[Recompensation of complications in patients with hepatitis B virus-related decompensated cirrhosis treated with entecavir antiviral therapy].

Objective: To analyze the occurrence of recompensation conditions in patients with chronic hepatitis B virus-related decompensated cirrhosis after entecavir antiviral therapy. Methods: Patients with hepatitis B virus-related decompensated cirrhosis with ascites as the initial manifestation were prospectively enrolled. Patients who received entecavir treatment for 120 weeks and were followed up every 24 weeks (including clinical endpoint events, hematological and imaging indicators, and others) were calculated for recompensation rates according to the Baveno VII criteria. Measurement data were compared using the Student t-test or Mann-Whitney U test between groups. Categorical data were compared by the χ (2) test or Fisher's exact probability method between groups. Results: 283 of the 320 enrolled cases completed the 120-week follow-up, and 92.2% (261/283) achieved a virological response (HBV DNA 20 IU/ml). Child-Pugh and MELD scores were significantly improved after treatment (8.33 ± 1.90 vs. 5.77 ± 1.37, t = 12.70, P < 0.001; 13.37 ± 4.44 vs. 10.45 ± 4.58, t = 5.963, P < 0.001). During the 120-week follow-up period, 14 cases died, two received liver transplants, 19 developed hepatocellular cancer, 11 developed gastroesophageal variceal bleeding, and four developed hepatic encephalopathy. 60.4% (171/283) (no decompensation events occurred for 12 months) and 56.2% (159/283) (no decompensation events occurred for 12 months and improved liver function) of the patients had achieved clinical recompensation within 120 weeks. Patients with baseline MELD scores > 15 after active antiviral therapy achieved higher recompensation than patients with baseline MELD scores ≤15 [50/74 (67.6%) vs. 109/209 (52.2%), χ (2) = 5.275, P = 0.029]. Conclusion: Antiviral therapy can significantly improve the prognosis of patients with hepatitis B virus-related decompensated cirrhosis. The majority of patients (56.2%) had achieved recompensation. Patients with severe disease did not have a lower probability of recompensation at baseline than other patients.

An expert consensus for the management of chronic hepatitis B in Asian Americans.

BACKGROUND: Hepatitis B virus (HBV) infection is common with major clinical consequences. In Asian Americans, the HBsAg carrier rate ranges from 2% to 16% which approximates the rates from their countries of origin. Similarly, HBV is the most important cause of cirrhosis, hepatocellular carcinoma (HCC) and liver related deaths in HBsAg positive Asians worldwide. AIM: To generate recommendations for the management of Asian Americans infected with HBV. METHODS: These guidelines are based on relevant data derived from medical reports on HBV from Asian countries as well as from studies in the HBsAg positive Asian Americans. The guidelines herein differ from other recommendations in the treatment of both HBeAg positive and negative chronic hepatitis B (CHB), in the approach to HCC surveillance, and in the management of HBV in pregnant women. RESULTS: Asian American patients, HBeAg positive or negative, with HBV DNA levels >2000 IU/mL (>104 copies/mL) and ALT values above normal are candidates for anti-viral therapy. HBeAg negative patients with HBV DNA >2000 IU/mL and normal ALT levels but who have either serum albumin <3.5 g/dL or platelet count <130 000 mm3 , basal core promoter (BCP) mutations, or who have first-degree relatives with HCC should be offered treatment. Patients with cirrhosis and detectable HBV DNA must receive life-long anti-viral therapy. Indications for treatment include pregnant women with high viraemia, coinfected patients, and those requiring immunosuppressive therapy. In HBsAg positive patients with risk factors, life-long surveillance for HCC with alpha-fetoprotein (AFP) testing and abdominal ultrasound examination at 6-month intervals is required. In CHB patients receiving HCC treatments, repeat imaging with contrast CT scan or MRI at 3-month intervals is strongly recommended. These guidelines have been assigned to a Class (reflecting benefit vs. risk) and a Level (assessing strength or certainty) of evidence. CONCLUSIONS: Application of the recommendations made based on a review of the relevant literature and the opinion of a panel of Asian American physicians with expertise in HBV treatment will inform physicians and improve patient outcomes.

BPGAP1 spatially integrates JNK/ERK signaling crosstalk in oncogenesis.

Simultaneous hyperactivation of stress-activated protein kinase/c-Jun N-terminal protein kinase (SAPK/JNK) and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling cascades has been reported in carcinogenesis. However, how they are integrated to promote oncogenesis remains unknown. By analyzing breast invasive carcinoma database (The Cancer Genome Altas), we found that the mRNA expression levels of both JNK1 and ERK2 are positively correlated with the mRNA level of EEA1, an endosome associated protein, indicating the potential JNK/ERK crosstalk at endosome. Unbiased screen of different endosome-associated Rab GTPases reveals that late endosome serves as a unique platform to integrate JNK/ERK signaling. Furthermore, we identify that BPGAP1 (a BCH domain-containing, Cdc42GAP-like Rho GTPase-activating protein) promotes MEK partner 1 (MP1)-induced ERK activation on late endosome through scaffolding MP1/MEK1 complex. This regulatory function requires phosphorylation of BPGAP1 by JNK at its C terminal tail (Ser424) to unlock its autoinhibitory conformation. Consequently, phosphorylated BPGAP1 facilitates endosomal ERK signaling transduction to the nucleus, driving cell proliferation and transformation via the ERK-Myc-CyclinA axis. BPGAP1 therefore provides a crucial spatiotemporal checkpoint where JNK and MP1/MEK1 work in concert to regulate endosomal and nuclear ERK signaling in cell proliferation control.

[Evaluation on efficacy of corticotomy-facilitated treatment in skeletal class â ¡ patients].

Objective: To investigate the effect of corticotomy-facilitated orthodontics on the treatment time and final outcome in skeletal class Ⅱ division 1 patients. Methods: Twenty adult skeletal class Ⅱ division 1 patients treated with two maxillary first premolar extractions were included and randomly divided into two groups (the corticotomy group and the control group). The treatment time was recorded and the changes of soft and hard tissue were compared by using three-dimensional measurement and analysis of cone-beam CT images before and after treatment. Results: There was no significant difference in the alignment time between two groups, while the time of maxillary space closure and the total treatment time in corticotomy group ([5.8±1.3] and [24.9±5.1] months, respectively) were shorter than that in the control group ([9.9±1.1] and [30.8±4.6] months, respectively) and the differences were significant (P<0.01). In the corticotomy group, the retraction amount of the upper central in cisal margin, apical tip, supradentale, labrale superius and the increase of nasolabial angle were greater than those in the control group (P<0.05). Conclusions: In patients with mild to moderate skeletal class Ⅱ division 1 malocclusion, corticotomy-facilitated orthodontics can not only shorten the treatment time, but is more conductive to the retraction of upper anterior teeth and improvement of the profile.

Lamivudine therapy during the second vs the third trimester for preventing transmission of chronic hepatitis B.

There are little data on the timing of initiating lamivudine therapy for preventing transmission of hepatitis B in highly viremic mothers. Between May 2008 and January 2015, we retrospectively enrolled mothers with HBV DNA >6 log10  copies/mL who received lamivudine during pregnancy, and we compared them to untreated mothers. The primary measurement was the vertical transmission rate. The secondary outcomes were the mothers' and infants' safety. Among 249 consecutive mothers enrolled, 66 and 94 received lamivudine during the second and third trimesters, respectively, and 89 were untreated. At delivery, maternal mean HBV DNA levels were significantly lower in mothers who received lamivudine (4.45 log10; vs 7.16 log10  copies/mL; P<.001). Lamivudine treatment was well tolerated. However, early treatment during the second trimester did not significantly increase the percentage of mothers achieving HBV DNA levels of <6 log10  copies/mL compared to those treated during the third trimester (98.5% vs 94.7%; P=.40). At the age of 28 weeks, the vertical transmission rates were significantly lower in the lamivudine-treated mothers vs in the untreated mothers (0% [0/160] vs 5.62% [5/89]; P<.001), but the rates were similar when comparing the two subgroups treated with lamivudine (0% [0/66] vs 0% [0/94], P>.05). The birth defect rates and mothers' and infants' adverse events were similar among the groups. Lamivudine treatment initiated in the second or third trimester for mothers with HBV DNA levels below 9 log10  copies/mL was equally safe and effective in preventing vertical transmission. Thus, lamivudine should be deferred until the third trimester to minimize foetal exposure and drug resistance.

Entecavir safety and effectiveness in a national cohort of treatment-naïve chronic hepatitis B patients in the US - the ENUMERATE study.

BACKGROUND: Entecavir (ETV) has been shown to be safe and efficacious in randomised controlled trials in highly selected patients with hepatitis B virus (HBV) infection. AIM: To determine the safety and effectiveness of ETV in 'real-world' HBV patients in the United States (US). METHODS: Treatment-naïve HBV patients ≥18 years old who received ETV for ≥12 months between 2005 and 2013 were included in a retrospective, cohort study. Rates of ALT normalisation, undetectable HBV DNA, HBeAg and HBsAg loss/seroconversion, adverse events (AE) and clinical outcomes were evaluated. RESULTS: Of 841 patients, 658 [65% male, 83% Asian; median age 47 years] met the inclusion criteria. 36% were HBeAg+ and 9.3% cirrhotic. 89% had abnormal ALT. Baseline median HBV DNA was 5.8 log 10 IU/mL. Median duration of ETV treatment was 4 years. Rates of ALT normalisation at 1, 3 and 5 years were 37.2%, 48.7% and 56.2% in HBeAg+ and 39.6%, 46.8% and 55.6% in HBeAg- patients. HBV DNA was undetectable at 1, 3 and 5 years in 34.6%, 64.7% and 84.6% in HBeAg+ patients, and 81.9%, 90.3% and 96.2% in HBeAg patients. Five-year cumulative probability of HBeAg loss and seroconversion was 46% and 33.7% and HBsAg loss was 4.6%. ETV was discontinued due to adverse events in 1.2% of patients. Hepatic decompensation occurred in 0.8%, liver cancer in 2.7% and death in 0.6%. CONCLUSION: Entecavir treatment was safe in a large cohort of US patients, but ALT normalisation and hepatitis B virus DNA suppression rates were lower than previously reported in clinical trials.

Tenofovir-based alternate therapies for chronic hepatitis B patients with partial virological response to entecavir.

Entecavir (ETV) is a first-line antiviral therapy for treating chronic hepatitis B (CHB); however, some patients have suboptimal response to ETV. Currently, there are limited data on how to approach these patients. Therefore, our aim was to compare the effectiveness of two alternate therapies--tenofovir (TDF) monotherapy and combination therapy of ETV+TDF--in CHB patients with ETV partial virological response. We conducted a retrospective study of 68 patients who had partial virological response to ETV, defined as having detectable HBV DNA following at least 12 months of ETV, and were switched to TDF monotherapy (n = 25) or ETV+TDF (n = 43). Patients were seen in seven US liver/community-based clinics and started on ETV between 2005 and 2009. The majority of patients were male; the vast majority were Asian and had positive hepatitis B e antigen (HBeAg). Patients in both groups had similar pretreatment characteristics. Complete viral suppression (CVS) rates with TDF monotherapy and ETV+TDF were similar after 6 months (71% vs 83%, P = 0.23) and 12 months (86% vs 84%, P = 0.85), and there was no statistically significant difference in CVS rates even when only patients with higher HBV DNA levels at switch (>1000 IU/mL) were evaluated. Multivariate analysis indicated that ETV+TDF was not an independent predictor of CVS compared to TDF monotherapy (OR = 1.19, P = 0.63). In conclusion, TDF monotherapy and ETV+TDF are comparable in achieving CVS in CHB patients with partial virological response to ETV. Long-term alternate therapy with one pill (TDF monotherapy) vs two pills (ETV+TDF) could lead to lower nonadherence rates and better treatment outcomes.

Cardiovascular effects of a PEGylated apelin.

Several studies have documented cardiovascular effects of apelin, including enhanced inotropy and vasodilation. However, these cardiovascular effects are short lived due to the predicted short circulating half-life of the apelin peptide. To address this limitation of apelin, we pursued N-terminal PEGylation of apelin and examined the cardiovascular effects of the PEGylated apelin. A 40kDa PEG conjugated apelin-36 (PEG-apelin-36) was successfully produced with N-terminal conjugation, high purity (>98%) and minimum reduction of APJ receptor binding affinity. Using an adenylate cyclase inhibition assay, comparable in vitro bioactivity was observed between the PEG-apelin-36 and unmodified apelin-36. In vivo evaluation of the PEG-apelin-36 was performed in normal rats and rats with myocardial infarction (MI). Cardiac function was assessed via echocardiography before, during a 20 min IV infusion and up to 100 min post peptide infusion. Similar increases in cardiac ejection fraction (EF) were observed during the infusion of PEG-apelin-36 and apelin-36 in normal rats. However, animals that received PEG-apelin-36 maintained significantly increased EF over the 100 min post infusion monitoring period compared to the animals that received unmodified apelin-36. Interestingly, EF increases observed with PEG-apelin-36 and apelin-36 were greater in the MI rats. PEG-apelin-36 had a prolonged circulating life compared to apelin-36 in rats. There were no changes in aortic blood pressure when PEG-apelin-36 or apelin-36 was administered. To our knowledge this is the first report of apelin PEGylation and documentation of its cardiovascular effects.

Response to tenofovir monotherapy in chronic hepatitis B patients with prior suboptimal response to entecavir.

Both entecavir (ETV) and tenofovir (TDF) are potent antiviral agents for hepatitis B virus (HBV). Suboptimal response (SOR) following antiviral therapy is associated with an increased risk of subsequent treatment failure and viral resistance. It remains unclear whether switching to TDF is a reasonable approach in patients with SOR to ETV treatment. This study was aimed to determine how HBV patients with SOR to ETV respond to TDF monotherapy. Data of patients with SOR to ETV (failure to achieve >1 log(10) HBV-DNA reduction during the last 24 weeks of ETV treatment) who were switched to TDF monotherapy during 2005 and 2010 were reviewed. Treatment adherence was assessed by pill-count. Fourteen patients (2.9%) were identified from a total cohort of 482 ETV-treated patients. All 14 patients were Chinese and were infected with HBV genotype C (71%) or B (29%). Nine patients were men, and the median age was 41.5 years (19-64). Twelve were treatment naïve (one lamivudine- and one peginterferon-experienced patient); 85.7% were HBeAg positive. The median baseline HBV-DNA was 7.55 (5.30-9.40) log(10) copies/mL, and 57% had abnormal serum alanine aminotransferase (ALT) levels. Precore and/or basal core promoter mutations were detected in four patients, whereas no genotypic resistance was detected at baseline and before switching to TDF. The median duration of ETV treatment was 64.5 (26-126) weeks. The median HBV-DNA at the time of switching to TDF was 3.69 (3.00-4.90) log(10) copies/mL. The median HBV-DNA reduction from baseline and during the last 6-month observation period prior to switching to TDF was 4.04 (0.51-6.06) log(10) and 0.43 (-0.09-1.13) log(10) copies/mL, respectively. After the switching to TDF, all 14 patients (100%) achieved undetectable HBV-DNA and ALT normalization within a median duration of 30 weeks. In 12 patients who were HBeAg positive, HBeAg seroconversion was observed in two patients after TDF treatment of 75- and 84-weeks duration. There was no virological breakthrough observed after switching to TDF with a median follow-up period of 50 (24-160) weeks. TDF treatment was safe and well tolerated. In conclusion, suboptimal response to ETV is rare (approximately 3%). TDF monotherapy is safe and very effective in the management of HBV patients with SOR to ETV.

Ca2+-dependent activity of human DNase I and its hyperactive variants.

We have recently constructed hyperactive human deoxyribonuclease I (DNase I) variants that digest double-stranded DNA more efficiently under physiological saline conditions by introducing positively charged amino acids at eight positions that can interact favorably with the negatively charged DNA phosphates. In this study, we present data from supercoiled DNA nicking, linear DNA digestion, and hyperchromicity assays that distinguish two classes of DNase I hyperactive variants based upon their activity dependence on Ca2+. Class A variants are highly dependent upon Ca2+, having up to 300-fold lower activity in the presence of Mg2+ alone compared to that in the presence of Mg2+ and Ca2+, and include Q9R, H44K, and T205K, in addition to wild-type DNase I. In contrast, the catalytic activity of Class B variants, which comprise the E13R, T14K, N74K, S75K, and N110R hyperactive variants, is relatively Ca2+ independent. A significant proportion of this difference in Ca2+-dependent activity can be attributed to one of the two structural calcium binding sites in DNase I. Compared to wild-type, the removal of Ca2+ binding site 2 by alanine replacements at Asp99, Asp107, and Glu112 decreased activity up to 26-fold in the presence of Mg2+ and Ca2+, but had no effect in the presence of Mg2+ alone. We propose that the rate-enhancing effect of Ca2+ binding at site 2 can be replaced by favorable electrostatic interactions created by proximal positively charged amino acid substitutions such as those found in the Class B variants, thus reducing the dependence on Ca2+.

Expression and characterization of a DNase I-Fc fusion enzyme.

Recombinant human deoxyribonuclease I (DNase I) is an important clinical agent that is inhaled into the airways where it degrades DNA to lower molecular weight fragments, thus reducing the viscoelasticity of sputum and improving the lung function of cystic fibrosis patients. To investigate DNases with potentially improved properties, we constructed a molecular fusion of human DNase I with the hinge and Fc region of human IgG1 heavy chain, creating a DNase I-Fc fusion protein. Infection of Sf9 insect cells with recombinant baculovirus resulted in the expression and secretion of the DNase I-Fc fusion protein. The fusion protein was purified from the culture medium using protein A affinity chromatography followed by desalting by gel filtration and was characterized by amino-terminal sequence, amino acid composition, and a variety of enzyme-linked immunosorbent assays (ELISA) and activity assays. The purified fusion contains DNase I, as determined by a DNase I ELISA and an actin-binding ELISA, and an intact antibody Fc region, which was quantified by an Fc ELISA, in a 2:1 stoichiometric ratio, respectively. The dimeric DNase I-Fc fusion was functionally active in enzymatic DNA digestion assays, albeit about 10-fold less than monomeric DNase I. Cleavage of the DNase I-Fc fusion by papain resulted in a specific activity comparable to the monomeric enzyme. Salt was inhibitory for wild type monomeric DNase I but actually enhanced the activity of the dimeric DNase I-Fc fusion. The DNase I-Fc fusion protein was also less Ca2+-dependent than DNase I itself. These results are consistent with a higher affinity of the dimeric fusion protein to DNA than monomeric DNase I. The engineered DNase I-Fc fusion protein described herein has properties that may have clinical benefits.

Cloning and characterization of an actin-resistant DNase I-like endonuclease secreted by macrophages.

We have cloned human and murine DNase I-like cDNAs, termed LS-DNase, which are expressed at high levels in liver and spleen tissues. LS-DNase expression is highly specific to macrophage populations within these and other tissues. Mature LS-DNase from both species is a secreted, non-glycosylated protein containing 285 residues, with a calculated molecular mass of 33 kDa and a basic isoelectric point. Human and murine LS-DNase are highly conserved and share 83% identity. Sequence analysis reveals that LS-DNase shares 46% amino acid sequence identity with DNase I. However, several residues identified as important for interaction of human DNase I with actin are not conserved in both human and murine LS-DNase. Consistent with this observation, recombinant human LS-DNase possesses a DNA hydrolytic activity which, unlike DNase I, is not inhibited by G-actin. The existence of a family of DNase I-like molecules that have tissue-specific expression patterns and the possible role of a macrophage specific DNase are discussed.

Improved potency of hyperactive and actin-resistant human DNase I variants for treatment of cystic fibrosis and systemic lupus erythematosus.

The ability of recombinant human DNase I (DNase I) to degrade DNA to lower molecular weight fragments is the basis for its therapeutic use in cystic fibrosis (CF) patients and its potential use as a treatment for systemic lupus erythematosus (SLE). To increase the potency of human DNase I, we have generated and characterized three classes of mutants: (a) hyperactive variants, which have from one to six additional positively charged residues (+1 to +6) and digest DNA much more efficiently relative to wild type, (b) actin-resistant variants, which are no longer inhibited by G-actin, a potent inhibitor of DNase I, and (c) combination variants that are both hyperactive and actin-resistant. For DNA scission in CF sputum where the DNA concentration and length are large, we measured a approximately 20-fold increase in potency relative to wild type for the +3 hyperactive variant Q9R/E13R/N74K or the actin-resistant variant A114F; the hyperactive and actin-resistant combination variant was approximately 100-fold more potent than wild type DNase I. For digesting lower concentrations of DNA complexed to anti-DNA antibodies in human serum, we found a maximal enhancement of approximately 400-fold over wild type for the +2 variant E13R/N74K. The +3 enzymes have approximately 4000-fold enhancement for degrading moderate levels of exogenous DNA spiked into human serum, whereas the +6 enzyme has approximately 30,000-fold increased activity for digesting the extremely low levels of endogenous DNA found in serum. The actin resistance property of the combination mutants further enhances the degree of potency in human serum. Thus, the human DNase I variants we have engineered for improved biochemical and pharmacodynamic properties have greater therapeutic potential for treatment of both CF and SLE.

Hyperactivity of human DNase I variants. Dependence on the number of positively charged residues and concentration, length, and environment of DNA.

Human DNase I, an enzyme used to treat cystic fibrosis patients, has been engineered to more effectively degrade double-stranded DNA to lower molecular weight forms by introducing positively charged amino acids at positions that can interact favorably with the proximal negatively charged phosphate groups of the DNA. A series of combination mutants having from one to six additional basic residues compared with the wild type has been constructed, expressed in human 293 cells, and characterized. The degree of hyperactivity for the mutants was highly dependent upon the conditions in various assays, including the concentration and length of the DNA substrate and the salt and divalent metal ion concentrations. The level of hyperactivity was inversely proportional to both DNA concentration and DNA length, consistent with the processive nicking mechanism for the hyperactive variants. Salt was inhibitory for wild type DNase I but actually enhanced the activity of the hyperactive variants. Under optimal conditions for wild type, variants with one additional positive charge possessed the highest activity, which was only severalfold greater than that for wild type. However, in the presence of low DNA concentrations and molecular weights, no Ca2+, and 150 mM NaCl, the variant with six engineered basic residues was most active, having >10,000-fold higher activity than the wild type enzyme. Therefore, any potential increase in potency for the hyperactive variants in vivo will be determined by the concentration, length, and environment of the DNA.

Mutational analysis of human DNase I at the DNA binding interface: implications for DNA recognition, catalysis, and metal ion dependence.

Human deoxyribonuclease I (DNase I), an enzyme used to treat cystic fibrosis patients, has been systematically analyzed by site-directed mutagenesis of residues at the DNA binding interface. Crystal structures of bovine DNase I complexed with two different oligonucleotides have implicated the participation of over 20 amino acids in catalysis or DNA recognition. These residues have been classified into four groups based on the characterization of over 80 human DNase I variants. Mutations at any of the four catalytic amino acids His 134, His 252, Glu 78, and Asp 212 drastically reduced the hydrolytic activity of DNase I. Replacing the three putative divalent metal ion-coordinating residues Glu 39, Asp 168, or Asp 251 led to inactive variants. Amino acids Gln 9, Arg 41, Tyr 76, Arg 111, Asn 170, Tyr 175, and Tyr 211 were also critical for activity, presumably because of their close proximity to the active site, while more peripheral DNA interactions stemming from 13 other positions were of minimal significance. The relative importance of these 27 positions is consistent with evolutionary relationships among DNase I across different species, DNase I-like proteins, and bacterial sphingomyelinases, suggesting a fingerprint for a family of DNase I-like proteins. Furthermore, we found no evidence for a second active site that had been previously implicated in Mn2+-dependent DNA degradation. Finally, we correlated our mutational analysis of human DNase I to that of bovine DNase I with respect to their specific activity and dependence on divalent metal ions.

Engineering hyperactive variants of human deoxyribonuclease I by altering its functional mechanism.

Human deoxyribonuclease I (DNase I), an enzyme used to treat cystic fibrosis patients, has been engineered to more effectively degrade double-stranded DNA to lower molecular weight fragments by altering its functional mechanism from the native single-stranded nicking pathway to a much more efficient one which results in increased double-stranded scission. By introducing positively charged amino acids at DNase I positions that can interact favorably with the proximal negatively charged phosphate groups of the DNA, we have created a hyperactive variant with approximately 35-fold higher DNA-degrading activity relative to wild type. This enhancement can be attributed to both a decrease in Km and an increase in Vmax. Furthermore, unlike wild-type DNase I, the hyperactive variants are no longer inhibited by physiological saline. Replacement of the same positions with negatively charged amino acids greatly reduced DNA cleavage activity, consistent with a repulsive effect with the neighboring DNA phosphates. In addition, these variants displayed similar activities toward a small synthetic substrate, p-nitrophenyl phenylphosphonate, suggesting that the difference in DNA cleavage activity is due to the interaction of the engineered charged residues with the DNA phosphate backbone rather than any change in catalytic machinery. Finally, experiments involving the repair of DNase I digested DNA with T4 DNA ligase and the Klenow fragment of DNA polymerase I suggest that single-stranded gaps are introduced by the hyperactive variants. Thus, the increased functional activity of the hyperactive variants may be explained in part by a shift toward a processive DNA nicking mechanism, which leads to a higher frequency of double-stranded breaks.

Variable structures of Fis-DNA complexes determined by flanking DNA-protein contacts.

The Fis protein from Escherichia coli and Salmonella typhimurium regulates many diverse reactions including recombination, transcription, and replication and is one of the most abundant DNA binding proteins present in the cell under certain physiological conditions. As a specific regulator, Fis binds to discrete sites that are poorly related in primary sequence. Analysis of DNA scission by a collection of Fis conjugates to 1,10-phenanthroline-copper combined with comparative gel electrophoresis has shown that the structures of Fis-DNA complexes are highly variable, displaying overall DNA curvatures that range from < or = 50 degrees to > or = 90 degrees. This variability is primarily determined by differential wrapping of flanking DNA around Fis. By contrast, DNA bending within the core recognition regions appears similar among the binding sites that were analyzed. Flanking DNA contacts by Fis depend on the nucleotide sequence and are mediated by an electrostatic interaction with arginine 71 and a hydrogen bond with asparagine 73, both of which are located outside of the helix-turn-helix DNA binding motif. These contacts strongly influence the kinetics of binding. These data, combined with the crystal structure of Fis, have enabled us to generate new models for Fis-DNA complexes that emphasize the variability in DNA structures within the flanking regions.