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H W Chen

Publications and source records attributed to H W Chen.

At least 55 records · Page 3Linked to original sources
PubMed2000

Structure-function analysis of yeast mRNA cap methyltransferase and high-copy suppression of conditional mutants by AdoMet synthase and the ubiquitin conjugating enzyme Cdc34p.

Here we present a genetic analysis of the yeast cap-methylating enzyme Abd1p. To identify individual amino acids required for Abd1p function, we introduced alanine mutations at 35 positions of the 436-amino acid yeast protein. Two new recessive lethal mutations, F256A and Y330A, were identified. Alleles F256L and Y256L were viable, suggesting that hydrophobic residues at these positions sufficed for Abd1p function. Conservative mutations of Asp-178 established that an acidic moiety is essential at this position (i.e. , D178E was viable whereas D178N was not). Phe-256, Tyr-330, and Asp-178 are conserved in all known cellular cap methyltransferases. We isolated temperature-sensitive abd1 alleles and found that abd1-ts cells display a rapid shut-off of protein synthesis upon shift to the restrictive temperature, without wholesale reduction in steady-state mRNA levels. These in vivo results are consistent with classical biochemical studies showing a requirement for the cap methyl group in cap-dependent translation. We explored the issue of how cap methylation might be regulated in vivo by conducting a genetic screen for high-copy suppressors of the ts growth defect of abd1 mutants. The identification of the yeast genes SAM2 and SAM1, which encode AdoMet synthase, as abd1 suppressors suggests that Abd1p function can be modulated by changes in the concentration of its substrate AdoMet. We also identified the ubiquitin conjugating enzyme Cdc34p as a high-copy abd1 suppressor. We show that mutations of Cdc34p that affect its ubiquitin conjugation activity or its capacity to interact with the E3-SCF complex abrogate its abd1 suppressor function. Moreover, the growth defect of abd1 mutants is exacerbated by cdc34-2. These findings suggest a novel role for Cdc34p in gene expression and engender a model whereby cap methylation or cap utilization is negatively regulated by a factor that is degraded when Cdc34p is overexpressed.

Alanine↗
PubMed2000

Suppression of altered hepatic foci development by a high fish oil diet compared with a high corn oil diet in rats.

Effects of low corn oil, high corn oil, and high fish oil diets on altered hepatic foci development in female Sprague-Dawley rats were investigated. Rats assigned to Groups 1-4 were initiated with saline as the control and those assigned to Groups 5-7 were initiated with diethylnitrosamine (DEN 15 mg/kg) at 24 hours of age. After weaning, all rats, except those in Group 1, received 500 ppm phenobarbital (PB) in their diet as tumor promoter for three months. Altered hepatic foci development was significantly lower in DEN-initiated rats fed the high fish oil + PB diet than in DEN-initiated rats fed the high corn oil + PB diets. Liver weight and relative liver weight were significantly greater in rats fed the high fish oil + PB diet than in rats fed the other diets, and hepatic biotransformation/detoxification enzyme activities were greater in rats fed the fish oil + PB diets than in rats fed the other diets. These results suggest that the effect of a high fish oil diet on altered hepatic foci may occur through regulation of hepatic biotransformation/detoxification enzyme activities, leading to alteration in the tumor-promoting action of PB. Dietary lipid significantly affected the hepatic phospholipid fatty acid composition of rats. n-3 polyunsaturated fatty acids were incorporated into membrane phospholipid at the expense of n-6 polyunsaturated fatty acids. A high fish oil diet caused greater oxidative stress in rats, as measured by plasma vitamin E level, red blood cell glutathione status, liver lipid peroxidation, and hepatic glutathione reductase activity. Pearson's correlation analysis indicated that the foci number was negatively correlated to the liver thiobarbituric acid-reactive substance and 7-pentoxyresorufin O-dealkylase activity, and the foci area was negatively correlated to the liver thiobarbituric acid-reactive substance activity (p < 0.05) in rats of groups that developed foci. These results suggest that the type of dietary lipid is the more important determinant for gamma-glutamyl transpeptidase-positive foci development than the amount of dietary lipid when rats consumed approximately the same amount of calories in all the dietary groups, and the underlying mechanisms may be partially ascribed to the antioxidant/oxidation status and biotransformation/detoxification system of rats.

Animals↗
PubMed2000

Attenuation of sepsis-induced apoptosis by heat shock pretreatment in rats.

Apoptosis is a process by which cells undergo a form of non-necrotic cellular suicide. Although it is a programmed process, apoptosis can be induced by various stressors. During sepsis, apoptosis has been regarded as an important cause of cell death in the immune system, leading to unresponsiveness to treatment. This study was designed to investigate how prior heat shock induction can influence the rate of apoptosis in animals that have experienced sepsis. Sprague-Dawley rats were used, and experimental sepsis was induced by cecal ligation and puncture (CLP). Animals in the heated group were anesthetized and received heat shock by whole-body hyperthermia. They were sacrificed 9 h and 18 h after CLP as early and late sepsis, respectively. Apoptosis was evaluated by "DNA ladder" detection in agarose electrophoresis and Tdt-mediated dUTP nick end-labeling (TUNEL) assay. Hsp72 was detected by Western blot analysis. The results showed that the DNA ladder was detected most clearly in the thymus at the late phase of sepsis with time course dependence, while it showed less clearly in heat shock treated animals. Histopathological study by TUNEL assay obtained similar results in the thymus, where the cortex was more susceptible to apoptosis than the medulla. The Western blot analysis showed that the heat shock induced Hsp72 concomitant with an increase in Bcl-2:Bax ratio. In conclusion, heat shock pretreatment prevents rats from sepsis-induced apoptosis that may account for the better outcome of experimental sepsis. An increase in the Bcl-2:Bax ratio may in part explain the molecular mechanism of the effect of heat shock pretreatment.

Animals↗
PubMed2000

[The medium optimization of xylitol fermentation based on neural networks and genetic algorithms].

Using genetic algorithms(GA) for medium optimization of xylitol fermentation, and coupling neural networks model for predicting xylitol concentration is introduced. The medium compose determined by GA is as input data of the neural networks, while the output data predicted by neural networks is as suitable value of GA for predicting. The optimum medium is further validated by experimentation. The good result, which save the experimental workload and charge, enhance the level of xylitol fermentation as well as reduced the medium consume is obtained.

Algorithms↗
PubMed2000

The role of free to total prostate-specific antigen ratio for prostate cancer in screening patients with total serum levels between 4 and 20 ng/ml.

BACKGROUND: We retrospectively tried to determine if the free to total prostate-specific antigen (f/t PSA) ratio could improve the specificity of PSA in prostate cancer screening of patients with total serum levels between 4 and 20 ng/ml. METHODS: Two hundred ninety-five patients with serum PSA levels from 4 to 20 ng/ml had undergone sextant prostate needle biopsy. Each patient had no prior history of prostate cancer, acute urine retention, or prostatitis. Prebiopsy free PSA values were measured in 155 patients. Total PSA levels were determined with the AxSYM enzyme-linked immunosorbent assay. Free PSA levels were measured with the AxSYM microparticle enzyme immunoassay. RESULTS: Mean f/t PSA ratios were 0.114+/-0.004 in men of the cancer group and 0.161+/-0.008 in men of the benign group (p<0.002). Based on the analysis of sensitivity and specificity in relation to f/t PSA ratios, use of the 18% cutoff point could detect 89% of cancer cases, and at the same time could avoid 35% of unnecessary prostate biopsies. The areas under the receiver-of-characteristic curve for f/t PSA ratio and total PSA were 0.649 and 0.545, respectively. CONCLUSION: Serum f/t PSA ratios were significantly lower in patients with prostate cancer than in patients with benign disease. The determination of an appropriate f/t PSA ratio should be based on the generated data such as that demonstrated in this study in order to improve diagnostic accuracy and specificity for patients with equivocal PSA values and to avoid conducting unnecessary prostate biopsies.

Aged↗
PubMed1999

Analysis of the steps involved in Dengue virus entry into host cells.

The initial steps of dengue viral entry have been divided into adsorption and penetration using acid glycine treatment to inactivate extracellular virus after attachment to baby hamster kidney (BHK) cells but prior to penetration. First, we showed that virus infection was accomplished within 2 h after adsorption. Second, the assay was used to examine the properties of dengue envelope E protein-specific monoclonal antibodies (MAbs), lectins, and heparin. We found that three MAbs, 17-2, 46-9, and 51-3, may neutralize dengue 2 virus (DEN-2) through inhibition of not only viral attachment but also of penetration. However, one MAb, 56-3.1, interfered specifically with attachment. Therefore, the functional domains of E protein involved in attachment and penetration may be different. Moreover, studies with lectins indicated that carbohydrates, especially alpha-mannose residues, present on the virion glycoproteins may contribute to binding and penetration of the virus into BHK and mosquito C6/36 cells. Finally, virus infectivity was inhibited by heparin through its blocking effects at both virus attachment and penetration. This suggests that cell surface heparan sulfate functions in both viral attachment and penetration of DEN-2 virus. In conclusion, our results further elucidated some aspects of the dengue virus entry process.

Adsorption↗
PubMed1999

Evidence of multi-step regulation of HSP72 expression in experimental sepsis.

Heat shock proteins (Hsps) are a family of highly conserved proteins induced in response to various stresses. Hsps protect cells against subsequent lethal circumstances. Previous work from our laboratory has indicated that Hsp72 is not induced during experimental sepsis in rats, but the regulation of the induction of Hsp72 synthesis in this disease cascade has not been investigated. In the present study, we evaluated the expression of the hsp72gene, focusing on the activation and DNA-binding ability of heat shock factor 1 (HSF1), hsp mRNA accumulation, and Hsp72 synthesis in animal sepsis models induced by cecal ligation and puncture procedure. The results were compared with those of sham-treated and heat-shocked rats. It was shown that the expression of the hsp72 gene in sepsis was a multi-step process, as previously documented in in vitro studies. Hsp72 synthesis was not induced during sepsis, whereas DNA binding of HSF was detectable, suggesting that the induction of Hsp72 is blocked downstream to HSF-DNA complex formation by the metabolic alteration occurring during sepsis. The dissociation failure of the constitutive heat shock element binding factor (CHBF) from the heat shock element may play an important role in this negative regulation.

Animals↗
PubMed1999

Screening of protective antigens of Japanese encephalitis virus by DNA immunization: a comparative study with conventional viral vaccines.

In this study, we evaluated the relative role of the structural and nonstructural proteins of the Japanese encephalitis virus (JEV) in inducing protective immunities and compared the results with those induced by the inactivated JEV vaccine. Several inbred and outbred mouse strains immunized with a plasmid (pE) encoding the JEV envelope protein elicited a high level of protection against a lethal JEV challenge similar to that achieved by the inactivated vaccine, whereas all the other genes tested, including those encoding the capsid protein and the nonstructural proteins NS1-2A, NS3, and NS5, were ineffective. Moreover, plasmid pE delivered by intramuscular or gene gun injections produced much stronger and longer-lasting JEV envelope-specific antibody responses than immunization of mice with the inactivated JEV vaccine did. Interestingly, intramuscular immunization of plasmid pE generated high-avidity antienvelope antibodies predominated by the immunoglobulin G2a (IgG2a) isotype similar to a sublethal live virus immunization, while gene gun DNA immunization and inactivated JEV vaccination produced antienvelope antibodies of significantly lower avidity accompanied by a higher IgG1-to-IgG2a ratio. Taken together, these results demonstrate that the JEV envelope protein represents the most critical antigen in providing protective immunity.

Animals↗
PubMed1999

Lack of effect of dietary alpha-tocopherol on chemically induced hepatocarcinogenesis in rats.

We investigated the effects of alpha-tocopherol on diethylnitrosamine (DEN) initiation-phenobarbital (PB) promotion of hepatic foci in female Sprague-Dawley rats. Groups of eight rats were initiated with DEN (15 mg/kg) at 24 hours of age. After weaning, they received diets containing 500 ppm PB and various concentrations of alpha-tocopherol, deficient (0 ppm), adequate (100 ppm), and supplemented (5,000 ppm), for 24 weeks. Rats fed alpha-tocopherol-supplemented diets had significantly greater hepatic alpha-tocopherol levels than those fed alpha-tocopherol-deficient or -adequate diets (p < 0.05). Liver lipid peroxidation (measured as thiobarbituric acid-reactive substances) was significantly greater in rats fed alpha-tocopherol-deficient diets than in those fed alpha-tocopherol-adequate or -supplemented diets (p < 0.05). The dietary alpha-tocopherol level had no significant effect on the ratios of reduced glutathione (GSH) to oxidized GSH or reduced GSH to total GSH in the liver or on the plasma prostaglandin E2 concentration or on the activities of hepatic cytosolic and particulate protein kinase C. Rats fed alpha-tocopherol-adequate or -supplemented diets had significantly greater hepatic glutathione S-transferase, GSH reductase, and GSH peroxidase activities than those fed alpha-tocopherol-deficient diets (p < 0.05). The dietary alpha-tocopherol level did not significantly affect the formation of hepatic gamma-glutamyl transpeptidase- and placental glutathione S-transferase-positive foci. These results suggest that alpha-tocopherol does not influence hepatic foci formation and that reactive oxygen species may not be the underlying mechanism of hepatic foci formation in this DEN initiation-PB promotion model of hepatocarcinogenesis.

Animals↗
PubMed1999

Effects of garlic oil and its organosulfur compounds on the activities of hepatic drug-metabolizing and antioxidant enzymes in rats fed high- and low-fat diets.

We examined the effects of garlic oil (GO) and two of its organosulfur compounds, diallyl sulfide (DAS) and diallyl disulfide (DADS), on the drug-metabolizing and antioxidant systems in rats and sought to determine whether these effects are associated with dietary fat. Rats were fed a high-fat diet and received GO or DADS (200 mg/kg body wt) or DAS (100 mg/kg) orally three times a week for seven weeks. Control animals received corn oil alone. Another group of rats was fed a low-fat diet, with or without GO. GO and DADS significantly reduced the body weight gain of rats (p < 0.05). GO, however, dramatically increased the spleen weight and spleen weight-to-body weight ratio (p < 0.05). DAS increased glutathione S-transferase (GST) and 7-pentoxyresorufin O-dealkylase activities, whereas DADS increased only GST activity (p < 0.05). Immunoblot assay showed GO-, DAS-, and DADS-enhanced expression of the placental form of GST and cytochrome P-450 IIBI but suppressed cytochrome P-450 IIEI expression. Hepatic antioxidant enzyme activities were also modulated by these garlic components. GO and DADS inhibited glutathione peroxidase activity (p < 0.05), and DADS and DAS enhanced glutathione reductase activity (p < 0.05). Only GO enhanced the superoxide dismutase activity (p < 0.05). All these garlic components increased glutathione levels in red blood cells (p < 0.05) but did not influence hepatic glutathione levels. Although the amount of fat in the diet modulated drug-metabolizing and antioxidant functions, no interactions between GO and dietary fat were observed. These results indicate that GO and its allyl sulfide components, as well as dietary lipid, modulate drug-metabolizing and antioxidant enzyme activities. The action of GO appears to be independent of dietary lipid content.

Allyl Compounds↗
PubMed1999

Prognostic factors and strategy of treatment in Fournier's gangrene: a 12-year retrospective study.

BACKGROUND: Fournier's gangrene (FG) is a fulminant and fatal infection of the genitalia. However, the clinical course is unpredictable. This study retrospectively analyzed the possible prognostic factors of FG. METHODS: Data obtained from 57 patients treated for FG from January 1985 through December 1996 were retrospectively analyzed. Possible prognostic factors including age, diagnostic delay, hospital stay, underlying diseases, clinical symptoms, origins, extents, bacteriologic findings, diverting colostomy and mortality rate were all considered in the analysis. RESULTS: Patients with extensive or localized FG had mortality rates of 31.3% and 16.0%, respectively (p = 0.227). The mortality rates of patients with FG of anorectal, urogenital and non-specific origin were 30.3%, 0% and 40.0%, respectively (p = 0.712). The mortality rates of patients with FG of anorectal origin who received primary or secondary diverting colostomy were 16.7% and 40.0%, respectively. However, the mortality rate of patients with FG of anorectal origin who did not undergo diversion was 29.4%. The mortality rate of patients with FG presenting with septic shock at emergency was 53.8% as compared with 0% in those without septic shock (p < 0.001). CONCLUSION: Fournier's gangrene is a rapidly progressive and life threatening infection of the genitalia. Age, underlying diseases, origin, extent and fecal diversion can not be regarded as prognostic factors of FG. Early primary diverting colostomy may reduce the mortality rate in those with severe infection of anorectal origin. Presence of septic shock in those with FG is the most important and the only factor related to death.

Adult↗
PubMed1999

Previous hyperthermic treatment increases mitochondria oxidative enzyme activity and exercise capacity in rats.

The study was designed to investigate the role of hyperthermia in the tolerance of exercise in rats and the possible mechanism was examined. The hyperthermic pretreatment was performed using an electric pad on the anesthesized rats 24 hours before exercise. Rats were exercised passively in a motor-controlled round treadmill in high temperature environment (36-37 degrees C) until exhaustion. The capacity of tolerance was calculated. Lymphocytes and gastrocnemius muscle were collected from both groups. The changes in muscular morphology, mitochondria oxidative enzyme activity and induction of Hsp72 were investigated. The results revealed that experimental rats were more tolerant to exercise at high temperature than the control group; the duration time were 89 +/- 17.8 min and 63.1 +/- 7.3 min, respectively. Hsp72 was induced markedly in the experimental group, both in muscle and lymphocytes, indicating a heat shock response. There was no significant change in morphology of the mitochondria, 24 hours after hyperthermic treatment, as shown by histopathological and electromicroscopic investigation. However, the activity of mitochondrial enzymes increased significantly in experimental group before exercise: 84.6 +/- 6.3 and 345 +/- 15.4 (nmole cytochrome c/min/mg total protein) respectively of NADH-cytochome c reductase and succinate-cytochome c reductase activity in experimental group compared to 58.9 +/- 4.7 and 269.0 +/- 24.0 in control group (p < 0.05, by student t-test). It is concluded that hyperthermic treatment enhances muscular mitochondrial oxidative enzyme activity in rats, and results in increasing tolerance to exercise at high temperature. The heat shock response, most probably the inducible Hsp72, is a crucial factor in this effect.

Adenosine Triphosphate↗
PubMed1999

Enterovesical fistula: experiences with 41 cases in 12 years.

BACKGROUND: A retrospective analysis of enterovesical fistula treated at Chang Gung Memorial Hospital was conducted to determine the optimal diagnosis and management of this disease. METHODS: The records of 41 patients who presented from 1984 to 1996 and had a final diagnosis of enterovesical fistula were retrospectively reviewed. The etiology, symptoms on presentation, diagnostic tools, and modality of treatment were analyzed. RESULTS: The majority of these cases were associated with malignancy (38, 92.7%), and the others with diverticulitis (2, 4.9%) and iatrogenic causes (1, 2.4%). In those with malignancy, 15 patients (39.5%) were found to have tumor recurrence. The most frequent symptom in enterovesical fistula was fecaluria (58.5%), followed by abdominal pain (22%) and dysuria (14.6%). Diagnostic tools included the barium enema, cystography, and cystoscopy; these methods could identify the fistula in 63.2%, 60%, and 53.8% of the patients, respectively. Methods of management included diversion only (39%), one-stage fistula repair (36.6%), and watchful surveillance (24.4%). CONCLUSION: Enterovesical fistula should be considered if fecaluria, pneumaturia, or persistent non-specific urinary tract infection present as the initial complaint. A thorough surgery for a possible underlying malignancy is mandatory when confronted with enterovesical fistula, since the incidence of inflammatory bowel disease is low in this area. An abdominal computer tomography (CT) scan, barium enema, and cystogram can be useful diagnostic tools. Treatment of this entity should be individualized according to each patients clinical status.

Adult↗
PubMed1999

Modulation of immune responses to DNA vaccines by codelivery of cytokine genes.

DNA vaccines containing genes for antigenic portions of viruses have recently been developed as a novel vaccination technology. Direct injection of plasmid DNA in vivo results in prolonged expression of viral proteins and may, thus, mimic the action of attenuated vaccines. An important advantage of this vaccination method is that in vivo-synthesized viral proteins can enter both major histocompatibility complex (MHC) class I and class II antigen-processing pathways to activate specific immunization. In many animal models for infectious diseases, DNA vaccines induced a broad range of immune responses, including antibody, CD8+ cytotoxic T lymphocytes (CTL) and CD4+ helper T (Th) lymphocyte responses, and protective immunity against challenge with the pathogen. The magnitude and nature of these immune responses to DNA vaccines can be further manipulated by codelivery of cytokine genes. Summarizing the many studies reported to date, we can draw conclusions regarding the adjuvant effects of these cytokine genes on DNA vaccines. Coadministration of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-2 genes induces higher antibody titers and T-cell proliferation responses than other cytokine genes tested to date. In contrast, the CTL activity is only modestly increased by the GM-CSF and IL-2 genes. The IL-12 gene polarizes the immune responses to DNA vaccines toward Th1 cell development and stimulates the strongest CTL activity. In contrast, co-injection of the IL-4 gene promotes the development of Th2 cells and increases production of antibodies, but suppresses CTL activity. Thus, the immune responses to DNA vaccines can be engineered by co-injection of an appropriate cytokine gene to favor the formation of either CTL or neutralization antibodies and, therefore, provide the best protection against a particular pathogen.

Cytokines↗
PubMed1998

Alterations of small-molecular-weight antioxidants in the blood of smokers.

Plasma alpha-tocopherol, ascorbate, retinol, uric acid, and lipid peroxides were investigated in 39 male smokers and 64 male non-smokers. The average level of plasma alpha-tocopherol of 35-45-year-old smokers (1.74+/-0.49 microg/mg total lipid) was significantly lower than that of age-matched non-smokers (2.55+/-0.88 microg/mg total lipid, P = 0.032). Similarly, the plasma alpha-tocopherol of smokers aged above 45 (1.66+/-0.29 microg/mg total lipid) was lower than that of the age-matched non-smokers (2.38+/-1.26 microg/mg total lipid, P = 0.014). However, no difference in plasma level of alpha-tocopherol was found between smokers and non-smokers below the age of 35. The average concentration of ascorbate in plasma was significantly decreased only in those smokers older than 45 (0.33+/-0.16 mg/dl vs non-smokers 0.53+/-0.19 mg/dl, P = 0.003). The average level of lipid peroxides (measured as malondialdehyde,MDA) in the plasma of smokers (2.77+/-0.51 nmol/ml) was higher than that of non-smokers (2.35+/-0.21 nmol/ml) aged above 45 (P = 0.034). No differences in the plasma levels of uric acid and retinol were noted between smokers and non-smokers in all age groups. Using partial correlation analysis under age control, we found that the plasma level of alpha-tocopherol was negatively correlated with the plasma level of MDA (r = -0.523, P = 0.038). In contrast, the plasma level of ascorbate was only weakly correlated with the plasma level of MDA (r = -0.341, P = 0.094). Moreover, we found a negative correlation between the plasma level of alpha-tocopherol and smoking index (r = -0.414, P = 0.006) under age control, but there was no correlation between plasma level of ascorbate and smoking index (r=0.221, P = 0.193). These results indicate that adequate levels of alpha-tocopherol and ascorbate may protect the plasma from oxidative damage elicited by smoking-mediated reactive oxygen species (ROS) and free radicals in young smokers. However, the antioxidant activities of alpha-tocopherol and ascorbate may be overwhelmed by the long-standing oxidative stress elicited by cigarette smoking in elderly subjects.

Adult↗
PubMed1998

Functions of the ATP hydrolysis subunits (RecB and RecD) in the nuclease reactions catalyzed by the RecBCD enzyme from Escherichia coli.

The RecBCD enzyme from Escherichia coli is an ATP-dependent nuclease and helicase. Two of its subunits, the RecB and RecD proteins, are DNA-dependent ATPases. We have purified RecB and RecD proteins with mutations in their consensus ATP binding sites to study the functions of these subunits in the ATP-dependent nuclease activities of RecBCD. Reconstituted heterotrimeric enzymes were prepared by mixing wild-type RecB or RecB-K29Q mutant protein (RecB*) with purified RecC protein, and with a histidine-tagged wild-type RecD (hD) or mutant hRecD-K177Q (hD*) protein. RecBCD and all four reconstituted enzymes (wild-type, two single mutants, and the double mutant) cleave a single-stranded DNA oligomer substrate (25-mer) in the absence of ATP at rates of 0.03 to 0.06 min-1. The nuclease reaction catalyzed by RecB*ChD* is not stimulated significantly by ATP, while the reactions catalyzed by RecBCD, RecBChD, RecBChD*, and RecB*ChD are 300 to 3000 fold faster in the presence of 0.5 mM ATP. RecB*ChD* also has very low ATP hydrolysis activity (approximately 10(3)-fold less than RecBCD), as do the individual mutant RecB* and hRecD* proteins (approximately 100-fold less than RecB or hRecD). The products from the ATP-stimulated nuclease reaction with the oligomer substrate suggest a mechanism where two DNA molecules bind to the enzyme in opposite orientations and are cleaved by the nuclease active site. Cleavage towards the 3'-end of one oligomer (observed with RecBChD*) depends on the wild-type RecB subunit, while RecD-dependent cleavage (observed with RecB*ChD) occurs towards the 5'-end of the second bound oligomer.

Adenosine Triphosphate↗