Evidence for chromosomal replicons as units of sister chromatid exchanges

Chromosomal replicons have been described as the cytological counterpart of DNA replicon clusters and have previously been studied in vitro using premature chromosome condensation-sister chromatid differentiation (PCC-SCD) techniques. Chromosomal replicons are visualized as small SCD segments in S-phase cells, and measurement of these segments can provide estimates of relative chromosomal replicon size corresponding to DNA replicon clusters functioning coordinately in S-phase. Current hypotheses of sister chromatid exchange (SCE) formation postulate that sites of SCE induction are associated with active replicons or replicon clusters. We have applied the PCC-SCD technique to in vivo studies of mouse bone marrow cells that have been treated with cyclophosphamide (CP) for two cell cycles. We have been able to visualize chromosomal replicons, as well as SCEs which have been induced in vivo by CP treatment, simultaneously in the same cells. Chromosomal replicons visualized as small SCD segments were measured in PCC cells classified at early or late S-phase based on SCD segment size prevalence. Early S-phase (E/S) PCC cells contained 90% of the SCD segments measured clustered in a segment size range of 0.1 to 0.8 m with a peak value around 0.3 to 0.6 m regardless of CP treatment. As the cells progressed through S-phase, late S-phase (L/S) PCC cells were characterized by the appearance of larger SCD segments and even whole SCD chromosomes in addition to small SCD segments. A concentration of units around 0.4 to 1.0 m was found for L/S SCD segment size distributions regardless of CP treatment with an apparent bimodal profile. Our in vivo data support the existence of a subunit organization of chromosomal replication with a basic functional unit being 0.3 to 0.6 m in size. In addition, we have found that this chromosomal unit of replication or chromosomal replicon does not seem to be functionally perturbed by the mutagen CP. We also found that small SCD segments of 0.4 to 0.7 m in length were involved in the formation of an SCE, suggesting that both spontaneous and CP-induced SCEs occur between chromosomal replicons. These findings provide direct cytogenetic evidence to support a replicon cluster/chromosomal replicon model for SCE formation.     

Late replicating bands of human chromosomes demonstrated by fluorochrome and Giemsa staining.

The addition of thymidine (TdR) to cells growing in a medium containing 5-bromodeoxyuridine (BUdR) at the end of the first replication cycle results in the incorporation of TdR into the late replicating DNA regions. These sites can be visualized by staining the metaphase chromosomes with the fluorescent dye "33258 Hoechst" or a "33258 Hoechst" Giemsa procedure. A sequence of late replication patterns has been established in metaphase chromosomes of cultured human peripheral lymphocytes. The patterns are in agreement with those obtained by the standard autoradiographic procedures, but are more accurate. As is known from autoradiography, late replicating bands are in the position of G or Q bands. The "33258 Hoechst" Giemsa staining procedure of chromosomes which have replicated in the presence of BUdR first and in TdR for the last 2 hrs of the S phase is preferable to the currently used Giemsa banding techniques: the method yields very well banded metaphases in all preparations examined, as the chromosome structure is not disrupted by the pretreatment. The bands are very distinct, even in the "difficult" chromosomes (e.g. No. 4, 5, 8 and X). In female cells the late replicating X chromosome can be identified by its size and staining pattern. In addition to the replication asynchrony, the sequence of replication within both X chromosomes in female cells is not absolutely identical. The phenomenon of a phase difference in replication between the homologues is not a peculiarity of the X chromosome, but can be found in all autosomes as well as in homologous positions on the chromatids of individual chromosomes.     

Detection of replication initiation by a replicon family in DNA of synchronized pea (Pisum sativum) root cells using benzoylated naphthoylated DEAE-cellulose chromatography

Fractionated replicating DNA from pea was obtained from both synchronized cells just starting replication and from carbohydrate-starved cells ending replication. Benzoylated naphthoylated DEAE-cellulose chromatography of pulse-labeled DNA digested with EcoR I gave evidence that a family of replicons initiated replication 45 to 60 min after synchronized cells were released from the G₁/S phase boundary. DNA from cells labeled in late S phase, on the other hand, showed no signs of additional replication initiations before entering G₂ phase. Results with DNA from both early and late S phase cells comply with a model based on the premise that with short pulses of [³H]-thymidine the isotope is localized at replication forks and that longer pulses label both replication forks and recently replicated segments of double-stranded DNA. The model applies only to DNA subjected to fragmentation before chromatography.The results also suggest that benzoylated naphthoylated DEAE-cellulose chromatography is a useful means to isolate origins and replication forks from synchronized plant cells.     

Replication timing in a single human chromosome 11 transferred into the Chinese hamster ovary (CHO) cell line

DNA replication in eukaryotes initiates from discrete genomic regions, termed origins, according to a strict and often tissue-specific temporal program. However, the genetic program that controls activation of replication origins has still not been fully elucidated in mammalian cells. Previously, we measured replication timing at the sequence level along human chromosomes 11q and 21q. In the present study, we sought to obtain a greater understanding of the relationship between replication timing programs and human chromosomes by analysis of the timing of replication of a single human chromosome 11 that had been transferred into the Chinese hamster ovary (CHO) cell line by chromosome engineering. Timing of replication was compared for three 11q chromosomal regions in the transformed CHO cell line (CHO(h11)) and the original human fibroblast cell line, namely, the R/G-band boundary at 11q13.5/q14.1, the centromere and the distal telomere. We found that the pattern of replication timing in and around the R/G band boundary at 11q13.5/q14.1 was similar in CHO(h11) cells and fibroblasts. The 11q centromeric region, which replicates late in human fibroblasts, replicated in the second half of S phase in CHO(h11) cells. By contrast, however, the telomeric region at 11q25, which is late replicating in fibroblasts (and in several other human cell lines), replicated in the first half of S phase or in very early S phase in CHO(h11) cells. Our observations suggest that the replication timing programs of the R/G-band boundary and the centromeric region of human chromosome 11q are maintained in CHO(h11) cells, whereas that for the telomeric region is altered. The replication timing program of telomeric regions on human chromosomes might be regulated by specific mechanisms that differ from those for other chromosomal regions.     

Regulation of replication at the R/G chromosomal band boundary and pericentromeric heterochromatin of mammalian cells

Mammalian chromosomes consist of multiple replicons; however, in contrast to yeast, the details of this replication process (origin firing, fork progression and termination) relative to specific chromosomal domains remain unclear. Using direct visualization of DNA fibers, here we show that the rate of replication fork movement typically decreases in the early-mid S phase when the replication fork proceeds through the R/G chromosomal band boundary and pericentromeric heterochromatin. To support this, fluorescence in situ hybridization (FISH)-based replication profiles at the human 1q31.1 (R-band)-32.1 (G-band) regions revealed that replication timing switched around at the putative R/G chromosomal band boundary predicted by marked changes in GC content at the sequence level. Thus, the slowdown of replication fork movement is thought to be the general property of the band boundaries separating the functionally different chromosomal domains. By simultaneous visualization of replication fork movement and pericentromeric heterochromatin sequences on DNA fibers, we observed that this region is duplicated by many replication forks, some of which proceed unidirectionally, that originate from clustered replication origins. We showed that histone hyperacetylation is tightly associated with changes in the replication timing of pericentromeric heterochromatin induced by 5-aza-2'-deoxycytidine treatment. These results suggest that, similar to the yeast system, histone modification is involved in controlling the timing of origin firing in mammals.     

Kinetics of DNA replication in the Indian muntjac chromosomes as studied by quantitative autoradiography

DNA replication patterns of individual chromosomes and their various euchromatic and heterochromatic regions were analyzed by means of quantitative autoradiography. The cultured cells of the skin fibroblast of a male Indian muntjac were pulse labeled with ³H-thymidine and chromosome samples were prepared for the next 32 h at 1–2 h intervals. A typical late replication pattern widely observed in heterochromatin was not found in the muntjac chromosomes. The following points make the DNA replication of the muntjac chromosomes characteristics: (1) Heterochromatin replicated its DNA in a shorter period with a higher rate than euchromatin. (2) Two small euchromatic regions adjacent to centromeric heterochromatin behaved differently from other portions of euchromatin, possessing shorter Ts, higher DNA synthetic rates and starting much later and ending earlier their DNA replication. (3) Segmental replication patterns were observed in the chromosomes 2 and 3 during the entire S phase. (4) Both homologues of the chromosome 3 showed a synchronous DNA replication pattern throughout the S phase except in the distal portion of the long arms during the mid-S phase.     

Cell fusion-induced quick change in replication time of the inactive mouse X chromosome: an implication for the maintenance mechanism of late replication.

It is unknown how and why the genetically inactivated mammalian X chromosome replicates late in S phase. There are also occasional inactive X chromosomes characterized by an opposite behavior replicating early in S phase. Two clonal cell lines, MTLB3 and MTLH8, isolated from a cultured murine T-cell lymphoma have an allocyclic X chromosome of the latter type. This precociously replicating X chromosome was judged to be genetically inactive as the late replicating one. Immediately after fusion with another cell line, the precociously replicating X chromosome from these cells starts to replicate late in S phase. This finding seems to suggest that late replication characterizing the inactive X chromosome is actively maintained by a trans-acting factor in female somatic cells, and that its lack entails a switch from late replication to precocious replication. It remains unknown whether this presumptive factor also modifies the autosomal replication pattern.     

Asynchrony of DNA replication and mitotic spiralization along heterochromatic portions of Chinese hamster chromosomes

Sequence of DNA synthesis and mitotic chromosome spiralization along heterochromatic portions of the sex (X₁X₂) and of some marker chromosomes in cultured Chinese hamster cells were studied, employing two methods: study of segmentation pattern caused in chromosomes with colcemid, and autoradiography with tritiated thymidine. The heterochromatic portions of all chromosomes studied were characterized by striking internal asynchrony of DNA replication. In particular, they had segments that replicated relatively early. The short arm of the X₂ chromosome, heterochromatic in female somatic cells, had at least three such segments. Replication patterns of the long arms of the X₁ and X₂ chromosomes were different. In X₁ this arm contains several segments showing relatively early replication. The long arm of X₂ had no similar segments. The possible significance of the data obtained is discussed with regard to the problem of genetic inertness of heterochromatin. At the terminal stage of the S period, H³-thymidine seems to be incorporated into condensed chromatin of interphase nuclei. On the basis of the data obtained, it is proposed that during replication of heterochromatin consecutive despiralization of parts of it takes place.     

Studies on mammalian chromosome replication : III. Organization and replication of diplochromosomes

The process of DNA synthesis in normal and endoreduplicating mammalian cells are very similar. Both types of chromosomes are replicated in defined units termed chromosomal replicons, and in the same sequences along their lengths. The sister chromosomes of the diplochromosomes are replicated synchronously in identical patterns. The present observations suggest that organization and sequences of chromosome replication are genetically programmed.     

Dynamics of replication foci and nuclear matrix during S phase in <Emphasis Type="Italic">Allium cepa</Emphasis> L. cells

The sequential organisation of replication foci during S phase in onion ( Allium cepa) and their relationship to the nuclear matrix were investigated. To discern their structural features and temporal firing sequence, immunodetection of 5-bromo-2'-deoxyuridine (BrdU) was carried out after in vivo feeding in synchronised cells released from a 14-h-long hydroxyurea block. Replication foci consisted of small replication granules, called replisomes, which clustered together. Analysis of synchronous binucleate cells that maintained in their two nuclei the specular symmetry of distribution of sister chromosomes in anaphase, showed that replication starts in small replication foci at the telomeric pole (pattern I), though the telomeres themselves formed large foci that were late-replicating. The rDNA replication foci (pattern II) also become replicated in early S phase. Replication of large foci, including the heterochromatin (IV), occurred in late S phase and finished at the centromeric nuclear pole (pattern V). Labelling of proliferating cell nuclear antigen (PCNA) in nuclear matrices, prepared from S-phase nuclei after extensive DNase digestion, demonstrated that replication foci were always stably anchored to the nuclear matrix. Thus, association with the nucleoskeleton is not exclusively mediated by the replicating or nascent DNA. The overlapping of patterns I, II and III in the nuclear matrix, in contrast to the results of BrdU localisation in nuclei, suggests that PCNA becomes associated with the nuclear matrix before the replication foci are operative, and remains bound during replication.     

Identification of Chromosomal Bands Replicating Early in the S Phase of Normal Human Fibroblasts

Normal human fibroblasts (NHF1) were released from confluence arrest (G₀) and replated in medium containing bromodeoxyuridine (BrdU) and aphidicolin. Despite severe reduction in the rate of DNA synthesis by aphidicolin, cells reentering the cell cycle incorporated BrdU at regions of the human genome that replicated very early in S phase. After removal of aphidicolin and BrdU from the tissue culture medium, cells were collected in mitosis. Q-banding with 4′,6-diamidino-2-phenylindole/actinomycin D was used to identify metaphase chromosomes. A monoclonal anti-BrdU antibody and a fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse antibody were used to identify the BrdU-labeled sites. The criterion for scoring DNA replication sites was the detection of FITC fluorescence at homologous regions of both sister chromatids. Early replicating regions mapped within R-bands, but not all R-bands incorporated BrdU. Chromosomal bands 1p36.1, 8q24.1, 12q13, 15q15, 15q22, and 22q13 were labeled in 53% or more of the copies of these chromosomes in the data set, suggesting that these sites replicated very early in S phase. Chromosomal band 15q22 was the most frequently labeled site (64%), which indicates that it contains some of the earliest replicating sequences in normal human fibroblasts.     

Orc mutants arrest in metaphase with abnormally condensed chromosomes

The origin recognition complex (ORC) is a six subunit complex required for eukaryotic DNA replication initiation and for silencing of the heterochromatic mating type loci in Saccharomyces cerevisiae. Our discovery of the Drosophila ORC complex concentrated in the centric heterochromatin of mitotic cells in the early embryo and its interactions with heterochromatin protein 1 (HP-1) lead us to speculate that ORC may play some general role in chromosomal folding. To explore the role of ORC in chromosomal condensation, we have identified a mutant of subunit 5 of the Drosophila melanogaster origin recognition complex (Orc5) and have characterized the phenotypes of both the Orc5 and the previously identified Orc2 mutant, k43. Both Orc mutants died at late larval stages and surprisingly, despite a reduced number of S-phase cells, an increased fraction of cells were also detected in mitosis. For this latter population of cells, Orc mutants arrest in a defective metaphase with shorter and thicker chromosomes that fail to align at the metaphase plate within a poorly assembled mitotic spindle. In addition, sister chromatid cohesion was frequently lost. PCNA and MCM4 mutants had similar phenotypes to Orc mutants. We propose that DNA replication defects trigger the mitotic arrest, due to the fact that frequent fragmentation was observed. Thus, cells have a mitotic checkpoint that senses chromosome integrity. These studies also suggest that the density of functional replication origins and completion of S phase are requirements for proper chromosomal condensation.     

MAMMALIAN CHROMOSOMES IN VITRO : XVIII. DNA Replication Sequence in the Chinese Hamster

The complete DNA replication sequence of the entire complement of chromosomes in the Chinese hamster may be studied by using the method of continuous H³-thymidine labeling and the method of 5-fluorodeoxyuridine block with H³-thymidine pulse labeling as relief. Many chromosomes start DNA synthesis simultaneously at multiple sites, but the sex chromosomes (the Y and the long arm of the X) begin DNA replication approximately 4.5 hours later and are the last members of the complement to finish replication. Generally, chromosomes or segments of chromosomes that begin replication early complete it early, and those which begin late, complete it late. Many chromosomes bear characteristically late replicating regions. During the last hour of the S phase, the entire Y, the long arm of the X, and chromosomes 10 and 11 are heavily labeled. The short arm of chromosome 1, long arm of chromosome 2, distal portion of chromosome 6, and short arms of chromosomes 7, 8, and 9 are moderately labeled. The long arm of chromosome 1 and the short arm of chromosome 2 also have late replicating zones or bands. The centromeres of chromosomes 4 and 5, and occasionally a band on the short arm of the X are lightly labeled.     

Premature replication of late S period DNA regions in early S nuclei transferred to late S cytoplasm by fusion in Physarum polycephalum.

Fusion of a late S period plasmodium of Physarum polycephalum to an early S period plasmodium causes premature replication of late S replicating regions in the nuclei of the early S plasmodium. The extent of ahead-of-schedule replication of late S replicating regions in early S period nuclei increases to a plateau of 16-20% for fusions with 40-70 min of phase difference, then declines for larger phase differences. The stimulatory factors for late S replicative units are present only in late S plasmodia and appear to act only on late S regions. Once replicated, early S replicating regions are not stimulated to replicate again by fusion to a plasmodium entering the S period. Our data do not discriminate between anti-termination of replication by factors of stop sites on long replicons, and a sequential initiation of replication on new, possibly non-adjacent regions, but does provide evidence that the stimulatory factors are distinct from one another and specific for certain target replicative units.     

Assembly and characterization of heterochromatin and euchromatin on human artificial chromosomes

Background

Human centromere regions are characterized by the presence of alpha-satellite DNA, replication late in S phase and a heterochromatic appearance. Recent models propose that the centromere is organized into conserved chromatin domains in which chromatin containing CenH3 (centromere-specific H3 variant) at the functional centromere (kinetochore) forms within regions of heterochromatin. To address these models, we assayed formation of heterochromatin and euchromatin on de novo human artificial chromosomes containing alpha-satellite DNA. We also examined the relationship between chromatin composition and replication timing of artificial chromosomes.

Results

Heterochromatin factors (histone H3 lysine 9 methylation and HP1α) were enriched on artificial chromosomes estimated to be larger than 3 Mb in size but depleted on those smaller than 3 Mb. All artificial chromosomes assembled markers of euchromatin (histone H3 lysine 4 methylation), which may partly reflect marker-gene expression. Replication timing studies revealed that the replication timing of artificial chromosomes was heterogeneous. Heterochromatin-depleted artificial chromosomes replicated in early S phase whereas heterochromatin-enriched artificial chromosomes replicated in mid to late S phase.

Conclusions

Centromere regions on human artificial chromosomes and host chromosomes have similar amounts of CenH3 but exhibit highly varying degrees of heterochromatin, suggesting that only a small amount of heterochromatin may be required for centromere function. The formation of euchromatin on all artificial chromosomes demonstrates that they can provide a chromosome context suitable for gene expression. The earlier replication of the heterochromatin-depleted artificial chromosomes suggests that replication late in S phase is not a requirement for centromere function.

     

Rate of DNA replication in the DNA synthetic period of the barley chromosomes

The rate of DNA replication, as judged by H³-thymidine incorporation, at the specific time of the S-period in chromosomes of barley (Hakata No. 2) is studied by means of autoradiography.In the barley chromosomes, two different DNA units with respect to replication-time are distinguishable. The early replicating DNA is replicated at least within 1 hour ab init. of the S-period, and the late replicating DNA within 1/2 to 1 hour before the end of the S-period. The replication scarcely occurs in the middle of the S-period. These evidences suggest that the replication of chromosomal DNA in the present material does, therefore, not proceed in a continuous time sequence. Topographically, the early replicating DNA is almost confined exclusively to the distal regions of the chromosomes 1 and 5, and this situation seems applicable to other chromosomes as well, whereas the late replicating DNA is close to the centromere on its both sides. Hence, the replication of chromosomal DNA does not proceed uniformly in a longitudinal sequence along the chromosomes. The interrelationships among chromosome structure in its cytological expression, replication -pattern and -time of chromosomes, and regulating mechanisms of DNA replication are discussed.     

Monitoring S phase progression globally and locally using BrdU incorporation in TK(+) yeast strains

Eukaryotic chromosome replication is initiated from numerous origins and its activation is temporally controlled by cell cycle and checkpoint mechanisms. Yeast has been very useful in defining the genetic elements required for initiation of DNA replication, but simple and precise tools to monitor S phase progression are lacking in this model organism. Here we describe a TK(+) yeast strain and conditions that allow incorporation of exogenous BrdU into genomic DNA, along with protocols to detect the sites of DNA synthesis in yeast nuclei or on combed DNA molecules. S phase progression is monitored by quantification of BrdU in total yeast DNA or on individual chromosomes. Using these tools we show that yeast chromosomes replicate synchronously and that DNA synthesis occurs at discrete subnuclear foci. Analysis of BrdU signals along single DNA molecules from hydroxyurea-arrested cells reveals that replication forks stall 8-9 kb from origins that are placed 46 kb apart on average. Quantification of total BrdU incorporation suggests that 190 'early' origins have fired in these cells and that late replicating territories might represent up to 40% of the yeast genome. More generally, the methods outlined here will help understand the kinetics of DNA replication in wild-type yeast and refine the phenotypes of several mutants.     

BrdU-Hoechst-Giemsa analysis of DNA replication in synchronized lymphocyte cultures

The combination of a technique of reversible methotrexate (MTX) imposed G₁/S block in cultures of human lymphocytes with the BrdU-Hoechst-Giemsa technique permitted the study of DNA replication patterns in individual chromosomes at different intervals of the S phase in a cell cohort with uniform S + G₂ duration. The procedure did not increase either the frequency of chromosomal breakage or SCE freqeuncy. The technique applied permitted visualization of the banding pattern in over 90% of mitoses. Examination of mitoses following different times of exposure to BrdU revealed a high degree of synchrony in the progression of the cell cohort examined through the S phase.The presence of two distinct late replication patterns of the allocyclic X chromosome was confirmed in studies on lymphocytes from normal human females by this technique. Interindividual and intercellular differences of the replication pattern have been demonstrated. The replicating patterns from one individual were relatively constant.The analysis of the Y chromosome has revealed marked differences of the termination of replication in individual cells. Euchromatic regions have been shown to complete DNA synthesis first, followed by the distal part of the long arm and, finally, by the region of Yq11/Yq12 junction. Lateral asymmetry was localised at this region.This paper was presented as a preliminary communication at the Helsinki Chromosome Conference Aug. 29–31, 1977, and, in its final form, at the 7th International Chromosome Conference, Oxford, Aug. 26–30, 1980.