Efficient synthesis of a supercoiled M13 DNA molecule containing a site specifically placed psoralen adduct and its use as a substrate for DNA replication

We report a simple method for the in vitro synthesis of large quantities of site specifically modified DNA. The protocol involves extension of an oligonucleotide primer annealed to M13 single-stranded DNA using part of the T4 DNA polymerase holoenzyme. The resulting nicked double-stranded circles are ligated and supercoiled in the same tube, producing good yields of form I DNA. When the oligonucleotide primer is chemically modified, the resultant product contains a site-specific lesion. In this study, we report the synthesis of an M13 mp19 form I DNA which contains a psoralen monoadduct or cross-link at the KpnI site. We demonstrate the utility of these modified substrates by assessing the ability of the bacteriophage T4 DNA replication complex to bypass the damage and show that the psoralen monoadduct poses a severe block to the holoenzyme when attached to the template strand.     

Evidence for structural deformation of the DNA helix by a psoralen diadduct but not by a monoadduct.

We have investigated the structural change in a double-stranded DNA helix caused by covalent addition of a psoralen. A synthetic double-stranded DNA was constructed to contain either a psoralen furan-side monoadduct or an interstrand diadduct at a specific site. When the unmodified and psoralen modified DNAs were examined by electron microscopy in the presence of distamycin, which stiffens the DNA helix, the DNA containing the psoralen interstrand diadduct appeared bent (or kinked), whereas the furan-side monoadducted DNA appeared similar to the unmodified DNA. RecA protein from E. coli has been shown to preferentially bind UV (ultra violet) irradiated DNA presumably due to alterations in the normal DNA helical structure. Using a nitrocellulose filter binding assay, we have found that the psoralen interstrand diadduct enhances the binding of recA protein to the double-stranded DNA, whereas a furan-side monoadduct has little effect. Thus both the recA protein binding and the electron microscopic data suggest that a psoralen diadduct causes deformation of a DNA helix, most likely by kinking the helix, and that a monoadduct has little effect on the DNA helix structure.     

Site-specific interaction of vaccinia virus topoisomerase I with duplex DNA. Minimal DNA substrate for strand cleavage in vitro.

Purified vaccinia virus DNA topoisomerase I forms a cleavable complex with duplex DNA at a conserved sequence element 5'(C/T)CCTTdecreases in the incised DNA strand. DNase I footprint studies show that vaccinia topoisomerase protects the region around the site of covalent adduct formation from nuclease digestion. On the cleaved DNA strand, the protected region extends from +13 to -13 (+1 being the site of cleavage). On the noncleaved strand, the protected region extends from +13 to -9. Similar nuclease protection is observed for a mutant topoisomerase (containing a Tyr ---- Phe substitution at the active site amino acid 274) that is catalytically inert and does not form the covalent intermediate. Thus, vaccinia topoisomerase is a specific DNA binding protein independent of its competence in transesterification. By studying the cleavage of a series of 12-mer DNA duplexes in which the position of the CCCTTdecreases motif within the substrate is systematically phased, the "minimal" substrate for cleavage has been defined; cleavage requires six nucleotides upstream of the cleavage site and two nucleotides downstream of the site. An analysis of the cleavage of oligomer substrates mutated singly in the CCCTT sequence reveals a hierarchy of mutational effects based on position within the pentamer motif and the nature of the sequence alteration.     

Identification of the different intermediates in the interaction of (A)BC excinuclease with its substrates by DNase I footprinting on two uniquely modified oligonucleotides

(A)BC excinuclease is the enzymatic activity resulting from the joint actions of UvrA, UvrB and UvrC proteins of Escherichia coli. The enzyme removes from DNA many types of adducts of dissimilar structures with different efficiencies. To understand the mechanism of substrate recognition and the basis of enzyme specificity, we investigated the interactions of the three subunits with two synthetic substrates, one containing a psoralen-thymine monoadduct and the other a thymine dimer. Using DNase I as a probe, we found that UvrA makes a 33 base-pair footprint around the psoralen-thymine adduct and that UvrA-UvrB make a 45 base-pair asymmetric footprint characterized by a hypersensitive site 11 nucleotides 5' to the adduct and protection mostly on the 3' side of the damage. Conditions that favor dissociation of UvrA from the UvrA-UvrB-DNA complex, such as addition of excess undamaged DNA to the reaction mixture, resulted in the formation of a 19 base-pair UvrB footprint. In contrast, a thymine dimer in a similar sequence context failed to elicit a UvrA, a UvrA-UvrB or UvrB footprint and gave rise to a relatively weak DNase I hypersensitive site typical of a UvrA-UvrB complex. Dissociation of UvrA from the UvrA-UvrB-DNA complex stimulated the rate of incision of both substrates upon addition of UvrC, leading us to conclude that UvrA is not a part of the incision complex and that it actually interferes with incision. The extent of incision of the two substrates upon addition of UvrC (70% for the psoralen adduct and 20% for the thymine dimer) was proportional to the extent of formation of the UvrA-UvrB-DNA (i.e. UvrB-DNA) complex, indicating that substrate discrimination occurs at the preincision step.     

Post-incision steps of nucleotide excision repair in Escherichia coli. Disassembly of the UvrBC-DNA complex by helicase II and DNA polymerase I.

UvrA, UvrB, and UvrC initiate nucleotide excision repair by incising a damaged DNA strand on each side of the damaged nucleotide. This incision reaction is substoichiometric with regard to UvrB and UvrC, suggesting that both proteins remain bound following incision and do not "turn over." The addition of only helicase II to such reaction mixtures turns over UvrC; UvrB turnover requires the addition of helicase II, DNA polymerase I, and deoxynucleoside triphosphates. Column chromatography and psoralen photocross-linking experiments show that following incision, the damaged oligomer remains associated with the undamaged strand, UvrB, and UvrC in a post-incision complex. Helicase II releases the damaged oligomer and UvrC from this complex, making repair synthesis possible; DNase I footprinting experiments show that UvrB remains bound to the resulting gapped DNA until displaced by DNA polymerase I. The specific binding of UvrB to a psoralen adduct in DNA inhibits psoralen-mediated DNA-DNA cross-linking, yet promotes the formation of UrvB-psoralen-DNA cross-links. The discovery of psoralen-UvrB photocross-linking offers the potential of active-site labeling.     

Nucleotide sequence from the genetic left end of bacteriophage T7 DNA to the beginning of gene 4

The nucleotide sequence running from the genetic left end of bacteriophage T7 DNA to within the coding sequence of gene 4 is given, except for the internal coding sequence for the gene 1 protein, which has been determined elsewhere. The sequence presented contains nucleotides 1 to 3342 and 5654 to 12,100 of the approximately 40,000 base-pairs of T7 DNA. This sequence includes: the three strong early promoters and the termination site for Escherichia coli RNA polymerase: eight promoter sites for T7 RNA polymerase; six RNAase III cleavage sites; the primary origin of replication of T7 DNA; the complete coding sequences for 13 previously known T7 proteins, including the anti-restriction protein, protein kinase, DNA ligase, the gene 2 inhibitor of E. coli RNA polymerase, single-strand DNA binding protein, the gene 3 endonuclease, and lysozyme (which is actually an N-acetylmuramyl-l-alanine amidase); the complete coding sequences for eight potential new T7-coded proteins; and two apparently independent initiation sites that produce overlapping polypeptide chains of gene 4 primase. More than 86% of the first 12,100 base-pairs of T7 DNA appear to be devoted to specifying amino acid sequences for T7 proteins, and the arrangement of coding sequences and other genetic elements is very efficient. There is little overlap between coding sequences for different proteins, but junctions between adjacent coding sequences are typically close, the termination codon for one protein often overlapping the initiation codon for the next. For almost half of the potential T7 proteins, the sequence in the messenger RNA that can interact with 16 S ribosomal RNA in initiation of protein synthesis is part of the coding sequence for the preceding protein. The longest non-coding region, about 900 base-pairs, is at the left end of the DNA. The right half of this region contains the strong early promoters for E. coli RNA polymerase and the first RNAase III cleavage site. The left end contains the terminal repetition (nucleotides 1 to 160), followed by a striking array of repeated sequences (nucleotides 175 to 340) that might have some role in packaging the DNA into phage particles, and an A · T-rich region (nucleotides 356 to 492) that contains a promoter for T7 RNA polymerase, and which might function as a replication origin.