Patterns of cigarette smoking and alcohol use among lesbians and bisexual women enrolled in a large health maintenance organization.

OBJECTIVES: This study compared the prevalence of cigarette smoking and alcohol use among lesbians and bisexual women with that among heterosexual women. METHODS: Logistic regression models were created with data from an extensive member health survey at a large health maintenance organization. Sexual orientation was the primary predictor, and alcohol consumption and cigarette smoking were outcomes. RESULTS: Lesbians and bisexual women younger than 50 years were more likely than heterosexual women to smoke cigarettes and drink heavily. Lesbians and bisexual women aged 20 to 34 reported higher weekly alcohol consumption and less abstinence compared with heterosexual women and older lesbians and bisexual women. CONCLUSIONS: Lesbians and bisexual women aged 20 to 34 years are at risk for alcohol use and cigarette smoking.

Increase in wound breaking strength in rats in the presence of positively charged dextran beads correlates with an increase in endogenous transforming growth factor-beta1 and its receptor TGF-betaRI in close proximity to the wound.

We have previously shown that positively charged beads (DEAE A25) increase wound breaking strength in linear incisions in rats and nonhuman primates at days 10-14 post-wounding. The increased wound strength may result in part from a stimulation of cells adjacent to the DEAE A25 beads to produce growth factors important for wound healing. In this report, we investigate this hypothesis by comparing the relative expression levels of transforming growth factor-beta1 and its receptor transforming growth factor-beta receptor type I in DEAE A25-treated and contralateral untreated rat linear incisions. DEAE A25-treated incisions were stronger than untreated control wounds at 3 days post-wounding, and the difference in breaking strength reached statistical significance at days 5, 7 and 10. Immunohistochemical analysis revealed a significant increase in transforming growth factor-beta1 and transforming growth factor-beta receptor type I expression in DEAE A25-treated incisions, up to 7 days post-wounding, as compared to untreated control wounds. FACS analysis revealed that macrophage cell lines exposed to DEAE A25 in vitro upregulate transforming growth factor-beta1 and transforming growth factor-beta receptor type I expression by 2-3 fold. Therefore, the increase in expression of transforming growth factor-beta1 and transforming growth factor-beta receptor type I in DEAE A25-treated incisions may be due to an increase in the concentration of macrophages adjacent to DEAE A25 beads, as well as the stimulation of individual macrophages to produce greater amounts of transforming growth factor-beta1 and transforming growth factor-beta receptor type I. This study also supports the significance of transforming growth factor-beta1 in wound healing.

Cost of care for patients in cancer clinical trials.

BACKGROUND: Information on the costs of medical care for patients enrolled in clinical trials is needed by policymakers evaluating ways to facilitate clinical research in a managed care environment. We examined the direct costs of medical care for patients enrolled in cancer clinical trials at a large health maintenance organization (HMO). METHODS: Costs for 135 patients who entered 22 cancer clinical trials (including 12 breast cancer trials) at Kaiser Permanente in Northern California, from 1994 through 1996 were compared with costs for 135 matched control subjects who were not enrolled in such trials. Cancer registry data and medical charts were used in matching the control subjects to the trial enrollees with respect to cancer site, stage, date of diagnosis, age, sex, and trial eligibility. The direct costs of medical care were compared between trial enrollees and the control subjects for a 1-year period, with data on costs and utilization of services obtained from Kaiser Permanente databases and medical charts. RESULTS: Mean 1-year costs for the enrollees in trials were 10% higher than those for the control subjects ($17 003 per enrollee compared with $15 516 per control subject; two-sided P =.011). The primary component of this difference was a $1376 difference in chemotherapy costs ($4815 per trial enrollee versus $3439 per control subject; two-sided P<.001). Costs for the 11 enrollees in trials that had a bone marrow transplant (BMT) arm were approximately double the costs for their matched control subjects (borderline significance: two-sided P=.054). The $15 041 mean cost for the enrollees in trials without BMT was similar to the $15 186 mean cost for their matched control subjects. CONCLUSIONS: Participation in cancer clinical trials at a large HMO did not result in substantial increases in the direct costs of medical care.

Stimulation of wound healing by positively charged dextran beads depends upon clustering of beads and cells in close proximity to the wound.

We have previously shown that positively charged dextran (DEAE A25) increases wound breaking strength in linear incisions in rats and nonhuman primates at days 10-14 postwounding. In this article, we examined the cellular responses to different types of charged dextran beads (DEAE A50 and Cytodex-1) in culture studies and in rat incisional wounds. We show that Cytodex 1 and DEAE A50 beads also increased wound breaking strength in a rat linear incisional model. However, the increase was approximately 30-40% less than that observed in wounds treated with DEAE A25 beads. The main distinction between the three types of beads was the presence of bead clusters observed in tissue sections. Wounds treated with DEAE A25 beads formed distinct clusters while both Cytodex 1 and DEAE A50 beads clustered to a lesser extent or failed to cluster at all. We propose that the different types of charged dextran beads improve healing by promoting cell adhesion and encouraging proliferation in close proximity to the wound. We also hypothesize that the 30-40% improvement in wound breaking strength seen with DEAE A25 beads compared to other types of charged dextran beads (DEAE A50 and Cytodex-1) originates from the unique characteristic of DEAE A25 beads in forming cell-bead aggregates adjacent to the wounded area. This clustering, in turn, affects the distribution of cells infiltrating the wounded area (such as macrophages) during the healing process and, as a consequence, alters the distribution of matrix molecules and growth factors secreted by these cells.

Effects of electrically charged particles in enhancement of rat wound healing.

BACKGROUND: Many studies have show that various growth factors enhance wound healing in animal models. However, growth factors are expensive and complex and their wound pharmacokinetics are unknown. The clinical trials with growth factors in the treatment of chronic wounds have produced unsuccessful results. A viable alternative to growth factors may be certain biomaterials such as hydrophilic, carbohydrate beads. MATERIALS AND METHODS: Positively charged, negatively charged, or uncharged beads were applied to paired 6-cm rat incisions. The following key aspects of the wound healing process were examined: wound breaking strength and histological analysis. RESULTS: Our data show that wounds treated with positively charged, DEAE Sephadez beads had a 46-50% (P < 0.001) increase in breaking strength over untreated control wounds. A variety of other positively charged, anion exchange materials also elicited a wound healing response, despite the fact that the positively charged chemical moieties as well as the bead matrix were different. In conjunction with the increase in wound breaking strength, an increase in wound macrophage was observed in wounds treated with anion exchangers (P < 0.01). Negatively charged or uncharged beads showed no significant difference from the untreated controls. CONCLUSION: We conclude from this study that the enhancement of wound healing seen with positively charged beads is due principally to the positive charge on the beads; we postulate that the anion exchange between the positively charged beads and tissue is responsible for this enhancement.

Transforming growth factor-beta1 fails to stimulate wound healing and impairs its signal transduction in an aged ischemic ulcer model: importance of oxygen and age.

Clinical trials of exogenous growth factors in treating chronic wounds have been less successful than expected. One possible explanation is that most studies used animal models of acute wounds in young animals, whereas most chronic wounds occur in elderly patients with tissue ischemia. We described an animal model of age- and ischemia-impaired wound healing and analyzed the wound-healing response as well as the transforming growth factor (TGF)-beta1 effect in this model. Rabbits of increasing ages were made ischemic in the ear where dermal ulcers were created. Histological analysis showed that epithelium ingrowth and granulation tissue deposition were significantly impaired with increased age under ischemia. TGF-beta1 stimulated wound repair under both ischemic and non-ischemic conditions in young animals, although it showed no statistical difference in aged animals. Procollagen mRNA expression decreased under ischemic conditions and with aging. Neither TGF-beta1 nor procollagen alpha1(I) mRNA expression increased in response to TGF-beta1 treatment under ischemia in aged animals. Therefore, the wound-healing process is impaired additively by aging and ischemia. The lack of a wound-healing response to TGF-beta1 in aged ischemic wounds may play a role in the chronic wounds.

Charged beads enhance cutaneous wound healing in rhesus non-human primates.

Enhanced cutaneous wound healing by positively charged cross-linked diethylaminoethyl dextran beads (CLDD) was studied in a standardized incisional wound model in 20 adult and 20 geriatric Macaca mulatta (rhesus) partitioned equally over five time periods. Physiologic saline served as a control. Soft-tissue linear incisions were prepared between and 1 cm inferior to the scapulae. There were four incisions per rhesus; each incision was 1.5 cm long with 1 cm of undisturbed tissue between incisions, and both the experimental CLDD and physiologic saline treatments were administered to each rhesus. The incision treatments were either CLDD and soft-tissue closure with 4-0 BioSyn sutures or sterile physiologic saline and closure with 4-0 BioSyn sutures. The hypothesis was CLDD would enhance cutaneous wound repair. Verification of the hypothesis consisted of clinical examinations and histologic and tensiometric evaluations on biopsy specimens at 10 and 15 days, whereas 5-day and 2- and 4-month groups were assessed clinically and biopsy specimens were assessed histologically. The clinical course of healing for all groups was unremarkable. At 10 days, incisions in adult rhesus treated with CLDD had a 30-percent greater tensile strength compared with the physiologic saline-treated incisions (p = 0.01), whereas for geriatric rhesus, the CLDD treatment proved to be 15 percent greater in tensile strength compared with the physiologic saline cohort (p = 0.11). By day 15, incisions in adult rhesus were 26 percent stronger than the saline treatment group (p = 0.07), and the difference was 36 percent (p = 0.02) for the geriatric rhesus. From 5 through 15 days, histologic observations revealed a gradual decrease in quantity and integrity of CLDD, with no remnants of CLDD at either 2 or 4 months. Macrophages and multinucleated giant cells were localized in the dermis and were associated with the CLDD. These cells decreased commensurately with the decrease of CLDD beads. The data suggest that CLDD can enhance significantly the tensile properties of healing cutaneous wounds in both adult and geriatric rhesus. Moreover, if the wound healing is enhanced in geriatric patients, this finding may be clinically germane to conditions where wound healing is compromised, such as in diabetics and patients on steroids.

Biodegradable positively charged ion exchange beads: a novel biomaterial for enhancing soft tissue repair.

Previous work in the area of vulnerary agents is extensive. One material of focus has been positively charged ion exchange beads, which have been shown to promote a variety of wound-healing responses in several models. The goal of this work was to improve upon the clinical utility of positively charged dextran beads by creating a biodegradable version that maintains the material's inherent efficacy. A chemical method consisting of a sodium periodate oxidation was used to create a degradable diethylaminoethanol crosslinked dextran bead. The ability of this process to create a degradable bead was verified in vitro and in vivo. Furthermore, efficacy was shown in a rat linear incision model for a variety of beads exhibiting different degradation rates. The results show that efficacy is maintained by a degradable bead, but there is a diminution of the magnitude of the response as the mass loss rate is increased. Efficacy also was investigated for a moderate but completely degrading bead material over time and dose. Wound breaking strength was evaluated at days 7, 10, 14, 21, and 28 for degradable beads at doses of 10 mg/mL and 50 mg/mL. Although little difference in efficacy was noted for the increased dose, statistically significant increases over control were seen at days 7, 10, and 14 for the 10 mg/mL dose and at days 10 and 14 for the 50 mg/mL dose. At days 21 and 28 there were no differences between treated and control wounds.

Assessment of an experimental bone wax polymer plus TGF-beta 1 implanted into calvarial defects.

The study reported describes an experimental biodegradable polymer ceramic composite with wax-like handling properties that was combined with 2.0 micrograms of recombinant human transforming growth factor beta (rhTGF-beta(1)). The polymer/rhTGF-beta(1) combination was introduced into standard-sized calvarial defects in rabbits to evaluate biodegradability, biocompatibility, hemostasis control, and bone promotion. The experimental wound model was a standard-size circular calvarial defect 8 mm in diameter. The experimental design included 24 skeletally mature New Zealand white rabbits divided evenly between two time periods (6 and 12 weeks) and among three experimental treatments (untreated defects and defects treated with polymer with or without rhTGF-beta(1)). Evaluations consisted of clinical examinations, standarized radiography, radiomorphometry, as well as histology and histomorphometry. Data were analyzed by an Analysis of Variance (ANOVA) and Fisher's Protected Least Significant Difference test at each time period (level of significance p < or = 0.05). Radiomorphometry data indicated that standard-sized defects treated with the wax-like polymer alone and the polymer plus 2.0 micrograms of TGF-beta(1) were significantly more radiopaque than control sites at both 6 and 12 weeks. Histomorphometric data revealed the amount of new bone was significantly greater at 6 weeks in the polymer plus 2.0 micrograms of TGF-beta(1) and in the control group than in the polymer alone. Moreover, at 12 weeks, there was significantly more new bone in the control than in either the polymer alone or the polymer plus 2.0 micrograms of TGF-beta(1). We speculate the incomplete biodegradation of the polymer ceramic composite contributed to the radiopacity and may have retarded osseous regeneration. It is important that the bone wax-like polymer material was biocompatible and acted as a hemostatic agent.

The effect of patient and provider reminders on mammography and Papanicolaou smear screening in a large health maintenance organization.

BACKGROUND: We evaluated the effectiveness of 2 reminder interventions to increase the use of screening mammograms and Papanicolaou (Pap) smears among female members of a large health maintenance organization. METHODS: Seven thousand seventy-seven female health maintenance organization members (aged 50-74 years with no prior mammogram in the previous 30 months or aged 20-64 years with no prior Pap smear in the previous 36 months) were randomized to receive one of the following: a letter inviting them to make an appointment for a mammogram or a Pap smear; in addition to the letter, a reminder manually placed in the patient's medical chart alerting providers of that member's need for screening; or their usual care. RESULTS: Compared with women who did not receive the reminder letter, women who did receive the letter were more likely to obtain mammograms (16.0% vs 25.5%, respectively; P < .001) or Pap smears (9.1% vs 19.5%, respectively; P < .001) in the 6 months following their entry into the study. Compared with women who received only the reminder letter, women who received a reminder letter and had a reminder placed in their medical chart were more likely to obtain mammograms (26.5% vs 30.9%, respectively; P = .02) and marginally more likely to receive Pap smears (19.5% vs 22.8%, respectively; P = .04). CONCLUSIONS: We recommend the use of patient reminder letters as a first step in a mammography or Pap smear screening outreach program. Further research is needed to evaluate a cost-effective provider reminder system and additional outreach strategies directed to women who do not use health care services.

Differential effects of platelet-derived growth factor BB in accelerating wound healing in aged versus young animals: the impact of tissue hypoxia.

Platelet-derived growth factor BB (PDGF-BB) plays a central role in wound healing. Platelet-derived growth factor has been shown to accelerate healing in preclinical and clinical studies, but its wound-healing effects in older animals have not been examined. An ischemic incisional model in young female (5 months) and aged male (60 months) rabbits was used to determine the influence of platelet-derived growth factor on day 14 postwounding with reduced blood supply. Wounds in aged ischemic animals were severely impaired in breaking strength compared with their nonischemic control wounds and wounds in young animals (p < 0.001). Topical platelet-derived growth factor (10 microg per wound) partially reversed the reduction in breaking strength in aged ischemic animals but was ineffective in young animals. Histologic studies showed that new granulation tissue deposition and mononuclear cell infiltration was dramatically lower in ischemic wounds of aged animals compared with their control wounds as well as wounds from young animals under both nonischemic and ischemic conditions. Platelet-derived growth factor partially reversed this deficit in old ischemic wounds but not in young ischemic wounds. Epithelial growth was reduced in wounds from aged animals compared with wounds from young animals after 14 days of healing. Platelet-derived growth factor treatment increased ischemic wound epithelial growth in aged ischemic animals about threefold compared with paired controls. In conclusion, wound healing in aged rabbits was severely impaired by ischemia, and a single topical platelet-derived growth factor treatment partially reversed this deficit.

Initial biocompatibility studies of a novel degradable polymeric bone substitute that hardens in situ.

Clinical management of osseous defects often requires bone grafts. The standard for treatment is autogenous bone harvested from sites such as either the iliac crest or the outer table of the calvaria. In addition to the problem of donor site morbidity and the limited supply of graft material, there is the additional operating time associated with harvesting procedures. A synthetic, bone graft substitute that can match the clinical performance of autogenous bone could alleviate these deficiencies. Therefore, a polymeric bone substitute was developed that consists of a four-armed star polymer of poly(dioxanone-co-glycolide) endcapped at each termini with a biocompatible lysine-based diisocyanate crosslinker. The polymer can be mixed with inorganic fillers such as either hydroxyapatite or tricalcium phosphate to form either injectable or moldable putty. The addition of a catalyst (for example, diethylaminoethanol and water) to the polymer produces a crosslinking reaction causing the combination to harden. This reaction is nontoxic, normo-thermic and can be performed in situ. During the course of the polymerization, carbon dioxide is liberated, producing an interconnected porous network within the implant, suitable for bone ingrowth. This paper will describe a preliminary biocompatibility assay of the bone substitute.

Differential expression of platelet-derived growth factor receptor-beta in an aging model of wound repair.

Impaired wound healing as a result of age is a well-documented phenomenon. However, the overall deficit in healing is substantially increased when the healing wound of an aged animal is ischemic. We hypothesized that both of these deficits are cytokine mediated. We have studied the messenger RNA expression of platelet-derived growth factor receptor-beta, using the rabbit dermal ulcer model of wound repair, in young (3 to 6 months) and aged (48 months and 60 months) rabbits under normal and ischemic conditions. Platelet-derived growth factor receptor-beta mRNA expression was measured with the use of quantitative reverse transcriptase-polymerase chain reaction with incorporation of a synthetic, nonhomologous DNA fragment complementary to platelet-derived growth factor receptor-beta primers as a competitive internal standard. Results in young rabbits showed a large upregulation of platelet-derived growth factor receptor-beta mRNA expression after wounding. In both aged animal groups, platelet-derived growth factor receptor-beta expression was found to be significantly decreased in nonischemic wounds relative to young nonischemic controls. Ischemia was found to have little effect on platelet-derived growth factor receptor-beta mRNA expression in young animals relative to matched controls. However, ischemia induced a large decrease in the platelet-derived growth factor receptor-beta mRNA levels of wounds of aged animals relative to paired aged nonischemic wounds. Results suggest an age-related delay in platelet-derived growth factor receptor-beta mRNA expression in healing wounds, as well as an age-related decline in responsiveness to confounding ischemia.

Differential effects of oxygen on human dermal fibroblasts: acute versus chronic hypoxia.

The observation that many chronic wounds are ischemic has spurred a series of studies evaluating the response of cells exposed to hypoxia. To date, these studies have shown largely beneficial effects from hypoxia, such as increased cellular replication and procollagen synthesis. These findings are counter-intuitive from a clinical standpoint because cellular growth and synthetic function are known to be retarded in chronic ischemic wounds. We have established an in vitro system in which human dermal fibroblasts grown chronically at 5 +/- 3 mm Hg will proliferate at a rate three times slower than those fibroblasts grown under standard culture conditions (namely an oxygen partial pressure of 150 mm Hg). No phenotypic changes are noted in chronically hypoxic cells, and the growth-retarding effects are reversible when the cells are returned to standard oxygen conditions. Competitive reverse transcription-polymerase chain reaction showed that acute exposure to hypoxia (up to 1 week) results in a 6.3-fold increase in the relative expression of transforming growth factor-beta1 messenger RNA, whereas chronic exposure to hypoxia leads to a 3.1-fold decrease in this message. Collagen production measured at both the mRNA and protein level is also decreased in the setting of chronic hypoxia. We propose that this system may be the most appropriate setting for studying the role of oxygen on dermal fibroblasts in ischemic, nonhealing wounds.

Role of bone substitutes.

Approximately 500 million years ago, the Paleozoic era heralded an evolutionary marvel: the skeleton. Unique to this evolutionary development was the capacity for regeneration: the physiologic renewal of embryologically derived tissue. Many of the cellular and molecular components for bone regeneration have been identified (bone morphogenetic proteins), and their therapeutic manipulation will become common clinical practice. Moreover, synthetic materials produced in the laboratory and novel bone derivatives will be used to exploit the skeleton's capacity to regenerate and repair. The concept of repair may be viewed as the restoration of form and function to deficient osseous tissue. Materials that provoke repair can be categorized broadly as bone substitutes. In this review, bone substitutes are grouped into 2 categories, polymers and ceramics, and each is subclassified as biodegradable or nonbiodegradable. Examples of these materials are provided as well as some of their liabilities and virtues.

B-DNA to Z-DNA structural transitions in the SV40 enhancer: stabilization of Z-DNA in negatively supercoiled DNA minicircles.

During replication and transcription, the SV40 control region is subjected to significant levels of DNA unwinding. There are three, alternating purine-pyrimidine tracts within this region that can adopt the Z-DNA conformation in response to negative superhelix density: a single copy of ACACACAT and two copies of ATGCATGC. Since the control region is essential for both efficient transcription and replication, B-DNA to Z-DNA transitions in these vital sequence tracts may have significant biological consequences. We have synthesized DNA minicircles to detect B-DNA to Z-DNA transitions in the SV40 enhancer, and to determine the negative superhelix density required to stabilize the Z-DNA. A variety of DNA sequences, including the entire SV40 enhancer and the two segments of the enhancer with alternating purine-pyrimidine tracts, were incorporated into topologically relaxed minicircles. Negative supercoils were generated, and the resulting topoisomers were resolved by electrophoresis. Using an anti-Z-DNA Fab and an electrophoretic mobility shift assay, Z-DNA was detected in the enhancer-containing minicircles at a superhelix density of -0.05. Fab saturation binding experiments demonstrated that three, independent Z-DNA tracts were stabilized in the supercoiled minicircles. Two other minicircles, each with one of the two alternating purine-pyrimidine tracts, also contained single Z-DNA sites. These results confirm the identities of the Z-DNA-forming sequences within the control region. Moreover, the B-DNA to Z-DNA transitions were detected at superhelix densities observed during normal replication and transcription processes in the SV40 life cycle.

Selective metal binding to Cys-78 within endonuclease V causes an inhibition of catalytic activities without altering nontarget and target DNA binding.

T4 endonuclease V is a pyrimidine dimer-specific DNA repair enzyme which has been previously shown not to require metal ions for either of its two catalytic activities or its DNA binding function by virtue of its ability to function in the presence of metal-chelating agents. However, we have investigated whether the single cysteine within the enzyme was able to bind metal salts and influence the various activities of this repair enzyme. A series of metals (Hg2+, Ag+, Cu+) were shown to inactivate both endonuclease Vs pyrimidine dimer-specific DNA glycosylase activity and the subsequent apurinic nicking activity. The binding of metal to endonuclease V did not interfere with nontarget DNA scanning or pyrimidine dimer-specific binding. The Cys-78 codon within the endonuclease V gene was changed by oligonucleotide site-directed mutagenesis to Thr-78 and Ser-78 in order to determine whether the native cysteine was directly involved in the enzyme's DNA catalytic activities and whether the cysteine was primarily responsible for the metal binding. The mutant enzymes were able to confer enhanced ultraviolet light (UV) resistance to DNA repair-deficient Escherichia coli at levels equal to that conferred by the wild type enzyme. The C78T mutant enzyme was purified to homogeneity and shown to be catalytically active on pyrimidine dimer-containing DNA. The catalytic activities of the C78T mutant enzyme were demonstrated to be unaffected by the addition of Hg2+ or Ag+ at concentrations 1000-fold greater than that required to inhibit the wild type enzyme. These data suggest that the cysteine is not required for enzyme activity but that the binding of certain metals to that amino acid block DNA incision by either preventing a conformational change in the enzyme after it has bound to a pyrimidine dimer or sterically interfering with the active site residue's accessibility to the pyrimidine dimer.

Molecular analysis of plasmid DNA repair within ultraviolet-irradiated Escherichia coli. I. T4 endonuclease V-initiated excision repair.

The process by which DNA-interactive proteins locate specific sequences or target sites on cellular DNA within Escherichia coli is a poorly understood phenomenon. In this study, we present the first direct in vivo analysis of the interaction of a DNA repair enzyme, T4 endonuclease V, and its substrate, pyrimidine dimer-containing plasmid DNA, within UV-irradiated E. coli. A pyrimidine dimer represents a small target site within large domains of DNA. There are two possible paradigms by which endonuclease V could locate these small target sites: a processive mechanism in which the enzyme "scans" DNA for dimer sites or a distributive process in which dimers are located by random three-dimensional diffusion. In order to discriminate between these two possibilities in E. coli, an in vivo DNA repair assay was developed to study the kinetics of plasmid DNA repair and the dimer frequency (i.e. the number of dimer sites on a given plasmid molecule) in plasmid DNA as a function of time during repair. Our results demonstrate that the overall process of plasmid DNA repair initiated by T4 endonuclease V (expressed from a recombinant plasmid within repair-deficient E. coli) occurs by a processive mechanism. Furthermore, by reducing the temperature of the repair incubation, the endonuclease V-catalyzed incision step has been effectively decoupled from the subsequent steps including repair patch synthesis, ligation, and supercoiling. By this manipulation, it was determined that the overall processive mechanism is composed of two phases: a rapid processive endonuclease V-catalyzed incision reaction, followed by a slower processive mechanism, the ultimate product of which is the dimer-free supercoiled plasmid molecule.

Molecular analysis of plasmid DNA repair within ultraviolet-irradiated Escherichia coli. II. UvrABC-initiated excision repair and photolyase-catalyzed dimer monomerization.

In this study, a novel approach to the analysis of DNA repair in Escherichia coli was employed which allowed the first direct determination of the mechanisms by which endogenous DNA repair enzymes encounter target sites in vivo. An in vivo plasmid DNA repair analysis was employed to discriminate between two possible mechanisms of target site location: a processive DNA scanning mechanism or a distributive random diffusion mechanism. The results demonstrate that photolyase acts by a distributive mechanism within E. coli. In contrast, UvrABC-initiated excision repair occurs by a limited processive DNA scanning mechanism. A majority of the dimer sites on a given plasmid molecule were repaired prior to the dissociation of the UvrABC complex. Furthermore, plasmid DNA repair catalyzed by the UvrABC complex occurs without a detectable accumulation of nicked plasmid intermediates despite the fact that the UvrABC complex generates dual incisions in the DNA at the site of a pyrimidine dimer. Therefore, the binding or assembly of the UvrABC complex on DNA at the site of a pyrimidine dimer represents the rate-limiting step in the overall process of UvrABC-initiated excision repair in vivo.

T4 endonuclease V promotes the formation of multimeric DNA structures.

Electron microscopy of UV-irradiated circular DNA molecules which had been treated with T4 endonuclease V revealed the formation of multimeric DNA structures in addition to the expected conversion of the superhelical DNA molecules into nicked circular and linear forms. The multimeric DNA molecules could be distinguished in electron micrographs from catenated molecules which were present in the original DNA preparation by a combination of rotary and single angle heavy metal shadowing. The complexity and frequency of these structures increased with time of reaction with endonuclease V. Their formation, as well as the endonuclease activity of enzyme, was dependent on UV irradiation of the DNA, and the complexes could be disrupted by prior phenol extraction and ethanol precipitation. Preparations of endonuclease V estimated to be 98% pure by mass promoted the same complex formation between DNA molecules as did preparations estimated to be only 5-10% pure. In addition to these intermolecular structures, the formation of complexes between regions on the same DNA molecules was manifest as discrete double-stranded 'loops' 200-300 base pairs in length. DNA 'bubble structures' were also observed and may represent folding of the 'loops' onto adjacent segments of DNA. These results suggest that at least one active form of T4 endonuclease V may be a multimeric complex of enzyme molecules in association with DNA.