14-3-3sigma is a cruciform DNA binding protein and associates in vivo with origins of DNA replication

A human cruciform binding protein (CBP) was previously shown to bind to cruciform DNA in a structure-specific manner and be a member of the 14-3-3 protein family. CBP had been found to contain the 14-3-3 isoforms beta, gamma, epsilon, and zeta. Here, we show by Western blot analysis that the CBP-cruciform DNA complex eluted from band-shift polyacrylamide gels also contains the 14-3-3sigma isoform, which is present in HeLa cell nuclear extracts. An antibody specific for the 14-3-3sigma isoform was able to interfere with the formation of the CBP-cruciform DNA complex. The effect of the same anti-14-3-3sigma antibody in the in vitro replication of p186, a plasmid containing the minimal replication origin of the monkey origin ors8, was also analyzed. Pre-incubation of total HeLa cell extracts with this antibody decreased p186 in vitro replication to approximately 30% of control levels, while non-specific antibodies had no effect. 14-3-3sigma was found to associate in vivo with the monkey origins of DNA replication ors8 and ors12 in a cell cycle-dependent manner, as assayed by a chromatin immunoprecipitation (ChIP) assay that involved formaldehyde cross-linking, followed by immunoprecipitation with anti-14-3-3sigma antibody and quantitative PCR. The association of 14-3-3sigma with the replication origins was maximal at the G(1)/S phase. The results indicate that 14-3-3sigma is an origin binding protein involved in the regulation of DNA replication via cruciform DNA binding.     

Limiting replication initiation factors execute the temporal programme of origin firing in budding yeast

Eukaryotic chromosomes are replicated from multiple origins that initiate throughout the S-phase of the cell cycle. Why all origins do not fire simultaneously at the beginning of S-phase is not known, but two kinase activities, cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), are continually required throughout the S-phase for all replication initiation events. Here, we show that the two CDK substrates Sld3 and Sld2 and their binding partner Dpb11, together with the DDK subunit Dbf4 are in low abundance in the budding yeast, Saccharomyces cerevisiae. Over-expression of these factors is sufficient to allow late firing origins of replication to initiate early and together with deletion of the histone deacetylase RPD3, promotes the firing of heterochromatic, dormant origins. We demonstrate that the normal programme of origin firing prevents inappropriate checkpoint activation and controls S-phase length in budding yeast. These results explain how the competition for limiting DDK kinase and CDK targets at origins regulates replication initiation kinetics during S-phase and establishes a unique system with which to investigate the biological roles of the temporal programme of origin firing.     

Regulatory mechanism of the initiation step of DNA replication by CDK in budding yeast

Cyclin-dependent kinases (CDKs) regulate the progression of the cell cycle in eukaryotes. At the onset of chromosomal DNA replication, CDKs phosphorylate two replication proteins, Sld2 and Sld3, in budding yeast. Phosphorylated Sld2 and Sld3 enhance the formation of complexes with the BRCT (BRCA1 C-terminal)-containing replication protein Dpb11. The formation of these complexes is essential and sufficient for the CDK-dependent activation of the initiation of chromosomal DNA replication. Multiple phosphorylation of Sld2 by CDKs fine-tunes the process of complex formation. Here, we discussed the regulation of the initiation step of chromosomal DNA replication via CDK-dependent phosphorylation.     

DNA replication joins the revolution: whole-genome views of DNA replication in budding yeast

Replication origins, which are responsible for initiating the replication of eukaryotic chromosomal DNAs, are spaced at intervals of 40 to 200 kb. Although the sets of proteins that assemble at replication origins during G(1) to form pre-replicative complexes are highly conserved, the structures of replication origins varies from organism to organism. The identification of replication origins has been a labor-intensive task, requiring the analysis of chromosomal DNA replication intermediates. As a result, only a few replication origins have been identified and studied. In a pair of recently published papers, Raghuraman and colleagues and Wyrick, Aparicio and colleagues provide complementary microarray-based approaches to the identification of replication origins. These genome-wide views of DNA replication in Saccharomyces cerevisiae provide new insights into the way that the genome is duplicated and hold promise for the analysis of other genomes.     

Analysis of the cruciform binding activity of recombinant 14-3-3zeta-MBP fusion protein,its heterodimerization profile with endogenous 14-3-3 isoforms,and effect on mammalian DNA replication in vitro

The human cruciform binding protein (CBP), a member of the 14-3-3 protein family, has been recently identified as an origin of DNA replication binding protein and involved in DNA replication. Here, pure recombinant 14-3-3zeta tagged with maltose binding protein (r14-3-3zeta-MBP) at its N-terminus was tested for binding to cruciform DNA either in the absence or presence of F(TH), a CBP-enriched fraction, by electromobility shift assay (EMSA), followed by Western blot analysis of the electroeluted CBP-cruciform DNA complex. The r14-3-3zeta-MBP was found to have cruciform binding activity only after preincubation with F(TH). Anti-MBP antibody immunoprecipitation of F(TH) preincubated with r14-3-3zeta-MBP, followed by Western blot analysis with antibodies specific to the beta, gamma, epsilon, zeta, and sigma 14-3-3 isoforms showed that r14-3-3zeta-MBP heterodimerized with the endogenous beta, epsilon, and zeta isoforms present in the F(TH) but not with the gamma or sigma isoforms. Immunoprecipitation of endogenous 14-3-3zeta from nuclear extracts (NE) of HeLa cells that were either serum-starved (s-s) or blocked at the G(1)/S or G(2)/M phases of the cell cycle revealed that at G(1)/S and G(2)/M, the zeta isoform heterodimerized only with the beta and epsilon isoforms, while in s-s extracts, the 14-3-3zeta/epsilon heterodimer was never detected, and the 14-3-3zeta/beta heterodimer was seldom detected. Furthermore, addition of r14-3-3zeta-MBP to HeLa cell extracts used in a mammalian in vitro replication system increased the replication level of p186, a plasmid bearing the minimal 186-bp origin of the monkey origin of DNA replication ors8, by approximately 3.5-fold. The data suggest that specific dimeric combinations of the 14-3-3 isoforms have CBP activity and that upregulation of this activity leads to an increase in DNA replication.     

Noc3p,a bHLH protein,plays an integral role in the initiation of DNA replication in budding yeast

Initiation of eukaryotic DNA replication requires many proteins that interact with one another and with replicators. Using a yeast genetic screen, we have identified Noc3p (nucleolar complex-associated protein) as a novel replication-initiation protein. Noc3p interacts with MCM proteins and ORC and binds to chromatin and replicators throughout the cell cycle. It functions as a critical link between ORC and other initiation proteins to effect chromatin association of Cdc6p and MCM proteins for the establishment and maintenance of prereplication complexes. Noc3p is highly conserved in eukaryotes and is the first identified bHLH (basic helix-loop-helix) protein required for replication initiation. As Noc3p is also required for pre-rRNA processing, Noc3p is a multifunctional protein that plays essential roles in two vital cellular processes.     

Self-regulating model for control of replication origin firing in budding yeast

A major research area concentrates on understanding the regulation of replication origin firing. It is now appreciated that checkpoint signaling participates in this controlled process and that defects in such signaling systems affect genome integrity. Inhibition of replication origin firing is most obviously apparent under conditions of replication stress, but origin firing must also be regulated on a minute-by-minute basis as cells progress normally through an unabated S-phase. Here we summarize a straightforward model to account for how origin firing could be controlled by a self-regulating system.     

Topoisomerase II inactivation prevents the completion of DNA replication in budding yeast

Type II topoisomerases are essential for resolving topologically entwined double-stranded DNA. Although anti-topoisomerase 2 (Top2) drugs are clinically important antibiotics and chemotherapies, to our knowledge, the mechanisms of cell killing by Top2 depletion and inactivation have never been directly compared. We show that depletion of Top2 protein from budding yeast cells prevents DNA decatenation during S phase. Cells complete DNA replication and enter the ensuing mitosis on schedule, suffering extensive chromosome missegregation. Cytokinesis through incompletely segregated chromosomes causes lethal DNA damage. By contrast, expression of catalytically inactive Top2 causes a stable G2 arrest requiring an intact DNA damage checkpoint. Checkpoint activation correlates with an inability to complete DNA replication, resulting in hypercatenated, gapped daughter DNA molecules. Thus, Top2 depletion and inactivation kill cells by different mechanisms, which has implications for understanding the nature of the catenation checkpoint, how DNA replication terminates, how anti-Top2 drugs work, and how new drugs might be designed.     

Analysis of DNA replication profiles in budding yeast and mammalian cells using DNA combing

DNA combing is a powerful method developed by Bensimon and colleagues to stretch DNA molecules on silanized glass coverslips. This technique provides a unique way to monitor the activation of replication origins and the progression of replication forks at the level of single DNA molecules, after incorporation of thymidine analogs, such as 5-bromo-2'-deoxyuridine (BrdU), 5-iodo-2'-deoxyuridine (IdU) and 5-chloro-2'-deoxyuridine (CldU) in newly-synthesized DNA. Unlike microarray-based approaches, this assay gives access to the variability of replication profiles in individual cells. It can also be used to monitor the effect of DNA lesions on fork progression, arrest and restart. In this review, we propose standard DNA combing methods to analyze DNA replication in budding yeast and in human cells. We also show that 5-ethynyl-2'-deoxyuridine (EdU) can be used as a good alternative to BrdU for DNA combing analysis, as unlike halogenated nucleotides, it can be detected without prior denaturation of DNA.     

Analyses of the interaction between the origin binding domain from simian virus 40 T antigen and single-stranded DNA provide insights into DNA unwinding and initiation of DNA replication

DNA helicases are essential for DNA metabolism; however, at the molecular level little is known about how they assemble or function. Therefore, as a model for a eukaryotic helicase, we are analyzing T antigen (T-ag) the helicase encoded by simian virus 40. In this study, nuclear magnetic resonance (NMR) methods were used to investigate the transit of single-stranded DNA (ssDNA) through the T-ag origin-binding domain (T-ag OBD). When the residues that interact with ssDNA are viewed in terms of the structure of a hexamer of the T-ag OBD, comprised of residues 131 to 260, they indicate that ssDNA passes over one face of the T-ag OBD and then transits through a gap in the open ring structure. The NMR-based conclusions are supported by an analysis of previously described mutations that disrupt critical steps during the initiation of DNA replication. These and related observations are discussed in terms of the threading of DNA through T-ag hexamers and the initiation of viral DNA replication.     

Deletion of the nitrate reductase N-terminal domain still allows binding of 14-3-3 proteins but affects their inhibitory properties

Nitrate reductase (NR) is post-translationally regulated by phosphorylation and binding of 14-3-3 proteins. Deletion of 56 amino acids in the amino-terminal domain of NR was previously shown to impair this type of regulation in tobacco (Nicotiana plumbaginifolia) (L. Nussaume, M. Vincentez, C. Meyer, J.-P. Boutin, M. Caboche [1995] Plant Cell 7: 611-621), although both full-length NR and deleted NR (DeltaNR) appeared to be phosphorylated in darkness (C. Lillo, S. Kazazaic, P. Ruoff, C. Meyer [1997] Plant Physiol 114: 1377-1383). We show here that in the presence of Mg(2+) and phosphatase inhibitors, NR and endogenous 14-3-3 proteins copurify through affinity chromatography. Assay of NR activity and western blots showed that endogenous 14-3-3 proteins copurified with both NR and DeltaNR. Electron transport in the heme-binding domain of DeltaNR was inhibited by Mg(2+)/14-3-3, whereas this was not the case for NR. This may indicate a different way of binding for 14-3-3 in the DeltaNR compared with NR. The DeltaNR was more labile than NR, in vitro. Lability was ascribed to the molybdopterin binding domain, and apparently an important function of the 56 amino acids is stabilization of this domain.     

Crystal structure of the DNA binding domain of the replication initiation protein E1 from papillomavirus

Papillomaviral infection causes both benign and malignant lesions and is a necessary cause of cervical carcinoma. Replication of this virus requires the replication initiation proteins E1 and E2, which bind cooperatively at the origin of replication (ori) as an (E1)2-(E2)2-DNA complex. This is a precursor to larger E1 complexes that distort and unwind the ori. We present the crystal structure of the E1 DNA binding domain refined to 1.9 A resolution. Residues critical for DNA binding are located on an extended loop and an alpha helix. We identify the E1 dimerization surface by selective mutations at an E1/E1 interface observed in the crystal and propose a model for the (E1)2-DNA complex. These and other observations suggest how the E1 DNA binding domain orchestrates assembly of the hexameric helicase on the ori.     

Tertiary initiation of replication in bacteriophage T4. Deletion of the overlapping uvsY promoter/replication origin from the phage genome

Tertiary initiation of bacteriophage T4 DNA replication is resistant to the RNA polymerase inhibitor rifampicin and apparently involved in the activity of recombination hot spots in the T4 genome (Kreuzer, K. N., and Alberts, B. M. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3345-3349). One of the origins that function by the tertiary mechanism maps at the promoter for gene uvs Y. A deletion and a linker-insertion mutation in the uvsY promoter/origin region were generated by in vitro manipulations and then placed into the T4 genome using the insertion/substitution system (Selick, H. E., Kreuzer, K. N., and Alberts, B. M. (1988) J. Biol. Chem. 263, 11336-11347). Both resulting phage strains are uvsY- mutants, but they differ in that one has a deletion of the minimal tertiary origin and the other does not. The effects of the uvsY mutations on tertiary origin activity were assayed by infecting tertiary origin plasmid-bearing Escherichia coli with the two phage mutants. The tertiary origin plasmids replicated extensively after infection by either uvsY- phage mutant, demonstrating that the uvsY protein is not required for tertiary initiation. The extent of plasmid replication was increased dramatically as a result of either mutation, indicating that the uvsY protein plays some negative role in either the initiation or subsequent processing of plasmid replicative intermediates. The phage strain with an origin deletion induced the replication of a tertiary origin plasmid with which it shared no homology. Therefore, plasmid-phage recombination is not required for the replication of tertiary origin plasmids. The replication of a tertiary origin plasmid is also shown to be independent of the phage genes uvsX, 59, and 46, but markedly reduced by mutations in the T4-induced topoisomerase.     

Role of the double-strand origin cruciform in pT181 replication

pT181 is a small rolling-circle plasmid from Staphylococcus aureus whose initiator protein, RepC, melts the plasmid's double-strand origin (DSO) and extrudes a cruciform involving IR II, a palindrome flanking the initiation nick site. We have hypothesized that the cruciform is required for initiation, providing a single-stranded region for the assembly of the replisome (R. Jin et al., 1997, EMBO J. 16, 4456-4566). In this study, we have tested the requirement for cruciform extrusion by disrupting the symmetry of the IR II palindrome or by increasing its length. The modified DSOs were tested for replication with RepC in trans. Rather surprisingly, disruption of the IR II symmetry had no detectable effect on replication or on competitivity of the modified DSO, though plasmids with IR II disrupted were less efficiently relaxed than the wild type by RepC. However, in conjunction with IR II disruption, modification of the tight RepC binding site IR III blocked replication. These results define two key elements of the pT181 initiation mechanism--the IR II conformation and the RepC binding site (IR III)--and they indicate that pT181 replication initiation is sufficiently robust to be able to compensate for significant modifications in the configuration of the DSO.     

DNA replication stress is a determinant of chronological lifespan in budding yeast

The chronological lifespan of eukaryotic organisms is extended by the mutational inactivation of conserved growth-signaling pathways that regulate progression into and through the cell cycle. Here we show that in the budding yeast S. cerevisiae, these and other lifespan-extending conditions, including caloric restriction and osmotic stress, increase the efficiency with which nutrient-depleted cells establish or maintain a cell cycle arrest in G1. Proteins required for efficient G1 arrest and longevity when nutrients are limiting include the DNA replication stress response proteins Mec1 and Rad53. Ectopic expression of CLN3 encoding a G1 cyclin downregulated during nutrient depletion increases the frequency with which nutrient depleted cells arrest growth in S phase instead of G1. Ectopic expression of CLN3 also shortens chronological lifespan in concert with age-dependent increases in genome instability and apoptosis. These findings indicate that replication stress is an important determinant of chronological lifespan in budding yeast. Protection from replication stress by growth-inhibitory effects of caloric restriction, osmotic and other stresses may contribute to hormesis effects on lifespan. Replication stress also likely impacts the longevity of higher eukaryotes, including humans.     

Abnormality in initiation program of DNA replication is monitored by the highly repetitive rRNA gene array on chromosome XII in budding yeast

We have shown previously that perturbation of origin firing in chromosome replication causes DNA lesions and triggers DNA damage checkpoint control, which ensures genomic integrity by stopping cell cycle progression until the lesions are repaired or by inducing cell death if they are not properly repaired. This was based on the observation that the temperature-sensitive phenotype of orc1-4 and orc2-1 mutants required a programmed action of the RAD9-dependent DNA damage checkpoint. Here, we report that DNA lesions in the orc mutants are induced much more quickly and frequently within the rRNA gene (rDNA) locus than at other chromosomal loci upon temperature shift. orc mutant cells with greatly reduced rDNA copy numbers regained the ability to grow at restrictive temperatures, and the checkpoint response after the temperature shift became weak in these cells. In orc2-1 cells, completion of chromosomal duplication was delayed specifically on chromosome XII, where the rDNA array is located, and the delay was partially suppressed when the rDNA copy number was reduced. These results suggest that the rDNA locus primarily signals abnormalities in the initiation program to the DNA damage checkpoint and that the rDNA copy number modulates the sensitivity of this monitoring function.     

14-3-3 cruciform-binding proteins as regulators of eukaryotic DNA replication

Cruciforms are secondary DNA structures, serving as recognition signals at or near eukaryotic (yeast and mammalian) origins of DNA replication. The cruciform-binding protein is a member of the 14-3-3 protein family and binds to origins of DNA replication in a cell cycle-dependent manner. Five 14-3-3 protein isoforms (beta, gamma, epsilon, zeta and sigma) have been identified as having cruciform binding activity.