XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis

In eukaryotic cells, chromosomal DNA replication begins with the formation of pre-replication complexes at replication origins. Formation and maintenance of pre-replication complexes is dependent upon CDC6 (ref. 1), a protein which allows assembly of MCM2-7 proteins, which are putative replicative helicases. The functional assembly of MCM proteins into chromatin corresponds to replication licensing. Removal of these proteins from chromatin in S phase is crucial in origins firing regulation. We have identified a protein that is required for the assembly of pre-replication complexes, in a screen for maternally expressed genes in Xenopus. This factor (XCDT1) is a relative of fission yeast cdt1, a protein proposed to function in DNA replication, and is the first to be identified in vertebrates. Here we show, using Xenopus in vitro systems, that XCDT1 is required for chromosomal DNA replication. XCDT1 associates with pre-replicative chromatin in a manner dependent on ORC protein and is removed from chromatin at the time of initiation of DNA synthesis. Immunodepletion and reconstitution experiments show that XCDT1 is required to load MCM2-7 proteins onto pre-replicative chromatin. These findings indicate that XCDT1 is an essential component of the system that regulates origins firing during S phase.     

三种ARS片段对水稻部分酶解小细胞团转化频率的影响

ＡＲＳ２,ＡＲＳ７,ＲＳ８是从水稻珍汕９７Ａ不育系统ＤＮＡ中的克隆的一组在酵母细胞中能直始ＤＮＡ复制的片段。其中ＡＲＳ８是单考贝而ＡＲＳ２,ＡＲＳ７却是高度重复顺序。将带有ＡＲＳ２,ＡＲＳ７,ＡＲＳ８的质粒用低压脉冲电泳法导入水稻部分酶解小细胞团中,经浅层培养,铺板,获得愈伤组织。     

DNA sequence at the end of IS1 required for transposition

The insertion sequence IS1 belongs to a class of bacterial transposable genetic elements that can form compound transposons in which two copies of IS1 flank an otherwise non-transposable segment of DNA. IS1 differs from other known elements of this class (such as IS10, IS50 and IS903) in several respects. It is one of the smallest known insertion elements, exhibits a relatively complex array of open reading frames, is present in the chromosomes of various Enterobacteria, in some cases in many copies, and its insertion can result in the duplication of either 8 or 9 base pairs (bp) in the target DNA. Furthermore, although, like other members of the compound class, it seems to undergo direct transposition, IS1 also promotes replicon fusion (co-integrate formation) at a relatively high frequency. Like all other elements studied to date, the integrity of the extremities of IS1 are essential for efficient transposition. We have constructed a test system to determine the minimal DNA sequences at the extremities of IS1 required for transposition. Sequential deletions of the end sequences reveal that 21-25 bp of an isolated extremity are sufficient for transposition. A specific sequence 13-23 bp from the ends, defining the edge of the minimal sequence, is implicated as an essential site. The sites, symmetrically arrayed at both ends of IS1, correspond to the apparent consensus sequence of the known binding sites for the Escherichia coli DNA-binding protein (called integration host factor or IHF) which is required for the site-specific recombination that leads to integration of bacteriophage lambda into the bacterial genome. The sites at the ends of IS1 may thus bind a host protein, such as JHF or a related protein, that is involved in regulating the transposition apparatus.     

Replication fork movement sets chromatin loop size and origin choice in mammalian cells

Genome stability requires one, and only one, DNA duplication at each S phase. The mechanisms preventing origin firing on newly replicated DNA are well documented, but much less is known about the mechanisms controlling the spacing of initiation events(2,3), namely the completion of DNA replication. Here we show that origin use in Chinese hamster cells depends on both the movement of the replication forks and the organization of chromatin loops. We found that slowing the replication speed triggers the recruitment of latent origins within minutes, allowing the completion of S phase in a timely fashion. When slowly replicating cells are shifted to conditions of fast fork progression, although the decrease in the overall number of active origins occurs within 2 h, the cells still have to go through a complete cell cycle before the efficiency specific to each origin is restored. We observed a strict correlation between replication speed during a given S phase and the size of chromatin loops in the next G1 phase. Furthermore, we found that origins located at or near sites of anchorage of chromatin loops in G1 are activated preferentially in the following S phase. These data suggest a mechanism of origin programming in which replication speed determines the spacing of anchorage regions of chromatin loops, that, in turn, controls the choice of initiation sites.     

Nucleosome positioning can affect the function of a cis-acting DNA element in vivo

Positioning of nucleosomes has been proposed as one mechanism whereby the activity of DNA is regulated: cis-acting elements located in linker DNA might be more accessible for interaction with trans-acting protein factors than they would be if they were directly associated with histones in nucleosome core particles. The eleven base pairs constituting the autonomously replicating sequence (ARS) of the high-copy-number TRP1ARS1 plasmid of Saccharomyces cerevisiae are located in a linker region near the edge of a positioned nucleosome and form an origin of replication. Could nucleosome positioning render the ARS accessible for interaction with the proteins necessary for its function? I have tested this hypothesis by making deletions and an insertion to move the ARS into the nucleosome DNA and then examining the effects on ARS function. There is a marked decrease in copy number when the ARS is moved into the central DNA region of the nucleosome core particle, a region known to differ in structure and stability from the peripheral segments of nucleosome DNA.     

Reduction of the minimal sequence for initiation of DNA synthesis by qualitative or quantitative changes of an initiator protein

Initiation of DNA synthesis at an origin of DNA replication involves complex protein-DNA interactions that are still poorly understood. Some of these interactions are highly specific and involve proteins (initiator proteins) thought to be essential for regulation of the initiation process because of their rate-limiting activity. We show here that both qualitative and quantitative changes in one of these proteins have profound effects on protein-DNA interactions at an origin of DNA replication, and are sufficient to reduce to less than one-third the minimal sequence required for initiation. The general implications of these findings are discussed.     

Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast

Cyclin-dependent kinases (CDKs) drive major cell cycle events including the initiation of chromosomal DNA replication. We identified two S phase CDK (S-CDK) phosphorylation sites in the budding yeast Sld3 protein that, together, are essential for DNA replication. Here we show that, when phosphorylated, these sites bind to the amino-terminal BRCT repeats of Dpb11. An Sld3-Dpb11 fusion construct bypasses the requirement for both Sld3 phosphorylation and the N-terminal BRCT repeats of Dpb11. Co-expression of this fusion with a phospho-mimicking mutant in a second essential CDK substrate, Sld2, promotes DNA replication in the absence of S-CDK. Therefore, Sld2 and Sld3 are the minimal set of S-CDK targets required for DNA replication. DNA replication in cells lacking G1 phase CDK (G1-CDK) required expression of the Cdc7 kinase regulatory subunit, Dbf4, as well as Sld2 and Sld3 bypass. Our results help to explain how G1- and S-CDKs promote DNA replication in yeast.     

Cell-type-specific replication initiation programs set fragility of the FRA3B fragile site

Common fragile sites have long been identified by cytogeneticists as chromosomal regions prone to breakage upon replication stress. They are increasingly recognized to be preferential targets for oncogene-induced DNA damage in pre-neoplastic lesions and hotspots for chromosomal rearrangements in various cancers. Common fragile site instability was attributed to the fact that they contain sequences prone to form secondary structures that may impair replication fork movement, possibly leading to fork collapse resulting in DNA breaks. Here we show, in contrast to this view, that the fragility of FRA3B--the most active common fragile site in human lymphocytes--does not rely on fork slowing or stalling but on a paucity of initiation events. Indeed, in lymphoblastoid cells, but not in fibroblasts, initiation events are excluded from a FRA3B core extending approximately 700 kilobases, which forces forks coming from flanking regions to cover long distances in order to complete replication. We also show that origins of the flanking regions fire in mid-S phase, leaving the site incompletely replicated upon fork slowing. Notably, FRA3B instability is specific to cells showing this particular initiation pattern. The fact that both origin setting and replication timing are highly plastic in mammalian cells explains the tissue specificity of common fragile site instability we observed. Thus, we propose that common fragile sites correspond to the latest initiation-poor regions to complete replication in a given cell type. For historical reasons, common fragile sites have been essentially mapped in lymphocytes. Therefore, common fragile site contribution to chromosomal rearrangements in tumours should be reassessed after mapping fragile sites in the cell type from which each tumour originates.     

DNA sequences and structural homologies of the replication origins of lambdoid bacteriophages.

The DNA sequences for the origins of replication of the lambdoid bacteriophages phi80, 434, phi21, and lambdaimm21 (identical to phi21) have been determined and compared to the lambda structure. Two presumptive elaborate binding sites for two initiator proteins have been identified in their outer sections, while a replicational primer start site seems to be located in their centres.     

Recognition of hemi-methylated DNA by the SRA protein UHRF1 by a base-flipping mechanism

DNA methylation of CpG dinucleotides is an important epigenetic modification of mammalian genomes and is essential for the regulation of chromatin structure, of gene expression and of genome stability. Differences in DNA methylation patterns underlie a wide range of biological processes, such as genomic imprinting, inactivation of the X chromosome, embryogenesis, and carcinogenesis. Inheritance of the epigenetic methylation pattern is mediated by the enzyme DNA methyltransferase 1 (Dnmt1), which methylates newly synthesized CpG sequences during DNA replication, depending on the methylation status of the template strands. The protein UHRF1 (also known as Np95 and ICBP90) recognizes hemi-methylation sites via a SET and RING-associated (SRA) domain and directs Dnmt1 to these sites. Here we report the crystal structures of the SRA domain in free and hemi-methylated DNA-bound states. The SRA domain folds into a globular structure with a basic concave surface formed by highly conserved residues. Binding of DNA to the concave surface causes a loop and an amino-terminal tail of the SRA domain to fold into DNA interfaces at the major and minor grooves of the methylation site. In contrast to fully methylated CpG sites recognized by the methyl-CpG-binding domain, the methylcytosine base at the hemi-methylated site is flipped out of the DNA helix in the SRA-DNA complex and fits tightly into a protein pocket on the concave surface. The complex structure suggests that the successive flip out of the pre-existing methylated cytosine and the target cytosine to be methylated is associated with the coordinated transfer of the hemi-methylated CpG site from UHRF1 to Dnmt1.     

A whole genome approach to in vivo DNA-protein interactions in E. coli.

The increasingly rapid pace at which genomic DNA sequences are being determined has created a need for more efficient techniques to determine which parts of these sequences are bound in vivo by the proteins controlling processes such as gene expression, DNA replication and chromosomal mechanics. Here we describe a whole-genome approach to identify and characterize such DNA sequences. The method uses endogenous or artificially introduced methylases to methylate all genomic targets except those protected in vivo by protein or non-protein factors interfering with methylase action. These protected targets remain unmethylated in purified genomic DNA and are identified using methylation-sensitive restriction endonucleases. When the method was applied to the Escherichia coli genome, 0.1% of the endogenous adenine methyl-transferase (Dam methylase) targets were found to be unmethylated. Five foreign methylases were examined by transfection. Database-matched DNA sequences flanking the in vivo-protected Dam sites all fell in the non-coding regions of seven E. coli operons (mtl, cdd, flh, gut, car, psp and fep). In the first four operons these DNA sequences closely matched the consensus sequence that binds to the cyclic AMP-receptor protein. The in vivo protection at the Dam site upstream of the car operon was correlated with a downregulation of car expression, as expected of a feedback repressor-binding model.     

ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex.

A multiprotein complex that specifically recognizes cellular origins of DNA replication has been identified and purified from the yeast Saccharomyces cerevisiae. We observe a strong correlation between origin function and origin recognition by this activity. Interestingly, specific DNA binding by the origin recognition complex is dependent upon the addition of ATP. We propose that the origin recognition complex acts as the initiator protein for S. cerevisiae origins of DNA replication.