Formation of noncanonical DNA structures mediated by human ORC4, a protein component of the origin recognition complex.

Many genomic sequences, DNA replication origins included, contain specific structural motifs prone to alternative base pairing. Structural rearrangements of DNA require specific environmental conditions and could be favored by chemical agents or proteins. To improve our understanding of alternative conformations of origins and the manner in which they form, we have investigated the effect of DNA-binding, AAA+ protein human ORC4 on single-stranded origin DNA or various oligonucleotides. Here we demonstrate that human ORC4 stimulated formation of inter- and intramolecular T.A.T triplexes and created novel structures, such as homoadenine duplexes. Adenine-based structures were held together by Hoogsteen hydrogen bonds, as demonstrated on 7-deaza-dAMP- or dAMP-containing substrates, and characterized by increased thermal stability. Adenine pairing occurred only in the presence of human ORC4, in a neutral buffer supplemented with ATP and Mg (2+) ions. The protein mutant that could not bind ATP was inactive in this reaction. Since the action of human ORC4 could be biologically important, its potential impact on DNA replication is discussed.

The CFTR M470V gene variant as a potential modifier of COPD severity: study of Serbian population.

Chronic obstructive pulmonary disease (COPD) is a complex disease influenced by genetic and environmental factors. Cystic fibrosis transmembrane conductance regulator (CFTR) protein is an important component of the lung tissue homeostasis, involved in the regulation of the rate of mucociliary clearance. As it is known that certain CFTR variants have consequences on the function of CFTR protein, the aim of this study was to examine the possible role of F508del, M470V, Tn locus, and R75Q variants in COPD development and modulation. Total number of 86 COPD patients and 102 control subjects were included in the study. Possible association between COPD susceptibility, severity, and onset of the disease and allele or genotype of four analyzed CFTR variants was examined. No associations were detected between COPD development, onset of the disease and tested CFTR alleles and genotypes. However, VV470 genotype was associated with mild/moderate COPD stages in comparison to severe/very severe ones (OR = 0.29, 95%CI = 0.11-0.80, p = 0.016). Our study showed that patients with VV470 genotype had a 3.4-fold decreased risk for the appearance of severe/very severe COPD symptoms, and the obtained results indicate that this genotype may have a protective role. These results also suggest the importance of studying CFTR gene as a modifier of this disease.

Analysis of Y chromosome microdeletions and CFTR gene mutations as genetic markers of infertility in Serbian men.

BACKGROUND/AIM: Impaired fertility of a male partner is the main cause of infertility in up to one half of all infertile couples. At the genetic level, male infertility can be caused by chromosome aberrations or gene mutations. The presence and types of Y chromosome microdeletions and cystic fybrosis transmembrane conductance regulator (CFTR) gene mutations as genetic cause of male infertility was tested in Serbian men. The aim of this study was to analyze CFTR gene mutations and Y chromosome microdelations as potential causes of male infertility in Serbian patients, as well as to test the hypothesis that CFTR mutations in infertile men are predominantly located in the several last exons of the gene. METHODS: This study has encompassed 33 men with oligo- or azoospermia. The screening for Y chromosome microdeletions in the azoospermia factor (AZF) region was performed by multiplex PCR analysis. The screening of the CFTR gene was performed by denaturing gradient gel electrophoresis (DGGE) method. RESULTS: Deletions on Y chromosome were detected in four patients, predominantly in AZFc region (four of total six deletions). Mutations in the CFTR gene were detected on eight out of 66 analyzed chromosomes of infertile men. The most common mutation was F508del (six of total eight mutations). CONCLUSION: This study confirmed that both Y chromosome microdeletions and CFTR gene mutations played important role in etiology of male infertility in Serbian infertile men. Genetic testing for Y chromosome microdeletions and CFTR gene mutations has been introduced in routine daignostics and offered to couples undergoing assisted reproduction techniques. Considering that both the type of Y chromosome microdeletion and the type of CFTR mutation have a prognostic value, it is recomended that AZF and CFTR genotyping should not only be performed in patients with reduced sperm quality before undergoing assisted reproduction, but also for the purpose of preimplantation and prenatal diagnostics in couples in which in vitro fertilization has been performed successfully.

Noncanonical DNA elements in the lamin B2 origin of DNA replication.

DNA replication origins of eukaryotes lack linear replicator elements but contain short (dT)(n) (dA)(n) sequences that could build mutually equivalent unorthodox structures. Here we report that the lamin B2 origin of DNA replication adopts an alternative form characterized by unpaired regions CTTTTTTTTTTCC/GGAAAAAAAAAAG (3900-3912) and CCTTTTTTTTC/GAAAAAAAAGG (4141-4151). Both unpaired regions are resistant to DNase and except in central parts of their homopyrimidine strands are sensitive to single strand-specific chemicals. Interactions that protect central pyrimidines probably stabilize the bubble-like areas. Because DNA fragments containing either one or both bubbles migrate in TBM (89 mm Tris base, 89 mm boric acid, and 2 mm MgCl(2)) PAGE even faster than expected from their linear size, interacting regions are expected to belong to the same molecule. In an origin fragment containing a single bubble, free homopyrimidine strand can only interact with Hoogsteen hydrogen bonding surfaces from a complementary double stranded sequence. Indeed, this origin fragment reacts with triplex preferring antibody. In competition binding experiments control double stranded DNA or single stranded (dT)(40) do not affect origin-antibody interaction, whereas TAT and GGC triplexes exert competitive effect. Because the chosen fragment does not contain potential GGC forming sequences, these experiments confirm that the lamin B2 origin adopts a structure partly composed of intramolecular TAT triads.

[Mutations and polymorphisms in CFTR genes in infertile men with oligospermia or azoospermia]. / Mutacije i polimorfizmi u genu CFTR kod infertilnih muskaraca s oligospermijom ili azospermijom.

INTRODUCTION: Impaired infertility of the male partner is causative or contributOry to in up to one half of all couples unable to conceive spontaneously. A considerable number of genes are now known that have an essential function in human reproduction and which, when deleted or mutated, can cause pathologic changes in the male reproductive system. Congenital bilateral absence of the vas deferens (CBAVD) is an important cause of obstructive azoospermia in otherwise healthy men. It is also present in 95% of men with an autosomal recessive systematic disease--cystic fibrosis. However, clinically affected CF patients present a spectrum of genital phenotypes ranging from normal fertility to severely impaired spermatogenesis and CBAVD. Cystic fibrosis and most cases of CBAVD are caused by mutations in CFTR (cystic fibrosis transmembrane conductance regulator) gene. The aim of this study was to test the possible involvement of the CFTR gene in the aetiology of male infertility other than CBAVD. METHODS: Twenty one infertile men with oligo or azoospermia were analysed for the presence of mutations and polymorphisms in the CFTR gene. Patients were divided in two groups according to the spermatogram: 1) patients with obstructive azoospermia (V < 2 mL, pH < 7.2, low level of a-glucosidase and fructose and absence of spermatozoa; 2) patients with impaired spermatogenesis or sperm maturation. We performed direct detection for the following mutations: delta F508 and delta 1507 (heteroduplex analysis), 621 + 1 G-->T, and N1303K (PSM--PCRmediated site-specific mutagenesis), A455E, 1717-1 G-->A, S549N, R560T, W1282X, R334W, R347P, R117H, 3849 + 10 kb C-->T and Tn, F508C, 1507V, 1506V polymorphisms (reverse dot blot method). G542X, R553X and GSS1D mutations were tested by SSCP (Single Strand Conformation Polymorphism). We also performed indirect detection of mutations and polymorphisms in 3, 5, 6a, 8, 9, 11, 12, 14a, 14b, 15, 17b, 18, 20, 21 and 23 exons by DGGE (Denaturant Gradient Gel Electrophoresis). Differences between frequencies were tested by chi-square statistic, p values of less than 0.05 were considered statistically significant. RESULTS: Among 42 chromosomes from infertile men with oligo or azoospermia we detected 7 mutations in CFTR gene (16.7%), which was significantly (p = 0.0319) more frequent than in general population (2%). Frequency of 5T allele in analysed group was high (11.9%) compared to general population (5%), but not statistically significant (0.0938). The most common mutation in the group of 10 men with obstructive azoospermia was delta F508. It was detected on one chromosome in five patients. In three of these patients with 4F508 mutation on the other chromosome we found 5T allele on polymorphic Tn locus. In one patient, heterozygous for delta F508 mutation, 711 + 3 A-->G mutation on the other chromosome was detected. In the group of 11 infertile men with impaired spermatogenesis or sperm maturation we detected one mutation--delta F508. Two patients from this group had 5T variant on one chromosome. DISCUSSION: We analysed 21 infertile men with oligo or azoospermia not caused by endocrine or inflammatory character, or chromosome mutations. Within this group frequency of CFTR mutations was increased compared to general population (p = 0.0319), suggesting that CFTR gene may be involved in the aetiology of infertility in men with oligo or azOospermia. In the group of patients with obstructive azoospermia 50% had at least one mutation, but only 10% had mutations in both chromosomes. One of the possible explanations would be that mutations are in the promoter region, introns or exons that were not included in analyses. The second explanation could be that some cases of obstructive azoospermia are only partially (or not) related to CFTR gene. In the group of patients with impaired spermatogenesis or sperm maturation, the frequencies of CFTR mutations and 5T allele were also increased compared to general population, but lower than in the group with obstructive azoospermia. This fact could mean that the influence of some other genes is higher in this condition than in the case of obstructive azoospermia. CONCLUSION: We concluded that CFTR gene plays a role in the aetiology of obstructive azoospermia and that is also could be involved in some cases of impaired spermatogenesis and sperm maturation. Due to the high incidence of CFTR mutations in patients with obstructive azoospermia we suggest screening of CFTR mutations before assisted reproduction.