Cell cycle regulation of the licensing activity of Cdt1 in Xenopus laevis

Cdt1 is a conserved replication factor required in licensing the chromosome for a single round of DNA synthesis. The activity of Cdt1 is inhibited by geminin. The mechanism by which geminin interferes with Cdt1 activity is unknown. It is thought that geminin binds to and sequestrate Cdt1. We show that geminin does not interfere with the chromatin association of Cdt1 and that inhibition of DNA synthesis by geminin is observed following its accumulation on chromatin. The binding of geminin to chromatin has been investigated during S phase. We demonstrate that loading of geminin onto chromatin requires Cdt1, suggesting that geminin is targeted at replication origins. We also show that geminin binds chromatin at the transition from the pre-replication to pre-initiation complexes, which overlaps with the release of Cdt1. This regulation is strikingly different from that observed in somatic cells where the chromatin binding of these proteins is mutually exclusive. In contrast to somatic cells, we further show that geminin is stable during the early embryonic cell cycles. These results suggest a specific regulation of origin firing adapted to the rapid cell cycles of Xenopus and indicate that periodic degradation of geminin is not relevant to licensing during embryonic development.     

Replicative activity of isolated chromatin from proliferating and quiescent early passage and aging cultured mouse cells

Replicative activity of isolated chromatin from late passage cultured mouse cells has been compared to the activities of chromatin preparaions from dividing and quiescent early passage cells. Rates of endogenous DNA synthesis are similar for chromatin from growing or resting cells but this activity is stimulated 2.5-fold in senescent cell chromatin. Chromatin from growing young cells copies exogenously added single stranded DNA at the highest efficiency. Chromatin of senescent cells copies this template at a lower rate and resting young cell chromatin replicates single stranded DNA at the lowest efficiency. Similar relative rates are obtained when activated DNA is copied by the various chromatin preparations. Total activity of DNA polymerase extracted by salt from chromatin is similar for dividing and quiescent young cells but the proportion of DNA polymerase beta is higher in the latter. Elevated activities of DNA polymerases are extracted from chromatin of old cells. It is concluded, therefore, that chromatin-directed replication is differently arrested in non-dividing senescent cells and in quiescent early passage cells. The possible regulatory mechanisms of DNA replication in quiescence and aging are discussed.     

Xenopus egg extracts: A model system for chromatin replication

A cell-free system derived from Xenopus eggs enables in vitro reproduction of the steps occurring during eukaryotic DNA replication. With a circular single-stranded DNA template, extracts obtained from high-speed centrifugation perform complementary DNA strand synthesis coupled to chromatin assembly. Nucleosomes are formed on the newly replicated DNA and the overall reaction mimics the events occuring during chromosomal replication on the lagging strand at the replication fork. ATP is necessary at all steps examined individually, including RNA priming, elongation of DNA strands and chromatin assembly. Although not required for nucleosome formation, ATP is involved in the correct spacing of nucleosomes and the stability of the assembled chromatin. Replication of double-stranded DNA was observed only with extracts obtained from low-speed centrifugation using demembraned sperm nuclei as substrate. Nuclei are reconstituted around the DNA and then undergo a series of events characteristic of a cell cycle. In contrast, neither DNA elongation or chromatin assembly require formation of the nucleus, and both are independent of the cell cycle.     

Ligation and synthesis of chromatin deoxyribonucleic acid in vitro in neuronal, glial and liver nuclei isolated from adult guinea pig

Neuron-rich and glial nuclear preparations and liver nuclei were isolated from adult guinea pigs. These nuclei were incubated to carry out DNA-ligation and -synthesis reactions. Before and after incubation, the sizes of single-standed DNA and DNA-synthesis patterns in single strands were analysed by using alkaline sucrose-density-gradient centrifugation. Isolation of nuclei by cell-fractionation technique shortened chromatin DNA and decreased markedly the number-average molecular weight of DNA strands. Chromatin DNA in neuronal and glial nuclei was ligated at the nicks during incubation in a reaction mixture containing ATP, Mg(2+), dithiothreitol and four deoxyribonucleotides. The number-average molecular weights were estimated to increase 1.1-and 2.1-fold in neuronal and glial nuclei respectively. DNA strands in liver nuclei were shortened during incubation, but elongated under conditions that inhibit deoxyribonuclease. Since the endogenous deoxyribounuclease activity was conspicuously higher in liver nuclei than in neuronal and glial nuclei, the shortening and elongation were thought to depend on the balance between DNA ligase and deoxyribonuclease reactions. DNA synthesis occurred at the gaps in chromatin DNA and about 50% of the total synthesized DNA was found in the shorter strands having 6 to 297 bases in all species of nuclei. Based on these results, it was concluded that in nuclei isolated from non-dividing cells (neurons) and slowly dividing cells (glial and liver cells) DNA-ligation and -synthesis reactions proceeded in parallel at the breaks in single-stranded DNA, which was produced mainly by endogenous deoxyribonuclease during isolation and incubation processes.     

The arrangement of proteins on the deoxyribonucleic acid in chromatin

The arrangement of the protein component on the DNA of the chromatin complex was studied by comparing the rate of release of oligonucleotides and of protein after addition of deoxyribonuclease I and deoxyribonuclease II to rat thymus chromatin. Also the action of deoxyribonuclease I on normal chromatin and on chromatin depleted of non-histone protein was compared, to elucidate the role of the latter protein in chromatin structure. As a preliminary to the above, the rate of action of deoxyribonuclease I on DNA and on chromatin at the same DNA concentration, and the dependence of the action of this enzyme on the Mg(2+) concentration, were studied. It was found that: (1) little if any DNA in chromatin is present in extensive, truly ;free' zones, i.e. completely uncovered by protein; (2) at relatively low concentrations of added Mg(2+), deoxyribonuclease I degrades chromatin more rapidly than DNA; (3) the non-histone protein is not attached directly to the DNA in chromatin.     

Chromatin maturation depends on continued DNA-replication

The structure of [3H]thymidine pulse-labeled chromatin in lymphocytes differs from that of non-replicating chromatin by several operational criteria which are related to the higher nuclease sensitivity of replicating chromatin. These structural features of replicating chromatin rapidly disappear when the [3H]thymidine pulse is followed by a chase in the presence of an excess of non-radioactive thymidine. However, when the rate of DNA replication is reduced, as in cycloheximide-treated lymphocytes, chromatin maturation is retarded. No chromatin maturation is observed when nuclei from pulse-labeled lymphocytes are incubated in vitro in the absence of DNA precursors. In contrast, when these nuclei are incubated under conditions known to be optimal for DNA replication, the structure of replicating chromatin is efficiently converted to that of 'mature', non-replicating chromatin. We conclude that the properties of nascent DNA and/or the distance from the replication fork are important factors in chromatin maturation.     

Selective degradation of integrated murine leukemia proviral DNA by deoxyribonucleases

The sensitivity to micrococcal nuclease and DNAase I of the integrated proviral DNA sequences in Swiss mouse cells infected with Moloney murine leukemia virus has been studied. Chromatin was separated into micrococcal nuclease-sensitive and -resistant regions, and the amount of proviral sequences in these DNA preparations was estimated by kinetic hybridization with single-stranded complementary DNA of Moloney murine leukemia virus. At least two thirds of the proviral DNA sequences were found in the open regions of chromatin, and only one third was resistant to nuclease. The proviral DNA sequences are even more sensitive to deoxyribonuclease I. When intact nuclei were treated with limited amounts of enzyme, only 5% of the nuclear DNA was digested, whereas 48% of the proviral DNA was degraded.The proviral DNA sequences in cells which do not produce virus are more resistant to nuclease digestion, as compared to virus producer cells. Thus the endogenous proviral sequences, in normal uninduced Swiss mouse cells, are randomly distributed between resistant and sensitive portions of chromatin when tested with either micrococcal nuclease or pancreatic deoxyribonuclease I. The effect of cell cycle synchronization on the accessibility of the proviral sequences to pancreatic deoxyribonuclease I was investigated with rat cells infected with Moloney murine leukemia virus. The amount of proviral DNA sensitive to pancreatic deoxyribonuclease I is higher in actively dividing cells than in cells arrested at Go phase, which produce only small amounts of virus.     

A distinct first replication cycle of DNA introduced in mammalian cells

Many mutation events in microsatellite DNA sequences were traced to the first embryonic divisions. It was not known what makes the first replication cycles of embryonic DNA different from subsequent replication cycles. Here we demonstrate that an unusual replication mode is involved in the first cycle of replication of DNA introduced in mammalian cells. This alternative replication starts at random positions, and occurs before the chromatin is fully assembled. It is detected in various cell lines and primary cells. The presence of single-stranded regions increases the efficiency of this alternative replication mode. The alternative replication cannot progress through the A/T-rich FRA16B fragile site, while the regular replication mode is not affected by it. A/T-rich microsatellites are associated with the majority of chromosomal breakpoints in cancer. We suggest that the alternative replication mode may be initiated at the regions with immature chromatin structure in embryonic and cancer cells resulting in increased genomic instability. This work demonstrates, for the first time, differences in the replication progression during the first and subsequent replication cycles in mammalian cells.     

Pea chloroplast DNA primase: characterization and role in initiation of replication

A DNA primase activity was isolated from pea chloroplasts and examined for its role in replication. The DNA primase activity was separated from the majority of the chloroplast RNA polymerase activity by linear salt gradient elution from a DEAE-cellulose column, and the two enzyme activities were separately purified through heparin-Sepharose columns. The primase activity was not inhibited by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase, or by polyclonal antibodies prepared against purified pea chloroplast RNA polymerase, while the RNA polymerase activity was inhibited completely by either tagetitoxin or the polyclonal antibodies. The DNA primase activity was capable of priming DNA replication on single-stranded templates including poly(dT), poly(dC), M13mp19, and M13mp19_+ 2.1, which contains the AT-rich pea chloroplast origin of replication. The RNA polymerase fraction was incapable of supporting incorporation of ³H-TTP in in vitro replication reactions using any of these single-stranded DNA templates. Glycerol gradient analysis indicated that the pea chloroplast DNA primase (115–120 kDa) separated from the pea chloroplast DNA polymerase (90 kDa), but is much smaller than chloroplast RNA polymerase. Because of these differences in size, template specificity, sensitivity to inhibitors, and elution characteristics, it is clear that the pea chloroplast DNA primase is an distinct enzyme form RNA polymerase. In vitro replication activity using the DNA primase fraction required all four rNTPs for optimum activity. The chloroplast DNA primase was capable of priming DNA replication activity on any single-stranded M13 template, but shows a strong preference for M13mp19+2.1. Primers synthesized using M13mp19+2.1 are resistant to DNase I, and range in size from 4 to about 60 nucleotides.     

Studies on the chromatin of barley leaves during senescence

1. The activity of soluble ribonuclease and deoxyribonuclease first declined during senescence, but later increased during advanced stages of senescence. 2. Young leaves had very low ribonuclease or deoxyribonuclease activity associated with the chromatin, but the activity of these enzymes increased progressively during senescence until the leaves died. 3. No significant changes in the composition of chromatin from first seedling leaves of barley plants during aging (from 7 to 25 days) were noted. 4. The amount of RNA synthesized by chromatin in vitro declined as the leaf aged. However, if the loss of RNA due to chromatin-associated ribonuclease was taken into account, the RNA-synthesizing activity of chromatin from senescing (15-16-day-old) leaves appeared to be somewhat higher than that of chromatin from young (7-8-day-old) leaves. In leaves at the terminal stages of senescence (23 days old) the estimates of RNA synthesis by chromatin could not be made owing to complications created by high nuclease activities. 5. It is suggested that senescence may be triggered by a decline in some hormonal factor in leaves, and that the resulting production of chromatin-associated deoxyribonuclease and ribonuclease in increasing proportions may progressively cause increased degradation of DNA and newly synthesized RNA, so that ultimately the cellular functions are impaired and the cells die.     

Human DNA helicase B (HDHB) binds to replication protein A and facilitates cellular recovery from replication stress

Maintenance of genomic stability in proliferating cells depends on a network of proteins that coordinate chromosomal replication with DNA damage responses. Human DNA helicase B (HELB or HDHB) has been implicated in chromosomal replication, but its role in this coordinated network remains undefined. Here we report that cellular exposure to UV irradiation, camptothecin, or hydroxyurea induces accumulation of HDHB on chromatin in a dose- and time-dependent manner, preferentially in S phase cells. Replication stress-induced recruitment of HDHB to chromatin is independent of checkpoint signaling but correlates with the level of replication protein A (RPA) recruited to chromatin. We show using purified proteins that HDHB physically interacts with the N-terminal domain of the RPA 70-kDa subunit (RPA70N). NMR spectroscopy and site-directed mutagenesis reveal that HDHB docks on the same RPA70N surface that recruits S phase checkpoint signaling proteins to chromatin. Consistent with this pattern of recruitment, cells depleted of HDHB display reduced recovery from replication stress.     

RNA-Dependent DNA Polymerase Activity of RNA Tumor Viruses I. Directing Influence of DNA in the Reaction

The template requirements and deoxyribonucleic acid (DNA) products of the DNA polymerases isolated from Rauscher leukemia and avian myeloblastosis viruses have been examined. All DNA preparations or synthetic polydeoxynucleotides which are active as primers possess a duplex structure containing single-stranded regions with a 3'-hydroxyl terminus. Native DNA and fully single-stranded DNA are inactive; moreover, their activity is not enhanced by sonic oscillation or treatment with micrococcal nuclease, Neurospora nuclease, or low levels of deoxyribonuclease I. Poor DNA templates are activated by treatment with exonuclease III, large amounts of deoxyribonuclease I, or an endonuclease isolated from Rauscher viral preparations. In reactions primed with deoxyadenylate-deoxythymidylate copolymer, the product formed is covalently attached to primer strands, indicating that no new strands are initiated. DNA polymerase products formed with exonuclease III- or deoxyribonuclase I-treated DNA are duplex structures. Short single-stranded regions are completely filled in, whereas long single-stranded regions are only partly repaired. DNA preparations containing extensive single-stranded regions are poorly utilized as templates.     

Fine Structural in Situ Analysis of Nascent DNA Movement Following DNA Replication

Nascent DNA (newly replicated DNA) was visualized in situ with regard to the position of the previously replicated DNA and to chromatin structure. Localization of nascent DNA at the replication sites can be achieved through pulse labeling of cells with labeled DNA precursors during very short periods of time. We were able to label V79 Chinese Hamster cells for as shortly as 2 min with BrdU; Br-DNA, detected by immunoelectron microscopy, occurs at the periphery of dense chromatin, at individual dispersed chromatin fibers, and within dispersed chromatin areas. In these regions DNA polymerase α was also visualized. After a 5-min BrdU pulse, condensed chromatin also became labeled. When the pulse was followed by a chase, a larger number of gold particles occurred on condensed chromatin. Double-labeling experiments, consisting in first incubating cells with IdU for 20 min, chased for 10 min and then labeled for 5 min with CldU, reveal CldU-labeled nascent DNA on the periphery of condensed chromatin, while previously replicated IdU-labeled DNA has been internalized into condensed chromatin. Altogether, these results show that the sites of DNA replication correspond essentially to perichromatin regions and that the newly replicated DNA moves rapidly from replication sites toward the interior of condensed chromatin areas.     

组蛋白修饰在染色质复制过程中的作用

真核生物中的DNA复制,不但要保证DNA编码的基因组信息高保真复制,也要保证染色质结构所蕴含的表观遗传组稳定传递,这个过程对于维持基因组的完整性和稳定性至关重要。时至今日,人们对DNA复制的机制已经有了深入的认识,但是对染色质复制以及表观遗传信息传递的了解才刚刚开始。组蛋白是染色质结构中最主要的蛋白组成部分,其上面丰富的转录后修饰是表观遗传调控的核心方式之一。从最近几年组蛋白的修饰研究进展入手,主要综述在DNA复制过程中组蛋白修饰如何参与染色质复制的调控。     

Mitotic remodeling of the replicon and chromosome structure

Animal cloning by nuclear-transfer experiments frequently fails due to the inability of transplanted nuclei to support normal embryonic development. We show here that the formation of mitotic chromosomes in the egg context is crucial for adapting differentiated nuclei for early development. Differentiated erythrocyte nuclei replicate inefficiently in Xenopus eggs but do so as rapidly as sperm nuclei if a prior single mitosis is permitted. This mitotic remodeling involves a topoisomerase II-dependent shortening of chromatin loop domains and an increased recruitment of replication initiation factors onto chromatin, leading to a short interorigin spacing characteristic of early developmental stages. It also occurs within each early embryonic cell cycle and dominantly regulates initiation of DNA replication for the subsequent S phase. These results indicate that mitotic conditioning is crucial to reset the chromatin structure of differentiated adult donor cells for embryonic DNA replication and suggest that it is an important step in nuclear cloning.     

Sites in simian virus 40 chromatin which are preferentially cleaved by endonucleases.

Simian virus 40 (SV40) chromatin isolated from infected BSC-1 cell nuclei was incubated with deoxyribonuclease I, staphylococcal nuclease or an endonuclease endogenous to BSC-1 cells under conditions selected to introduce one doublestrand break into the viral DNA. Full-length linear DNA was isolated, and the distribution of sites of initial cleavage by each endonuclease was determined by restriction enzyme mapping. Initial cleavage of SV40 chromatin by deoxyribonuclease I or by endogenous nuclease reduced the recovery of Hind III fragment C by comparison with the other Hind III fragments. Similarly, Hpa I fragment B recovery was reduced by comparison with the other Hpa I fragments. When isolated SV40 DNA rather than SV40 chromatin was the substrate for an initial cut by deoxyribonuclease I or endogenous nuclease, the recovery of all Hind III or Hpa I fragments was approximately that expected for random cleavage. Initial cleavage by staphylococcal nuclease of either SV40 DNA or SV40 chromatin occurred randomly as judged by recovery of Hind III or Hpa I fragments. These results suggest that, in at least a portion of the SV40 chromatin population, a region located in Hind III fragment C and Hpa I fragment B is preferentially cleaved by deoxyribonuclease I or by endogenous nuclease but not by staphylococcal nuclease.Complementary information about this nuclease-sensitive region was provided by the appearance of clusters of new DNA fragments after restriction enzyme digestion of DNA from viral chromatin initially cleaved by endogenous nuclease. From the sizes of new fragments produced by different restriction enzymes, preferential endonucleolytic cleavage of SV40 chromatin has been located between map positions 0.67 and 0.73 on the viral genome.     

The isolation of Saccharomyces cerevisiae nuclear membranes with nuclease and high-salt treatment

Saccharomyces cerevisiae nuclear membranes were prepared from isolated nuclei by digesting chromatin with deoxyribonuclease and ribonuclease, washing of residual nuclei with 0.5 M MgCl₂, and discontinuous gradient centrifugation in buffered Ficoll solutions. Electron microscopic examination of the preparations showed single membrane and double membrane vesicles and membrane sheets. Pores or residual pores were often visible. In double membrane profiles the two unit membranes were often separated by the remains of the perinuclear cistern. The nuclear membrane fragments contained 58% protein, 23.8% phospholipid, 6% sterols, 7.1% neutral acylglycerols, 4.8% RNA, and 0.3% DNA. The phospholipid content of the membrane preparations was influenced by a phospholipase activity with acidic pH optimum.