Replication of alpha-satellite DNA arrays in endogenous human centromeric regions and in human artificial chromosome.

In human chromosomes, centromeric regions comprise megabase-size arrays of 171 bp alpha-satellite DNA monomers. The large distances spanned by these arrays preclude their replication from external sites and imply that the repetitive monomers contain replication origins. However, replication within these arrays has not previously been profiled and the role of alpha-satellite DNA in initiation of DNA replication has not yet been demonstrated. Here, replication of alpha-satellite DNA in endogenous human centromeric regions and in de novo formed Human Artificial Chromosome (HAC) was analyzed. We showed that alpha-satellite monomers could function as origins of DNA replication and that replication of alphoid arrays organized into centrochromatin occurred earlier than those organized into heterochromatin. The distribution of inter-origin distances within centromeric alphoid arrays was comparable to the distribution of inter-origin distances on randomly selected non-centromeric chromosomal regions. Depletion of CENP-B, a kinetochore protein that binds directly to a 17 bp CENP-B box motif common to alpha-satellite DNA, resulted in enrichment of alpha-satellite sequences for proteins of the ORC complex, suggesting that CENP-B may have a role in regulating the replication of centromeric regions. Mapping of replication initiation sites in the HAC revealed that replication preferentially initiated in transcriptionally active regions.

Identification of sequence elements regulating promoter activity and replication of a monopartite begomovirus-associated DNA beta satellite.

DNA beta is a circular single-stranded satellite DNA associated with certain monopartite begomoviruses (family Geminiviridae) which causes economically important diseases such as cotton leaf curl disease. DNA beta contains a single gene, betaC1, which encodes a pathogenicity protein responsible for symptom production. Transient expression studies in Nicotiana tabacum using the beta-glucuronidase reporter gene driven by a betaC1 promoter-deletion series of the DNA beta associated with cotton leaf curl Multan virus identified a 68 nt region (between -139 and -207) which is important for betaC1 transcription. This 68 nt region contains a G-box (CACGTG) located 143 nt upstream of the betaC1 start codon. Mutation of the G-box resulted in a significant reduction in betaC1 promoter activity and DNA beta replication efficiency. In addition, the G-box motif was found to bind specifically to a protein(s) in nuclear extracts prepared from tobacco leaf tissues. Our results indicate that interaction of the G-box motif with host nuclear factors is important for efficient gene expression and replication of DNA beta.

Replication promiscuity of DNA-beta satellites associated with monopartite begomoviruses; deletion mutagenesis of the Ageratum yellow vein virus DNA-beta satellite localizes sequences involved in replication.

Pseudorecombination studies in Nicotiana benthamiana demonstrate that Ageratum yellow vein virus (AYVV) and Eupatorium yellow vein virus (EpYVV) can functionally interact with DNA-beta satellites associated with AYVV, EpYVV, cotton leaf curl Multan virus (CLCuMV) and honeysuckle yellow vein virus (HYVV). In contrast, CLCuMV shows some specificity in its ability to interact with distinct satellites and HYVV is able to interact only with its own satellite. Using an N. benthamiana leaf disk assay, we have demonstrated that HYVV is unable to trans-replicate other satellites. To investigate the basis of trans-replication compatibility, deletion mutagenesis of AYVV DNA-beta has been used to localize the origin of replication to approximately 360 nt, encompassing the ubiquitous nonanucleotide/stem-loop structure, satellite conserved region (SCR) and part of the intergenic region immediately upstream of the SCR. Additional deletions within this intergenic region have identified a region that is essential for replication. The capacity for DNA-beta satellites to functionally interact with distinct geminivirus species and its implications for disease diversification are discussed.

Replicative intermediates of Tomato leaf curl virus and its satellite DNAs.

Several plant geminiviruses have been shown recently to utilize both rolling-circle replication (RCR) and recombination-dependent replication (RDR) strategies. A highly specific binding of the viral replication-associated protein (Rep) to its cognate DNA is essential for initiation of viral DNA replication and for the recognition of DNA components of the bipartite geminiviruses of the Begomovirus genus. We have extended the replication analysis to the monopartite Australian Tomato leaf curl virus (ToLCV), its Rep binding deficient mutants, and the satellite DNAs it supports. Analyses of viral DNA by two-dimensional agarose gel electrophoresis after fractionation by single-stranded (ss) DNA-selective cellulose chromatography revealed that DNA intermediates of ToLCV and its mutant were identical. Both RCR and RDR intermediates were identified. New ToLCV DNA forms were observed and characterized as subgenomic topoisomers, heterogeneous open circular double-stranded (ds) DNA, and degradation products. A 1350-nt DNA beta satellite associated with the unrelated Cotton leaf curl Multan virus (CLCuMV) was supported by ToLCV and produced intermediates of both RCR and RDR, suggesting that replication strategies of satellites are determined by the helper virus. Replicative intermediates of the 682 nt ToLCV satellite DNA could not be resolved; however, concatemers of up to octamer were detected, together with a field of hybridizing material suggestive of complementary strand replication on heterogeneous circular ssDNA templates.

Replication timing of homologous alpha-satellite DNA in Roberts syndrome.

Roberts syndrome (RS) is associated with a characteristic constitutive heterochromatin anomaly, namely, at metaphase the centromeres and heterochromatic segments appear split. In addition to this cytogenetic phenomenon, known as the RS effect, several other cytological features, especially affecting mitotic chromosome disjunction, are also observed. Applying FISH to interphase nuclei, we investigated the replication patterns of homologous alphoid centromeric DNA of chromosomes 9, 11, 16 and 17 in three patients showing the RS effect and in four normal individuals. A tendency for homologous centromeres to replicate asynchronously was observed in RS patients. This tendency was more evident in chromosomes 9 and 16, with large heterochromatic blocks and particularly subject to RS effect. This asynchrony could reflect a more generalized alteration in repetitive DNA replication timing that, in turn, would prevent the establishment of proper cohesion between sister chromatid heterochromatin, leading to the RS effect.

Regulated formation of extrachromosomal circular DNA molecules during development in Xenopus laevis.

Extrachromosomal circular DNA molecules of chromosomal origin have been detected in many organisms and are thought to reflect genomic plasticity in eukaryotic cells. Here we report a developmentally regulated formation of extrachromosomal circular DNA that occurs de novo in preblastula Xenopus embryos. This specific DNA population is not detected in the male or female germ cells and is dramatically reduced in later developmental stages and in adult tissues. The activity responsible for the de novo production of extrachromosomal circles is maternally inherited, is stored in the unfertilized egg, and requires genomic DNA as a template. The formation of circular molecules does not require genomic DNA replication but both processes can occur simultaneously in the early development. The production of extrachromosomal circular DNA does not proceed at random since multimers of the tandemly repeated sequence satellite 1 were over-represented in the circle population, while other sequences (such as ribosomal DNA and JCC31 repeated sequence) were not detected. This phenomenon reveals an unexpected plasticity of the embryonic genome which is restricted to the early developmental stage.

Clinicopathological significance of altered loci of replication error and microsatellite instability-associated mutations in gastric cancer.

Replication errors (RERs) judged by microsatellite instability and its associated mutations have been recognized as an important mechanism in tumorigenesis of gastric cancers (GCs). To gain a deeper insight into its significance, we examined the frequency of RERs using nine microsatellite markers and screened mutations in the polydeoxyadenine tract of the transforming growth factor beta type II receptor gene (TGF-betaRII) and polydeoxyguanine tracts of insulin-like growth factor II receptor and BAX genes. Twenty-four (30%) of 80 patients with GC had RERs, of which 3, 8, and 13 had one, two, and three or more loci, respectively. In 13 tumors with RERs in three or more loci, frameshift mutations of TGF-betaRII, insulin-like growth factor II receptor, and BAX were identified in 12, 3, and 2, respectively. Compared with GC with none, one or two RER-positive loci as a group, GC with RERs in three or more loci showed a significantly higher frequency of antral location (12 of 13 versus 35 of 67; P = 0.01), intestinal subtype (11 of 13 versus 30 of 67; P = 0.01), and previous Helicobacter pylori infection (12 of 13 versus 41 of 67; P = 0.05) and a lower incidence of lymph node metastasis (5 of 13 versus 49 of 67; P = 0.02) and tended to be in an advanced stage (12 of 13 versus 54 of 67; P = 0.28). These data indicate that GC with multiple RERs manifest distinct clinicopathological characteristics, and that a high frequency of frameshift mutations involving the TGF-betaRII gene may be causatively linked with tumorigenesis and progression.

Microsatellite instability in inherited and sporadic neoplasms.

Microsatellite instability (the replication error phenotype) is a new molecular assay that identifies a substantial fraction of human cancers. The microsatellite instability in these cancers arises from alterations in the tumors of the number of mono-, di-, and trinucleotide repeats that compose the microsatellites. Microsatellite instability is typical of colon and endometrial tumors arising in members of Lynch syndrome cancer families. Microsatellite instability is also found in a substantial percentage of sporadic cases of colon, endometrial, and gastric cancer, as well as in additional sporadic cancers, such as lung cancer, not usually seen in Lynch kindreds. Thus far, four different mutant genes, all homologous to bacterial DNA repair genes, have been identified as inherited in Lynch kindreds, and therefore are associated with the replication error phenotype. Clinical implications of the replication error phenotype include the demonstration that resistance to some alkylating agents appears to be directly altered in tumors with this phenotype.

Structural analysis of alpha-satellite DNA and centromere proteins using extended chromatin and chromosomes.

Human centromeres are characterized by distinct subsets of alpha-satellite DNA and by a number of centromeric proteins (CENPs) at least one of which, CENP-B, binds specifically to alpha-satellite DNA sequences. When the centromeres of metaphase chromosomes are mechanically stretched to five to 20 times their normal length, CENPs specifically recognized by CREST autoantibodies extend over the entire length of the linear alpha-satellite array. For higher resolution analysis we spread interphase chromatin across a slide resulting in highly extended chromatin fibers. By fluorescence in situ hybridization (FISH) with human alpha-satellite DNA and an oligomer specific for the CENP-B box sequence, the regular spacing of CENP-B binding motifs within arrays of alpha-satellite DNA was visualized directly. FISH with elongated chromatin structures released from interphase nuclei with the drug N-[4-(9-acridinylamino)-3-methoxyphenyl]methanesulfonamide shows that D7Z1 and D7Z2, two distinct alpha-satellite arrays on chromosome 7, are not interspersed with each other but are separated by as little as several hundred kilobases, consistent with previous long-range mapping data. The D7Z2 array, which does not bind detectable amounts of CENPs, can be assigned to the short arm side of the D7Z1 array using artificially stretched chromosomes. In interphase nuclei unreplicated segments give a singlet hybridization signal, whereas fully replicated loci appear as doublets. Although D7Z1 is replicated prior to D7Z2 in the majority of cells, the replication timing of one array relative to the other is variable. The replication of alpha-satellite arrays on homologous chromosomes is highly asynchronous. The newly replicated alpha-satellite lacks the CENP component.

Cloning and characterization of two satellite DNAs in the low-C-value genome of the nematode Meloidogyne spp.

Two highly reiterated StyI satellite DNAs have been cloned from two nematode species: one from Meloidogyne hapla and another from M. incognita. The monomeric units of these two satellites have a repeat length of 169 and 295 bp, respectively. These StyI repeated element families constitute 5% of the M. hapla and 2.5% of the M. incognita haploid genomes. The A + T content is elevated in both families (i.e., 68% and 77%, respectively). Nucleotide methylation and transcriptional activity are negative. No similarity was found between the two satellites, nor to other known highly repetitive elements. These StyI satellite DNAs are quite homogenous in sequence, showing on average 3% and 3.5% divergence from their respective calculated consensus sequence. An internal subrepeating unit of about 11 bp is observed in the StyI satellite monomer sequences of M. hapla, suggesting that it could have evolved from a shorter sequence. Because of the small size of the Meloidogyne genome (51 Mb) and the abundance of repeated sequences, this genus approaches a limit in terms of coding fraction.

Evidence for nucleosomal phasing and a novel protein specifically binding to cucumber satellite DNA.

The nucleosomal organization and the protein-binding capability of highly repeated and methylated satellite DNA of cucumber (Cucumis sativus L.), comprising approx. 30% of the genome, were analyzed. Nucleosomal core DNA from satellite type I was prepared after micrococcal nuclease digestion of chromatin and sequenced. Most of the core sequences obtained could be grouped in two main (A and B) and two minor groups (C and D) indicating a specific and complex phasing of nucleosomes on this satellite DNA. In vitro, gel retardation assays with cloned satellite DNA repeats (types I-IV) demonstrated a specific binding of nuclear proteins. These specific binding effects are also obtained with genomic, in vivo methylated and sequence heterogeneous (1 to 10% diversity) satellite type I DNA. For the first time in plants, a satellite DNA-binding protein with an apparent molecular weight of 14 kDa (SAT 14) was identified.

Timing of replication of beta satellite repeats of human chromosomes.

The beta satellite sequences of the human genome are a family of genetic elements consisting of 68-69 bp monomeric units repeated contiguously in long arrays up to 1 Mb in length. We have determined the timing of replication of beta satellite subgroups located in the heterochromatic portion of chromosome 9 and on the acrocentric chromosomes in regions both distal and proximal to the rDNA genes. We report that these dispersed subgroups of beta satellite sequences all replicate late during S phase of the cell cycle.

Retroelements in bacteria.

A peculiar type of satellite DNA, called msDNA, has been discovered in myxobacteria and some natural isolates of E. coli. These molecules are characterized by the presence of single-stranded DNA branching out from an internal guanosine residue of an RNA molecule by a unique 2',5'-phosphodiester linkage. Reverse transcriptase is required for the synthesis of msDNA. The discovery of retroelements in bacterial populations raises many intriguing questions concerning the evolutionary origin of reverse transcriptase, the function and the biosynthesis of msDNA, and the nature of the mechanisms generating the extensive diversity found in msDNA and reverse transcriptase genes among different bacterial strains.

Regulation of mouse satellite DNA replication time.

The satellite DNA sequences located near the centromeric regions of mouse chromosomes replicate very late in S in both fibroblast and lymphocyte cells and are heavily methylated at CpG residues. F9 teratocarcinoma cells, on the other hand, contain satellite sequences which are undermethylated and replicate much earlier in S. DNA methylation probably plays some role in the control of satellite replication time since 5-azacytidine treatment of RAG fibroblasts causes a dramatic temporal shift of replication to mid S. In contrast to similar changes accompanying the inactivation of the X-chromosome, early replication of satellite DNA is not associated with an increase in local chromosomal DNase I sensitivity. Fusion of F9 with mouse lymphocytes caused a dramatic early shift in the timing of the normally late replicating lymphocyte satellite heterochromatin, suggesting that trans-activating factors may be responsible for the regulation of replication timing.

Satellite versus total DNA replication in relation to endopolyploidy of decidual cells in the mouse.

In rodents, decidualization produces large endopolyploid cells. Amongst the various endocycles which have been demonstrated in animals and plants, different modes of DNA replication have been characterized: either total reproduction of all DNA types, or else, underreplication or amplified synthesis affecting specific parts of the genome. A double labelling method was used to determine to which of these categories the case of decidual cells belongs. A mixture of purified DNA from hormonally-stimulated control endometrium labelled by 3H-thymidine and from decidua labelled by 14C-thymidine was ultra-centrifuged to equilibrium in a Cs2 SO4-Ag gradient. Optical density at 260 nm and 14C/3H ratio were evaluated in serial fractions along the gradient. Since the 14C/3H ratio did not significantly vary along the gradient, it may be concluded that in the case of decidual cells, endopolyploidy corresponds to uniform replication of all nuclear DNA.