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Free radical species generated through fluorescence photobleaching have been reported to effectively couple a water-soluble species to surfaces containing electron-rich sites . In this report, we expand upon this strategy to control the patterned attachment of antibodies and peptides to surfaces for biosensing and tissue engineering applications. In the first application, we compare hydrophobic attachment and photobleaching methods to immobilize FITC-labeled anti-M13K07 bacteriophage antibodies to the SiO2 layer of a differential capacitive biosensor and to the polyester filament of a feedback-controlled filament array. On both surfaces, antibody attachment and function were superior to the previously employed hydrophobic attachment. Furthermore, a laser scanning confocal microscope could be used for automated, software-guided photoattachment chemistry. In a second application, the cell-adhesion peptide RGDS was site-specifically photocoupled to glass coated with fluorescein-conjugated poly(ethylene glycol). RGDS attachment and bioactivity were characterized by a fibroblast adhesion assay. Cell adhesion was limited to sites of RGDS photocoupling. These examples illustrate that fluorophore-based photopatterning can be achieved by both solution-phase fluorophores or surface-adhered fluorophores. The coupling preserves the bioactivity of the patterned species, is amenable to a variety of surfaces, and is readily accessible to laboratories with fluorescence imaging equipment. The flexibility offered by visible light patterning will likely have many useful applications in bioscreening and tissue engineering where the controlled placement of biomolecules and cells is critical, and should be considered as an alternative to chemical coupling methods.
The role of replication blockage by the exocyclic DNA adducts propanodeoxyguanosine (PdG) and pyrimido[1,2-alpha]purin-10(3H)-one (M1G) was determined through the use of site-specifically adducted M13MB102 genomes containing a C:C-mismatch approximately 3000 base-pairs from the site of adduct incorporation. Genomes containing either dG, PdG, or M1G positioned at site 6256 of the (-)-strand were transformed into repair-proficient and repair-deficient Escherichia coli strains and the percent template utilization was determined by hybridization analysis. Unmodified genomes containing a C:C-mismatch resulted in a percent template utilization of approximately 60 and 40% for the (-)- and (+)-strands, respectively. Transformation of PdG- or M(1)G-adducted genomes resulted in approximately a 60-40% and 50-50% (-)-strand to (+)-strand ratio, respectively, indicating that PdG and M(1)G are negligible blocks to replication in repair-proficient E. coli. This is in contrast to previous studies using (PdG:T)- and (M1G:T)-mismatched M13MB102 genomes, which resulted in a majority of the replication events using the unadducted (+)-strand and suggested that both adducts were significant blocks to replication [J. Biol. Chem. 272 (1997) 11434; Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 8652]. The C:C-mismatch results, though, indicate that the large strand bias detected in the earlier studies is due to repair of the adducts and resynthesis of the (-)-strand using the (+)-strand as a template for repair synthesis. Transformation of adducted C:C-mismatched genomes into E. coli strains deficient in nucleotide excision repair did result in an increased strand bias with only approximately 20 and 34% of the replication events using the (-)-strand for PdG- and M1G-adducted genomes, respectively. The increased strand bias indicates the importance of nucleotide excision repair in the removal of PdG and M1G.
Repair of the exocyclic DNA adduct propanodeoxyguanosine (PdG) was assessed in both in vivo and in vitro assays. PdG was site-specifically incorporated at position 6256 of M13MB102 DNA, and the adducted viral genome was electroporated into repair-proficient and repair-deficient Escherichia coli strains. Comparable frequencies of PdG --> T and PdG --> A mutations at position 6256 were detected following replication of the adducted genomes in wild-type E. coli strains. A 4-fold increase in the frequencies of transversions and transitions was observed in E. coli strains deficient in Uvr(A)BC-dependent nucleotide excision repair. A similar increase in the replication of the adduct containing strand was observed in the repair-deficient strains. No change in the frequency of targeted mutations was observed in strains deficient in one or both of the genes coding for 3-methyladenine glycosylase. Incubation of purified E. coli Uvr(A)BC proteins with a duplex 156-mer containing a single PdG adduct resulted in removal of a 12-base oligonucleotide containing the adduct. Incubation of the same adducted duplex with Chinese hamster ovary cell-free extracts also resulted in removal of the adduct. PdG was a better substrate for repair by the mammalian nucleotide excision repair complex than the bacterial repair complex and was approximately equal to a thymine-thymine dimer as a substrate for the former. The results of these in vivo and in vitro experiments indicate that PdG, a homolog of several endogenously produced DNA adducts, is repaired by the nucleotide excision repair pathway.
The role of cytosine deamination as a possible mechanism for PdG-->A transitions induced by propanodeoxyguanosine (PdG) was investigated by site-specific mutagenesis techniques. PdG was placed at position 6256 in the (-)-strand of M13MB102 by ligating the oligodeoxynucleotide 5'-GGT(PdG)TCCG-3' into a gapped-duplex derivative of the vector. Unmodified and PdG-modified M13MB102 genomes containing either uracil or thymine in the (+)-strand were transformed into Escherichia coli strains differing in their ability to excise uracil bases from DNA. After replication of the site specifically modified M13MB102, base-pair substitutions were detected by in situ hybridization using [32P]-labeled probes containing each of the possible mismatched bases opposite position 6256 in the (+)-strand. The ratio of PdG-->A and PdG-->T was unchanged in strains defective in the repair of uracil residues, which suggests that uracil is not an intermediate in the generation of PdG-->A mutations. Similar results were obtained when PdG-M13MB102 was incubated for 14 days prior to transformation in an attempt to increase the extent of deamination. As a control experiment to test the sensitivity of the assay to detect deaminations opposite PdG, uracil-containing M13MB102 with a PdG.T mismatch at position 6256 was transformed into E. coli JM105. Hybridization analysis indicated that approximately 80% of the phage plaques generated after genome replication contained T in the (+)-strand at position 6256. Thus, any deamination of cytosine to uracil would have been easily detected. Adducted and unadducted genomes were also transformed into E. coli LM114 or LM113, which carry a mutant umuD or umuC gene, respectively. Significant and comparable reductions in PdG-->A and PdG-->T were observed, suggesting that both mutations require the active participation of the UmuD and the UmuC proteins in the replication complex. The results of our experiments suggest that the PdG-->A mutations induced by PdG are not caused by cytosine deamination, but arise coincident with PdG-->T mutations during replication of the PdG-containing genomes. Also, the uracil-containing (+)-strand does not appear to be degraded, as is commonly assumed in site-specific mutagenesis experiments, and serves as a template for DNA synthesis when replication of the (-)-strand is blocked by an adduct such as PdG.
Frameshift mutations demonstrate a high degree of sequence specificity. In order to provide a vector for site-specific frameshift mutagenesis experiments, a recombinant M13 phage (M13MB102) was constructed by substitution of 27 base pairs of the Salmonella typhimurium hisD3052 sequence for 27 base pairs of the polylinker region of M13mp19. The inserted sequence contains most of the hotspot for frameshift mutations in hisD3052 and its derivative strain TA98. Structural elements of the insert include reiterated bases, direct repeats, and palindromes, and four unique restriction endonuclease cleavage sites. The recombinant phage produced blue plaques when grown in Escherichia coli strain JM105 on X-Gal indicator plates and exhibited a spontaneous mutation frequency similar to that of M13mp19. Methodology is described for preparation, isolation, and purification of closed circular duplex M13MB102 genomes containing an adduct between the SphI and BssHII cleavage sites in the (-)-strand and uracil residues in the (+)-strand. The latter modification decreases replication of the (+)-strand by 4 orders of magnitude and maximizes use of the adducted (-)-strand for in vivo replication. The structure of M13MB102 and the procedures described for introducing adducts at defined positions in its hisD3052 insert provide a convenient approach for evaluating the potential of individual carcinogen-DNA adducts to induce frameshift mutations.
The spectrum of mutations induced upon in vivo replication of an M13 genome containing a site-specifically located propanodeoxyguanosine (PdG) adduct was determined. PdG was used as a model for the major deoxyguanosine adduct produced on reaction of DNA with the endogenous genotoxin malondialdehyde. PdG was introduced at position 6256 of M13MB102 by ligating the oligodeoxynucleotide 5'-GGT(PdG)TCCG-3' into an 8-base gap in the (-)-strand of duplex M13MB102. Replication of the adducted strand was maximized by incorporation of uracil into the unadducted (+)-strand. Following replication of dG-containing and PdG-containing M13MB102 genomes in Escherichia coli JM105, frameshift mutations were detected as phenotypic changes in the lacZ alpha marker gene. Base pair substitutions were detected by differential hybridization using 32P-labeled 13-mers bearing different bases opposite position 6256. Neither frameshift nor base pair substitution mutations were detected following replication of PdG-adducted genomes in non-SOS-induced JM105. However, PdG-->T transversions and PdG-->A transitions were detected following transformation of PdG-adducted M13MB102 into SOS-induced JM105. Both types of mutations were detected at comparable frequencies, and the total mutation frequency was approximately 2%. The results indicate that PdG is an efficient premutagenic lesion in E. coli strains in which the SOS response is induced.
The mutagenicity of the lipid peroxidation product, malondialdehyde (MDA), was measured in the lacZ alpha forward mutation assay using a recombinant M13 phage, M13MB102. Single-stranded M13MB102 DNA was reacted with MDA at neutral pH and the modified DNA was transformed into strains of Escherichia coli induced for the SOS response. Increasing concentrations of MDA led to an increase in lacZ alpha-mutations coincident with an increase in the level of the major MDA-deoxyguanosine adduct. Spontaneous and MDA-induced M13MB102 mutants were collected and the lacZ alpha target region was subjected to automated DNA sequence analysis. The most common sequence changes induced by MDA were base-pair substitutions (76%). Of these, 43% (29/68) were transversions, most of which were G-->T (24/29). Transitions account for 57% of the base-pair substitutions (39/68) and were comprised exclusively of C-->T (22/39) and A-->G (17/39). Frameshift mutations were identified in 16% of the induced mutants and were comprised of mainly single base additions occurring in runs of reiterated bases (11/14). The diversity of base-pair substitution and frameshift mutations induced by MDA at low levels of adduction suggests it may be an important contributor to endogenous mutagenesis and carcinogenesis in aerobic organisms.
Malondialdehyde induces frameshift mutations in Salmonella typhimurium strain hisD3052. The ability of propanodeoxyguanosine (PdG), a structural analog of the major malondialdehyde-deoxyguanosine adduct, to induce site-specific frameshift mutations was tested in the (CpG)4 hot-spot of hisD3052 carried on an M13 vector (M13MB102). PdG was introduced at position 6248 of duplex M13MB102 by ligation of the oligonucleotide 5'-CGC(PdG)CGGCATG-3' into a heteroduplex containing an 11-nucleotide gap in the (-)-strand between the SphI and BssHII restriction sites and deoxyuridine in place of thymidine in the (+)-strand. Ligation proceeded with 70% efficiency, and closed circular duplex DNA molecules were isolated in 40% yield. The adducted genome was sensitive to cleavage by SphI but resistant to cleavage by BssHII. Transformation of Escherichia coli strain JM105 with adducted M13MB102 led to 25% reduced survival relative to unadducted M13MB102 and produced frameshift mutations in 2.5% of the progeny phage. All of the mutations were deletions, and 70% occurred by deletion of CpG. Unadducted genomes exhibited a 40-fold lower mutation frequency, and all the mutations were single-base deletions at the sites of ligation of the 11-mer. These results illustrate that PdG, a structural analog of the major malondialdehyde-deoxyguanosine adduct, induces frameshift mutations in M13MB102 and that single-stranded nicks are efficient premutagenic lesions in this recombinant bacteriophage.