Disruption of autophagy by the histone deacetylase inhibitor MGCD0103 and its therapeutic implication in B-cell chronic lymphocytic leukemia.

Evading apoptosis is a hallmark of B-cell chronic lymphocytic leukemia (CLL) cells and an obstacle to current chemotherapeutic approaches. Inhibiting histone deacetylase (HDAC) has emerged as a promising strategy to induce cell death in malignant cells. We have previously reported that the HDAC inhibitor MGCD0103 induces CLL cell death by activating the intrinsic pathway of apoptosis. Here, we show that MGCD0103 decreases the autophagic flux in primary CLL cells. Activation of the PI3K/AKT/mTOR pathway, together with the activation of caspases, and to a minor extent CAPN1, resulting in cleavage of autophagy components, were involved in MGCD0103-mediated inhibition of autophagy. In addition, MGCD0103 directly modulated the expression of critical autophagy genes at the transcriptional level that may contribute to autophagy impairment. Besides, we demonstrate that autophagy is a pro-survival mechanism in CLL whose disruption potentiates cell death induced by anticancer molecules including HDAC and cyclin-dependent kinase inhibitors. In particular, our data highlight the therapeutic potential of MGCD0103 as not only an inducer of apoptosis but also an autophagy suppressor in both combination regimens with molecules like flavopiridol, known to induce protective autophagy in CLL cells, or as an alternative to circumvent undesired immunomodulatory effects seen in the clinic with conventional autophagy inhibitors.

Molecular typing of the actin gene of Trichomonas vaginalis isolates by PCR-restriction fragment length polymorphism.

Human trichomoniasis, caused by the protozoan Trichomonas vaginalis, is a highly prevalent sexually transmitted infection. However, little is known about the degree of strain variability of T. vaginalis. A reliable classification method for T. vaginalis strains would be a useful tool in the study of the epidemiology, pathogenesis and transmission of T. vaginalis. A PCR-restriction fragment length polymorphism typing method was designed and evaluated using T. vaginalis isolates obtained after culture of vaginal specimens collected in the Democratic Republic of Congo and in Zambia. The variation of the actin gene of T. vaginalis was determined for three ATCC reference strains and 151 T. vaginalis isolates. Eight different types were identified, on the basis of the digestion patterns of the amplified actin gene, with each of the restriction enzymes HindII, MseI and RsaI. It was determined that the ATCC reference strains 30001, 30240 and 50141 were of actin genotypes G, H and E, respectively. The actin genotype type E was more common in the Democratic Republic of Congo, whereas type G was the commonest type in Zambia. Translation of the nucleotide sequence showed up to three amino acid substitutions. We developed a reproducible, sensitive and specific typing method for T. vaginalis, and were able to distinguish at least eight T. vaginalis actin genotypes. Further studies are needed to evaluate the method using clinical specimens and to determine the utility of the typing method for the genotypic characterization of T. vaginalis.

Comparison of culture and different PCR assays for detection of Trichomonas vaginalis in self collected vaginal swab specimens.

OBJECTIVES: DNA amplification techniques have become widely used for the diagnosis of sexually transmitted infections. For the detection of Trichomonas vaginalis, PCR techniques are not yet widely used despite the publication of several assays. The sensitivity and specificity of five independent primer sets were determined on self collected vaginal specimens obtained from female commercial sex workers. METHODS: Self collected specimens were obtained from symptomatic and asymptomatic women attending a female sex workers clinic in Abidjan, Côte d'Ivoire. Two vaginal specimens were collected, the first one was processed for culture and the second was processed for PCR analysis. PCR techniques for trichomonads were performed, using the primers as reported by Riley (TVA5/TVA6), Kengne (TVK3/TVK7), Madico (BTUB 9/BTUB 2), Shiao (IP1/IP2), and Mayta (TV1/TV2). An EIA amplicon detection method was designed for each of the primer sets. RESULTS: True positive specimens were defined as culture positive and/or two positive PCR results with EIA amplicon detection in any combination. According to this definition a prevalence of 20% was obtained compared to 7% obtained by culture. The PCR primer set TVK3/TVK7 gave the highest sensitivity (89.2%). Poor sensitivities were obtained with the primer sets TV1/TV2 (60.2%) and TVA5/TVA6 (63.9%). PCR showed a sensitivity improvement of 2.4% up to 12% when EIA was used for amplicon detection. CONCLUSIONS: Overall, the sensitivities of the different PCR assays resulting from this study were lower than those previously described. These findings could be the result of the nature of the specimen population and suggests a strain variability.

The epidemiology of gonorrhoea, chlamydial infection and syphilis in four African cities.

OBJECTIVES: To compare the epidemiology of gonorrhoea, chlamydial infection and syphilis in four cities in sub-Saharan Africa; two with a high prevalence of HIV infection (Kisumu, Kenya and Ndola, Zambia), and two with a relatively low HIV prevalence (Cotonou, Benin and Yaoundé, Cameroon). DESIGN: Cross-sectional study, using standardized methods, including a standardized questionnaire and standardized laboratory tests, in four cities in sub-Saharan Africa. METHODS: In each city, a random sample of about 2000 adults aged 15-49 years was taken. Consenting men and women were interviewed about their socio-demographic characteristics and their sexual behaviour, and were tested for HIV, syphilis, herpes simplex virus type 2 (HSV-2), gonorrhoea, chlamydial infection, and (women only) Trichomonas vaginalis infection. Risk factor analyses were carried out for chlamydial infection and syphilis seroreactivity. RESULTS: The prevalence of gonorrhoea ranged between 0% in men in Kisumu and 2.7% in women in Yaoundé. Men and women in Yaoundé had the highest prevalence of chlamydial infection (5.9 and 9.4%, respectively). In the other cities, the prevalence of chlamydial infection ranged between 1.3% in women in Cotonou and 4.5% in women in Kisumu. In Ndola, the prevalence of syphilis seroreactivity was over 10% in both men and women; it was around 6% in Yaoundé, 3-4% in Kisumu, and 1-2% in Cotonou. Chlamydial infection was associated with rate of partner change for both men and women, and with young age for women. At the population level, the prevalence of chlamydial infection correlated well with reported rates of partner change. Positive syphilis serology was associated with rate of partner change and with HSV-2 infection. The latter association could be due to biological interaction between syphilis and HSV-2 or to residual confounding by sexual behaviour. At the population level, there was no correlation between prevalence of syphilis seroreactivity and reported rates of partner change. CONCLUSION: Differences in prevalence of chlamydial infection could be explained by differences in reported sexual behaviour, but the variations in prevalence of syphilis seroreactivity remained unexplained. More research is needed to better understand the epidemiology of sexually transmitted infections in Africa.

The epidemiology of trichomoniasis in women in four African cities.

OBJECTIVES: To describe the epidemiology of Trichomonas vaginalis infection and its association with HIV infection, in women in four African cities with different levels of HIV infection. DESIGN: Cross-sectional study, using standardized methods, including a standardized questionnaire and standardized laboratory tests, in four cities in sub-Saharan Africa: two with a high prevalence of HIV infection (Kisumu, Kenya and Ndola, Zambia), and two with a relatively low prevalence of HIV (Cotonou, Benin and Yaoundé, Cameroon). METHODS: In each city, a random sample of about 2000 adults aged 15-49 years was taken. Consenting men and women were interviewed about their socio-demographic characteristics and their sexual behaviour, and were tested for HIV, syphilis, herpes simplex virus type 2 (HSV-2), gonorrhoea, chlamydial infection, and (women only) T. vaginalis infection. Risk factor analyses were carried out for trichomoniasis for each city separately. Multivariate analysis, however, was only possible for Yaoundé, Kisumu and Ndola. RESULTS: The prevalence of trichomoniasis was significantly higher in the high HIV prevalence cities (29.3% in Kisumu and 34.3% in Ndola) than in Cotonou (3.2%) and Yaoundé (17.6%). Risk of trichomoniasis was increased in women who reported more lifetime sex partners. HIV infection was an independent risk factor for trichomonas infection in Yaoundé [adjusted odds ratio (OR) = 1.8, 95% confidence interval (CI) = 0.9-3.7] and Kisumu (adjusted OR = 1.7, 95% CI = 1.1-2.7), but not in Ndola. A striking finding was the high prevalence (40%) of trichomonas infection in women in Ndola who denied that they had ever had sex. CONCLUSION: Trichomoniasis may have played a role in the spread of HIV in sub-Saharan Africa and may be one of the factors explaining the differences in levels of HIV infection between different regions in Africa. The differences in prevalence of trichomoniasis between the four cities remain unexplained, but we lack data on the epidemiology of trichomoniasis in men. More research is required on the interaction between trichomoniasis and HIV infection, the epidemiology of trichomoniasis in men, and trichomonas infections in women who deny sexual activity.

The epidemiology of HSV-2 infection and its association with HIV infection in four urban African populations.

OBJECTIVES: To estimate age- and sex-specific herpes simplex virus type-2 (HSV-2) prevalence in urban African adult populations and to identify factors associated with infection. DESIGN AND METHODS: Cross-sectional, population-based samples of about 2000 adults interviewed in each of the following cities: Cotonou, Benin; Yaoundé, Cameroon; Kisumu, Kenya and Ndola, Zambia. Consenting study participants were tested for HIV, HSV-2 and other sexually transmitted infections. RESULTS: HSV-2 prevalence was over 50% among women and over 25% among men in Yaoundé, Kisumu and Ndola, with notably high rates of infection among young women in Kisumu and Ndola (39% and 23%, respectively, among women aged 15-19 years). The prevalence in Cotonou was lower (30% in women and 12% in men). Multivariate analysis showed that HSV-2 prevalence was significantly associated with older age, ever being married, and number of lifetime sexual partners, in almost all cities and both sexes. There was also a strong, consistent association with HIV infection. Among women, the adjusted odds ratios for the association between HSV-2 and HIV infections ranged from 4.0 [95% confidence interval (CI) = 2.0-8.0] in Kisumu to 5.5 (95% CI = 1.7-18) in Yaoundé, and those among men ranged from 4.6 (95% CI = 2.7-7.7) in Ndola to 7.9 (95% CI = 4.1-15) in Kisumu. CONCLUSIONS: HSV-2 infection is highly prevalent in these populations, even at young ages, and is strongly associated with HIV at an individual level. At a population level, HSV-2 prevalence was highest in Kisumu and Ndola, the cities with the highest HIV rates, although rates were also high among women in Yaoundé, where there are high rates of partner change but relatively little HIV infection. The high prevalence of both infections among young people underlines the need for education and counselling among adolescents.

Visualization of recombination intermediates produced by RAD52-mediated single-strand annealing.

Double-strand breaks (DSBs) occur frequently during DNA replication. They are also caused by ionizing radiation, chemical damage or as part of the series of programmed events that occur during meiosis. In yeast, DSB repair requires RAD52, a protein that plays a critical role in homologous recombination. Here we describe the actions of human RAD52 protein in a model system for single-strand annealing (SSA) using tailed (i.e. exonuclease resected) duplex DNA molecules. Purified human RAD52 protein binds resected DSBs and promotes associations between complementary DNA termini. Heteroduplex intermediates of these recombination reactions have been visualized by electron microscopy, revealing the specific binding of multiple rings of RAD52 to the resected termini and the formation of large protein complexes at heteroduplex joints formed by RAD52-mediated annealing.

Antimicrobial susceptibilities of Neisseria gonorrhoeae in Kigali, Rwanda, and trends of resistance between 1986 and 2000.

BACKGROUND: Plasmid-mediated and chromosomal-mediated resistance of Neisseria gonorrhoeae to penicillin, tetracycline, thiamphenicol, and trimethoprim-sulfamethoxazole has spread dramatically in Africa. Monitoring of antimicrobial susceptibility is a key element in the control of sexually transmitted diseases. GOAL: To document antimicrobial susceptibilities of gonococci isolated during the past 15 years in Kigali, Rwanda. STUDY DESIGN: Minimal inhibitory concentrations of recently collected gonococcal isolates of eight antimicrobials were determined. The results were compared with data collected for isolates obtained since 1986. RESULTS: In 1986, 35% of the gonococcal isolates were penicillinase-producing N gonorrhoeae. Tetracycline-resistant N gonorrhoeae appeared in 1989. The prevalence of penicillinase-producing N gonorrhoeae and tetracycline-resistant N gonorrhoeae increased significantly to 70.5% and 89.2%, respectively. Chromosomal resistance to penicillin, tetracycline, and thiamphenicol increased temporarily, then decreased significantly. Chromosomal resistance to trimethoprim-sulfamethoxazole appeared in 1988 and increased to 21.6%. All the isolates were susceptible to ceftriaxone, ciprofloxacin, spectinomycin, and kanamycin. CONCLUSIONS: This study illustrated the rapidly increasing frequencies of penicillinase-producing N gonorrhoeae and tetracycline-resistant N gonorrhoeae. Chromosomal resistance to thiamphenicol and trimethoprim-sulfamethoxazole excludes these drugs as alternative treatment. Programs for antimicrobial susceptibility surveillance of N gonorrhoeae should urgently be established in Africa.

Effect of adverse storage conditions of antigen reagent on performance of the rapid plasma reagin test.

The aim of this study was to evaluate the performance of rapid plasma reagin (RPR) testing using expired and adversely stored antigen reagent. The sensitivity of RPR using antigen stored at 36 degrees C was compared at 3-monthly intervals with RPR using fresh antigen on 116 sera reactive by RPR and by Treponema pallidum particle agglutination (TPPA). After multiple phases of freezing and thawing, 8.3% of initial RPR reactive sera seroreverted. After storage at 36 degrees C for one year and 24 weeks after expiration the overall sensitivity of the adversely stored antigen was 93.8% compared with fresh antigen; the sensitivity was 100% for sera with RPR titres > or = 1:4 and 85.4% for sera with RPR titres of 1:1 and 1:2. The high stability of the reagent may increase the feasibility of the RPR test for use in poorly-equipped healthcare centres in developing countries.

Evaluation of the Roche Neisseria gonorrhoeae 16S rRNA PCR for confirmation of AMPLICOR PCR-positive samples and comparison of its diagnostic performance according to storage conditions and preparation of endocervical specimens.

The AMPLICOR PCR was used to detect Neisseria gonorrhoeae in endocervical specimens. A 16S rRNA PCR performed on N. gonorrhoeae-positive samples showed sensitivities of 73.2, 64.3, and 94.4% for samples treated directly with AMPLICOR lysis buffer, samples suspended in 2-sucrose phosphate, and samples suspended in diluted phosphate-buffered saline, respectively.

Detection of Chlamydia trachomatis and Neisseria gonorrhoeae by enzyme immunoassay, culture, and three nucleic acid amplification tests.

The purpose of this study was to evaluate and compare three commercially available nucleic acid amplification tests (NAATs) for the detection of Neisseria gonorrhoeae and Chlamydia trachomatis. Roche PCR and Becton Dickinson strand displacement amplification (SDA) were performed on 733 endocervical swab specimens from commercial sex workers. Abbott ligase chain reaction (LCR) was performed on a subset of 396 samples. Endocervical specimens from all women were also tested by culture for N. gonorrhoeae and by Syva MicroTrak enzyme immunoassay (EIA) for C. trachomatis. A positive N. gonorrhoeae result was defined as a positive result by culture or by two NAATs, and a positive C. trachomatis result was defined as a positive result by two tests. According to these definitions, the sensitivities and specificities for the subsample of 396 specimens of N. gonorrhoeae culture, PCR, SDA, and LCR were 69.8, 95.2, 88.9, and 88.9% and 100, 99.4, 100, and 99.1%, respectively; the sensitivities and specificities of C. trachomatis EIA, PCR, SDA, and LCR were 42.0, 98.0, 94.0, and 90.0% and 100, 98.0, 100, and 98.6%, respectively. The performance characteristics of N. gonorrhoeae culture, PCR, and SDA and C. trachomatis EIA, PCR, and SDA for all 733 specimens were defined without inclusion of LCR results and by discrepant analysis after resolution of discordant N. gonorrhoeae PCR results and of discordant C. trachomatis EIA and PCR results by LCR testing. The sensitivities of N. gonorrhoeae culture, PCR, and SDA before and after LCR resolution were 67.8, 95.7, and 93.9% and 65, 95.8, and 90.0%, respectively. The sensitivities of C. trachomatis EIA, PCR, and SDA decreased from 39.4, 100, and 100% to 38.7, 98.7, and 94.7%, respectively. All three NAATs proved to be superior to N. gonorrhoeae culture and to C. trachomatis EIA. The accuracies of the different NAATs were quite similar. SDA was the only amplification assay with 100% specificity for detection of both N. gonorrhoeae and C. trachomatis in endocervical specimens.

Antimicrobial susceptibilities and plasmid patterns of Neisseria gonorrhoeae in Bénin.

This study describes antimicrobial susceptibility patterns of Neisseria gonorrhoeae isolates obtained from female sex workers in Cotonou, Bénin. All isolates were susceptible to spectinomycin, ceftriaxone and ciprofloxacin, and susceptible to moderately susceptible to kanamycin; 9.8% of isolates were resistant to thiamphenicol; 9%, 87.5% and 3.5% were susceptible, moderately susceptible, resistant to trimethoprim-sulfamethoxazole, respectively; 94.4% and 99.3% were resistant to penicillin and tetracycline, respectively. All isolates with a minimal inhibitory concentration of tetracycline of >8 mg/l carried the 'American type' tetM plasmid; 94% and 6% of penicillinase-producing isolates possessed a 3.2 MDa and a 4.4MDa beta-lactamase plasmid, respectively. Surveillance of antimicrobial susceptibility of N. gonorrhoeae isolates to currently used drugs in Africa should become part of sexually transmitted diseases (STDs) control programmes.

Development of a heminested polymerase chain reaction assay for the detection of Haemophilus ducreyi in clinical specimens.

Detection of Haemophilus ducreyi in genital ulcer specimens by culture lacks sensitivity. To enhance detection, a heminested polymerase chain reaction (PCR) assay was developed targeting the nucleotide sequence of a gene, designated p27, which encodes for a 27 kDa H. ducreyi-specific protein. The p27 PCR assay detected all (37/37) H. ducreyi strains tested and gave no amplified product from DNA extracts of any of 31 other microorganisms, from 30 non-genital ulcer specimens, or from 29 urethral and vaginal swab specimens collected from non-chancroid STD patients. In genital ulcer disease specimens, compared to combined positive results obtained by culture and a previously described PCR assay, the p27 PCR assay showed a sensitivity of 91% (48/53). The p27 PCR assay provides a specific and a sensitive detection of H. ducreyi in clinical specimens.

Diagnostic performance of the Roche AMPLICOR PCR in detecting Neisseria gonorrhoeae in genitourinary specimens from female sex workers in Cotonou, Benin.

The objective of this study was to evaluate the diagnostic performance of the Roche multiplex AMPLICOR Chlamydia trachomatis/Neisseria gonorrhoeae PCR test for the detection of Neisseria gonorrhoeae infection in female urine specimens and wet and dry endocervical swabs. Endocervical swabs and urine specimens were collected from 342 female sex workers from Cotonou, Benin, and were tested using the AMPLICOR C. trachomatis/N. gonorrhoeae test (Roche Diagnostic Systems, Inc., Branchburg, N.J.) with internal control detection. Endocervical swabs were also cultured on Thayer-Martin medium. A series of alternate standards that included a combination of all the tests but not the test being evaluated was used to assess the performance of the test with each type of specimen. The sensitivity, specificity, and positive and negative predictive values for the urine were 53.8, 98.9, 93.5, and 87.5%, respectively. Corresponding figures for the wet swab were 91.5, 100, 100, and 97.4%, respectively. Those for the dry swab were 96.3, 96.2, 88.5, and 98.8%, respectively. Based on this study, the AMPLICOR PCR assay showed a low sensitivity for detection of N. gonorrhoeae infection in urine specimens, whereas the test was found to be highly sensitive and specific with endocervical specimens.

Reconstitution of the strand invasion step of double-strand break repair using human Rad51 Rad52 and RPA proteins.

The human Rad51 recombinase is essential for the repair of double-strand breaks in DNA that occur in somatic cells after exposure to ionising irradiation, or in germ line cells undergoing meiotic recombination. The initiation of double-strand break repair is thought to involve resection of the double-strand break to produce 3'-ended single-stranded (ss) tails that invade homologous duplex DNA. Here, we have used purified proteins to set up a defined in vitro system for the initial strand invasion step of double-strand break repair. We show that (i) hRad51 binds to the ssDNA of tailed duplex DNA molecules, and (ii) hRad51 catalyses the invasion of tailed duplex DNA into homologous covalently closed DNA. Invasion is stimulated by the single-strand DNA binding protein RPA, and by the hRad52 protein. Strikingly, hRad51 forms terminal nucleoprotein filaments on either 3' or 5'-ssDNA tails and promotes strand invasion without regard for the polarity of the tail. Taken together, these results show that hRad51 is recruited to regions of ssDNA occurring at resected double-strand breaks, and that hRad51 shows no intrinsic polarity preference at the strand invasion step that initiates double-strand break repair.

Precise binding of single-stranded DNA termini by human RAD52 protein.

The human RAD52 protein, which exhibits a heptameric ring structure, has been shown to bind resected double strand breaks (DSBs), consistent with an early role in meiotic recombination and DSB repair. In this work, we show that RAD52 binds single-stranded and tailed duplex DNA molecules via precise interactions with the terminal base. When probed with hydroxyl radicals, ssDNA-RAD52 complexes exhibit a four-nucleotide repeat hypersensitivity pattern. This unique pattern is due to the interaction of RAD52 with either a 5' or a 3' terminus of the ssDNA, is sequence independent and is phased precisely from the terminal nucleotide. Hypersensitivity is observed over approximately 36 nucleotides, consistent with the length of DNA that is protected by RAD52 in nuclease protection assays. We propose that RAD52 binds DNA breaks via specific interactions with the terminal base, leading to the formation of a precisely organized ssDNA-RAD52 complex in which the DNA lies on an exposed surface of the protein. This protein-DNA arrangement may facilitate the DNA-DNA interactions necessary for RAD52-mediated annealing of complementary DNA strands.

A phase I study of a novel potential intravaginal microbicide, PRO 2000, in healthy sexually inactive women.

BACKGROUND: Although the male condom provides a reliable means of preventing HIV transmission, a broader choice of methods is required particularly in circumstances where the negotiation of condom use is difficult. Development of new products that may be effective as topical vaginal microbicides is the focus of a great deal of research activity currently. The novel agent PRO 2000, a naphthalene sulphonate derivative with in vitro activity against HIV and other sexually transmissible pathogens, is one such compound. We have studied the local and systemic safety and tolerance of a vaginal gel formulation of this agent at two concentrations (0.5% and 4%) over a 2 week period of daily exposure in two cohorts of healthy sexually abstinent women (one in London, UK, and the other in Antwerp, Belgium). METHODS: This was a randomised, placebo controlled, double blind, three arm clinical trial conducted on two sites. Macroscopic evidence of genital epithelial changes was sought using colposcopy and evidence of microscopic inflammation was acquired using high vaginal biopsy from predetermined sites (UK cohort only). Blood levels of PRO 2000 were measured and laboratory safety tests, including coagulation screens, were performed. The impact on vaginal ecology was also assessed. RESULTS: 73 women were enrolled across both sites (36 UK, 37 Belgium); 24, 24, 25 in the 4%, 0.5%, and placebo groups respectively. Of these, 70 completed 2 weeks' exposure to the study gel. Three (all in the 4% group) withdrew owing to adverse events which were possibly or probably gel related. Cervicovaginal abrasion was seen colposcopically in three subjects after 14 days of gel use (two in the 4% group and one in the placebo group). Genital ulceration was not seen during gel use in any of the subjects who completed the study. Histological evaluation of vaginal biopsy samples (36 women only) showed evidence of increased inflammatory signs in one participant of the 4.0% group. One volunteer in the placebo group had moderate inflammation at screening and at follow up. Severe inflammation was not seen among any of the subjects tested. Plasma levels of PRO 2000 and laboratory safety tests showed no evidence of systemic absorption. No impact was seen on normal vaginal ecology in the UK cohort where samples were taken 12 hours after the last gel application. CONCLUSION: In this phase I study PRO 2000 gel was found to be generally well tolerated with promising local and systemic safety profiles. The 0.5% gel was better tolerated than the 4% gel as fewer genital epithelial adverse events were seen in the former. Phase II studies are about to begin in sexually active women.

The human Rad52 protein exists as a heptameric ring.

The RAD52 epistasis group was identified in yeast as a group of genes required to repair DNA damaged by ionizing radiation [1]. Genetic evidence indicates that Rad52 functions in Rad51-dependent and Rad51-independent recombination pathways [2] [3] [4]. Consistent with this, purified yeast and human Rad52 proteins have been shown to promote single-strand DNA annealing [5] [6] [7] and to stimulate Rad51-mediated homologous pairing [8] [9] [10] [11]. Electron microscopic examinations of the yeast [12] and human [13] Rad52 proteins have revealed their assembly into ring-like structures in vitro. Using both conventional transmission electron microscopy and scanning transmission electron microscopy (STEM), we found that the human Rad52 protein forms heptameric rings. A three-dimensional (3D) reconstruction revealed that the heptamer has a large central channel. Like the hexameric helicases such as Escherichia coli DnaB [14] [15], bacteriophage T7 gp4b [16] [17], simian virus 40 (SV40) large T antigen [18] and papilloma virus E1 [19], the Rad52 rings show a distinctly chiral arrangement of subunits. Thus, the structures formed by the hexameric helicases may be a more general property of other proteins involved in DNA metabolism, including those, such as Rad52, that do not bind and hydrolyze ATP.

The meiosis-specific recombinase hDmc1 forms ring structures and interacts with hRad51.

Eukaryotic cells encode two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, which are required for meiotic recombination. Rad51, like E.coli RecA, forms helical nucleoprotein filaments that promote joint molecule and heteroduplex DNA formation. Electron microscopy reveals that the human meiosis-specific recombinase Dmc1 forms ring structures that bind single-stranded (ss) and double-stranded (ds) DNA. The protein binds preferentially to ssDNA tails and gaps in duplex DNA. hDmc1-ssDNA complexes exhibit an irregular, often compacted structure, and promote strand-transfer reactions with homologous duplex DNA. hDmc1 binds duplex DNA with reduced affinity to form nucleoprotein complexes. In contrast to helical RecA/Rad51 filaments, however, Dmc1 filaments are composed of a linear array of stacked protein rings. Consistent with the requirement for two recombinases in meiotic recombination, hDmc1 interacts directly with hRad51.