoxyribopolymer, except [(3)H]poly(U).poly(dA). The degradation products formed from [(3)H]poly(A).poly(dT) were identified as oligonucleotides containing 3'-hydroxyl and 5'-phosphate termini, while AMP was not detected. The nuclease has been characterized as a processive exonuclease that requires ends of poly(A) chains for activity. Exonucleolytic attack occurs in both 5' to 3' and 3' to 5' directions.RNase H has also been purified from E. coli. This nuclease degrades all homoribopolymers tested in the presence of their complementary deoxyribopolymers to yield oligonucleotides with 5'-phosphate and 3'-hydroxyl termini. E. coli RNase H has been characterized as an endonuclease.     

Stepwise biosynthesis in vitro of globin genes from globin mRNA by DNA polymerase of avian myeloblastosis virus.

Two approaches have been explored for the synthesis of double-stranded DNA from single-stranded DNA template complementary to rabbit 9S globin mRNA (cDNA). (i) cDNA was elongated with dCMP or dTMP homopolymeric tracts using terminal deoxynucleotidyltransferase (EC 2.7.7.31; nucleosidetriphosphate:DNA deoxynucleotidylexotransferase). cDNA-dC, in the presence of an oligo(dG)10 primer, was an efficient template with either DNA polymerase of Escherichia coli (EC 2.7.7.7; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase) or RNA-directed DNA polymerase of avian myeloblastosis virus. cDNA-dT [ with an oligo(dA)10 primer] functioned as template only with E. coli polymerase. (ii) cDNA, without homopolymeric tails, was also efficiently copied in the absence of oligonucleotide primer, by DNA polymerase of avian myeloblastosis virus or of E. coli. The product of the reaction consisted of long hairpin molecules which could be converted into DNA duplex (melting temperature, 93 degrees) by digestion with single-strand nuclease S1. The data indicate that a loop structure on the 3' end of cDNA allowed DNA synthesis to take place by a "self-priming" mechanism. Some of the double-stranded DNA synthesized corresponded to the entire sequence of the 9S mRNA template. The synthesis of full-length double-stranded DNA from mouse globin mRNA and immunoglobulin light chain mRNA is also discussed.     

A Unique Form of Terminal Redundancy in Adenovirus DNA Molecules

A unique form of terminal redundancy has been observed in DNA molecules extracted from several human adenovirus serotypes. Electron microscopic studies reveal that single-stranded circular molecules are formed when native DNA is denatured and then annealed. Temperatures approaching the T(m) of native DNA are required to convert circles to linear molecules, indicating a high degree of self-complementarity between terminal base sequences of DNA strands. Single-stranded circles are not generated if a limited number of nucleotides (2-4%) are removed from the 3' ends of native DNA by digestion with Escherichia coli exonuclease III before denaturation and annealing. The lenght of the redundant segment appears to differ among major serotypic groups, and a possible association between increased length of the redundant segment and increased oncogenic capability of virus serotype is suggested. Evidence for the configuration of the duplex closure region of circular molecules is also presented.