Specific hydrolysis of the cohesive ends of bacteriophage lambda DNA by three single strand-specific nucleases.

Procedures have been worked out for Aspergillus nuclease S1 and mung been nuclease to quantitatively cleave off both of the 12-nucleotide long, single-stranded cohesive ends of lambdaDNA. This cleavage is indicated by the almost complete elimination of the repair incorporation of radioactive nucleotides by DNA polymerase into the digested DNA. With S1 nuclease, cleavage was complete at 10 degrees as well as at 30 degrees. Under the conditions for quantitative cleavage of the single-stranded regions there was no digestion of the double-stranded lambdaDNA. The mung bean nuclease cleaved off the cohesive ends completely at 30 degrees but at 5 degrees, the cleavage was not complete even at high enzyme concentration. The nearest neighbor analysis of the repaired DNA indicates that at 5 degrees about four nucleotides remained undigested. The mung bean nuclease also introduced, under the conditions used, some nicks into double-stranded DNA as determined by the repair incorporation. The Escherichia coli exonuclease VII cleaved off part of the cohesive ends of lambdaDNA, leaving two nucleotides on each end as single-stranded tails.     

Nucleotide sequence analysis of DNA. XI. The 3' terminal sequences of bacteriophage lambda and phi 80 DNA

Two procedures have been developed and applied to the determination of the 3′ terminal sequences of λ DNA and φ80 DNA. In the first procedure, each 3′ terminus was specifically labeled with a single ³²P-nucleotide. Radioactive oligonucleotides of different lengths were obtained by partial pancreatic deoxyribonuclease digestion. From the characteristic mobilities of these oligonucleotides in two dimensional fractionation systems, the 3′ terminal sequence -ACCCGCG for the r-strand and -GGTTACG for the l-strand of λ DNA have been determined. In the second procedure, approximately six nucleotides were removed from each 3′ terminus with exonuclease III, and they were replaced with radioactive nucleotides by partial repair synthesis. After enzymatic digestion and sequence analysis, the above sequences have been confirmed. The 3′ terminal sequences in φ80 DNA are identical to those in λ DNA at least up to the fifth nucleotide from the 3′ ends.     

Nucleotide Sequence Analysis of DNA

There are three major obstacles to the analysis of the nucleotide sequence in a DNA molecule starting from a known location in the DNA molecule. First, it is difficult to obtain large quantities of homogeneous DNA. Second, even the smallest DNA molecules contain several thousand nucleotides which make sequence analysis prohibitive. Third, there are no highly base-specific DNAases available for degrading DNA for sequence analysis. We have overcome some of these obstacles; first, by incorporating highly labelled deoxynucleotides into DNA in vitro, small amounts of material can be used for sequence analysis. Second, the nucleotide sequence of DNA molecules can now be determined from the 5′-terminal. Thus, two dodecanucleotide sequences corresponding to the two cohesive ends of λ DNA have been determined¹ and a nona-decanucleotide sequence corresponding to one cohesive end of phage 186 DNA has been completed². So far, our approach is limited to starting the analysis from the 5′-ends of a DNA molecule. A more general approach is being developed for starting the analysis from other selected parts of a DNA molecule with the use of specifically designed primers.     

Mechanism and regulation of DNA end resection in eukaryotes

The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is initiated by nucleolytic degradation of the 5′-terminated strands in a process termed end resection. End resection generates 3′-single-stranded DNA tails, substrates for Rad51 to catalyze homologous pairing and DNA strand exchange, and for activation of the DNA damage checkpoint. The commonly accepted view is that end resection occurs by a two-step mechanism. In the first step, Sae2/CtIP activates the Mre11–Rad50–Xrs2/Nbs1 (MRX/N) complex to endonucleolytically cleave the 5′-terminated DNA strands close to break ends, and in the second step Exo1 and/or Dna2 nucleases extend the resected tracts to produce long 3′-ssDNA-tailed intermediates. Initiation of resection commits a cell to repair a DSB by HR because long ssDNA overhangs are poor substrates for non-homologous end joining (NHEJ). Thus, the initiation of end resection has emerged as a critical control point for repair pathway choice. Here, I review recent studies on the mechanism of end resection and how this process is regulated to ensure the most appropriate repair outcome.     

Use of a new retrieving adaptor in the cloning of a synthetic human insulin A-chain gene

A DNA duplex encoding the A-chain of human insulin was constructed from eight chemically synthesized oligomers by enzymatic ligation to form a partial duplex followed by repair synthesis to complete the complementary strands. After sequential addition of translation start and stop signal adaptors the assembly was cloned in pBR322. To regenerate the end of the coding sequence by precise removal of extraneous nucleotides a new method using a synthetic retrieval adaptor was developed. The procedure included filling in the cohesive ends of the EcoRI site by repair synthesis, ligating a symmetrical adaptor having an MboII recognition sequence to the resulting blunt end, cutting with MboII and removing the single protruding 3′-nucleotide using the 3′ exonuclease activity of DNA polymerase I. Synthetic oligomers useful for ligation to a synthetic insulin C-chain gene were added to the retrieved end of the gene. Sequence analysis established that retrieval adaptors of this type may be used for precise excision of up to eight nucleotides from the end of a cloned DNA fragment.     

Evidence for two nucleotide sequence orientations within the terminal repetition of adeno-associated virus DNA.

Duplex adeno-associated virus (AAV) DNA, produced by annealing plus and minus virion single strands, has been digested with several bacterial restriction endonucleases. These studies reveal the existence of alternate secondary structures at the termini of duplex AAV DNA. Analysis of the sites of endo R-Hpa II cleavage, the products of complete endo R-Hpa II digestion, and the multiple terminal secondary structures leads to the conclusion that there are two possible nucleotide sequences at each end of AAV DNA. A model that attributes the terminal nucleotide sequence heterogeneity to two possible orientations of the first 120 nucleotides at each end of the DNA is proposed; in one case the sequence is 1 to 120; in the other case the sequence is inverted. An origin of the inversion is suggested based on previously described intermediates in AAV DNA replication.     

Nucleotide sequence of bacteriophage f1 DNA.

The nucleotide sequence of the DNA of the filamentous coliphage f1 has been determined. In agreement with earlier conclusions, the genome was found to comprise 6,407 nucleotides, 1 less than that of the related phage fd. Phage f1 DNA differs from that of phage M13 by 52 nucleotide changes, which lead to 5 amino acid substitutions in the corresponding proteins of the two phages, and from phage fd DNA by 186 nucleotide changes (including the single-nucleotide deletion), which lead to 12 amino acid differences between the proteins of phages f1 and fd. More than one-half of the nucleotide changes in each case are found in the sequence of 1,786 nucleotides comprising gene IV and the major intergenic region between gene IV and gene II. The sequence of this intergenic region (nucleotides 5501 to 6005) of phage f1 differs from the sequence reported by others through the inclusion of additional single nucleotides in eight positions and of a run of 13 nucleotides between positions 5885 and 5897, a point of uncertainty in the earlier published sequence. The differences between the sequence of bacteriophage f1 DNA now presented and a complete sequence for the DNA previously published by others are discussed, and the f1 DNA sequence is compared with those of bacteriophages M13 and fd.     

Processing of bacteriophage lambda DNA during its assembly into heads

DNA purified from bacteriophage λ added to a cell-free extract derived from induced λ lysogens can be packaged into infectious phage particles (Kaiser & Masuda, 1973). In this paper the structure of the DNA which is the substrate for in vitro packaging and head assembly is described. The active precursor is a multichromosomal polymer that contains covalently closed cohesive end sites. Neither circular or linear DNA monomers nor polymers with unsealed cohesive ends are packaged efficiently into heads. The unit length monomer is packaged when it is either contained in the interior of a polymer (both of its ends are in cos sites) or when it has a free left end and a cos site on its right. The monomer unit with a free right end is not a substrate for packaging.A procedure is given for the purification of λ DNA fragments that contain either the left or the right cohesive end. The fragments are produced by digesting λ DNA with the site-specific Escherichia coli R₁ endonuclease; the left and right ends are separated by sedimentation through a sucrose gradient. These fragments are used to construct small polymers that have a unit length λ monomer with (1) a free left end and a closed right end, (2) a free right end and a closed left end, or (3) both ends closed in cos sites.     

The nucleotide sequence of oocyte 5S DNA in Xenopus laevis. II. The GC-rich region

The primary sequence of the GC-rich half of the repeating unit in X. laevis 5S DNA has been determined in both a single plasmid-cloned repeating unit and in the total population of repeatig units. The GC-rich half of the repeating unit contains a single long duplication of 174 nucleotides. The duplicated segment commences 73 nucleotides preceding the 5' end of the gene and terminates at nucleotide 101 of the gene. The duplicated portion of the gene, termed the pseudogene, differs by 10 nucleotides from the corresponding portion of the gene, and the remaining duplicated sequence of 73 nucleotides differs by 13 nucleotides. The plasmid-cloned repeating unit differs from the dominant sequence in the total population repeating units by 6 nucleotides in the GC-rich region. Evidence is provided that most of the CpG dinucleotides in 5S DNA are at least partially methylated.     

Recognition sequence of restriction endonuclease KpnI from Klebsiella pneumoniae.

We have determined the recognition sequence of the restriction endonuclease KpnI, previously isolated from Klebsiella pneumoniae. The enzyme cleaves the twofold rotationally symmetric sequence (see book for formula) at the positions indicated by the arrows, producing 3' protruding cohesive ends, four nucleotides in length. The specific cleavage site was unambiguously deduced using both 3' and 5' end analyses of KpnI generated restriction fragments of simian-virus 40 (SV40) DNA (1 site), adenovirus-2 (Ad-2) DNA (8 sites), and a plasmid (pCRI) DNA (2 sites).     

DNA sequence analysis. Terminal sequences of bacteriophage phi80.

Sequences of the cohesive ends and the 3'-terminal regions of phi80 DNA have been determined. Sequences of the cohesive ends were obtained through the use of two standard methods. The first method involved the incorporation of all four labeled deoxyribonucleotides into the phi80 cohesive ends using DNA polymerase I. The DNA was then partially digested with micrococcal nuclease or pancreatic DNase. The products were separated by two-dimensional electrophoresis and characterized by composition, 3'-terminal, and nearest neighbor analyses. The second method involved partial incorporation using one, two, or three labeled deoxyribonucleotides followed by similar analyses. Sequences of the double-stranded regions adjacent to the cohesive ends were determined by three new methods. These methods were: (a) the DNA was specifically labeled at the 3' terminus and then partially degraded. Labeled oligonucleotide products were sequenced by their mobilities on various separation systems. (b) The cohesive ends were enlarged by limited degradation with exonuclease III. After this treatment, the DNA was partially repaired with labeled nucleotides, digested, and the products were analyzed. (c) A synthetic ologonucleotide primer was bound to phi80 DNA which had been repaired with DNA polymerase I, and then partially digested with lambda-exonuclease. The primer was extended into the region of interest by partial repair with labeled nucleotides. The extended primer was isolated and analyzed.     

178-Nucleotide sequence surrounding the cos site of bacteriophage lambda DNA.

A nucleotide sequence of 61 nucleotides at the left end and 117 nucleotides at the right end of DNA from bacteriophage lambdacI857Sam7 was determined by the Maxam and Gilbert method. A perfect inverted repeat sequence of 10 nucleotides is near the left end, and one of 15 nucleotides is near the right end. DNA from another closely related lambda strain, lambdacI857prm116Sam7, has about 10% divergence in the sequence of the first 110 nucleotides at the right end and has a 17-member perfect inverted repeat sequence.     

Mechanism of replication of human mitochondrial DNA. Localization of the 5' ends of nascent daughter strands

Human mitochondrial DNA contains two physically separate and distinct origins of DNA replication. The initiation of each strand (heavy and light) occurs at a unique site and elongation proceeds unidirectionally. Animal mitochondrial DNA is novel in that short nascent strands are maintained at one origin (D-loop) in a significant percentage of the molecules. In the case of human mitochondrial DNA, there are three distinct D-loop heavy strands differing in length at the 5' end. We report here the localization of the 5' ends of nascent daughter heavy strands originating from the D-loop region. Analyses of the map positions of 5' ends relative to known restriction endonuclease cleavage sites and 5' end nucleotides indicate that the points of initiation of D-loop synthesis and actual daughter strands are the same. In contrast, the second origin is located two-thirds of the way around the genome where light strand synthesis is presumably initiated on a single-stranded template. Mapping of 5' ends of daughter light strands at this origin relative to known restriction endonuclease cleavage sites reveals two distinct points of initiation separated by 37 nucleotides. This origin is in the same relative genomic position and shows a high degree of DNA sequence homology to that of mouse mitochondrial DNA. In both cases, the DNA region within and immediately flanking the origin of DNA replication contains five tightly clustered tRNA genes. A major portion of the pronounced DNA template secondary structure at this origin includes the known tDNA sequences.     

Primary organization of the nucleosome core particles sequential arrangement of histones along DNA

A high-resolution map for the arrangement of histones along DNA in the nucleosome core particles has been determined by a new sequencing procedure. The lysine groups of histones were crosslinked to partly depurinated DNA at neutral pH. One strand of DNA was split at the points of crosslinking, thus leaving the 5′-terminal DNA fragments bound to histones. The lengths of these crosslinked DNA fragments were measured to determine the position of histones on one strand of the core DNA from its 5′ end.The results demonstrate that histones are bound to regularly arranged, discrete DNA segments about six nucleotides long. These segments are separated by histone-free gaps about four nucleotides wide located at a distance of about 10n nucleotides from the 5′ end of DNA. The first 20 nucleotides from the 5′ ends of DNA seem to be free of histones. Histones appear to be arranged symmetrically and in a similar way on both DNA strands. Any one histone, being bound predominantly to discrete segments on one or other of the strands, can oscillate at the same time between the two strands across the major DNA groove. Two symmetrical models for the arrangement of two molecules of each core histone on linearized and folded DNA are proposed.     

DNA sequence analysis: a general, simple and rapid method for sequencing large oligodeoxyribonucleotide fragments by mapping

Several electrophoretic and chromatographic systems have been investigated and compared for sequence analysis of oligodeoxyribonucleotides. Three systems were found to be useful for the separation of a series of sequential degradation products resulting from a labeled oligonucleotide: (I) 2-D electrophoresis†; (II) 2-D PEI-cellulose; and (III) 2-D homochromatography. System (III) proved generally most informative regardless of base composition and sequence. Furthermore, only in this system will the omission of an oligonucleotide in a series of oligonucleotides be self-evident from the two-dimensional map. The sequence of up to fifteen nucleotides can be determined solely by the characteristic mobility shifts of its sequential degradation products distributed on the two-dimensional map. With this method, ten nucleotides from the double-stranded region adjacent to the left-hand 3′-terminus and seven from the right-hand 3′-terminus of bacteriophage λ DNA have been sequenced. Similarly, nine nucleotides from the double-stranded region adjacent to the left-hand 3′-terminus and five nucleotides from the right-hand terminus of bacteriophage 80 DNA have also been sequenced. The advantages and disadvantages of each separation system with respect to sequence analysis are discussed.