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Research has been conducted on the DNA polymerases which require tethering to the accessory factor, a ring-shaped clamp, to bind to DNA during replication. The authors review the structural studies on loading these clamps onto DNA by the clamp loader machine.

Research shows that FASCIATA1 and FASCIATA2 genes maintain cellular and functional organization of the shoot and root apical meristems in Arabidopsis. Data indicate that the FASCIATA gene products are analogous to chromatin assembly factor-1, which helps maintain stability of gene expression status during the apical meristems postembryonic development.

Formation of a heterochromatin-like structure results in transcriptional silencing at the HM mating-type loci and telomeres in Saccharomyces cerevisiae (1-3). Once formed, such epigenetically determined structures are inherited for many mitotic divisions (4). Here we show that mutations in the proliferating cell nuclear antigen (PCNA), an essential component at the DNA replication fork5, reduced repression of genes near a telomere and at the silent mating-type locus, HMR. The pol30-8 mutant displayed coexistence of both repressed (pink) and de-repressed (white) cells within a single colony when assayed with the ADE2 gene inserted at HMR. Unlike pol30-8,the pol30-6 and pol30-79 mutants partially reduced gene silencing at telomeres and the HMR and synergistically decreased silencing in cells lacking chromatin assembly factor 1 (CAF-1). All silencing defective mutants showed reduced binding to CAF-1 in vitro and altered chromatin association of the CAF-1 large subunit in vivo. Thus, PCNA participates in inheritance of both DNA and epigenetic chromatin structures during the S phase of the cell cycle, the latter by at least two mechanisms., PCNA is important in many aspects of DNA metabolism, including DNA replication and DNA repair in eukaryotes (5). Human PCNA interacts with CAF-1, a three-subunit protein (6),to couple DNA replication [...]

Research has shown that Cdc6p affects the structure and DNA-binding activity of the origin-recognition complex in vitro. The assembly of an origin-dependent complex containing ORC and Cdc6p from Saccharomyces cerevisiae has been described. CDC6p may be essential in determining origin specificity in metazoans.

Research was conducted to identify and characterize the autonomously replicating sequence binding factor 1 (ABF1) from yeast. Data indicate that ABF1 is a DNA-binding protein and plays a direct role in nucleotide excision repair in yeast.

The novel protein p62(super dok) was purified from a hematopoietic cell line expressing the chimeric p210(super bcr-abl) protein which is usually found in chronic myelogenous leukemia. A unique feature of the p62(super dok) protein is the presence of a signaling molecule. Association of p62(super dok) with the GTPase-activating protein correlated with its tyrosine phosphorylation. p62(super dok) is rapidly tyrosine-phosphorylated upon activation of the c-Kit receptor, indicating it to be a component of a signal transduction pathway downstream of receptor tyrosine kinases.

A chromatin assembly complex (CAC) that contains chromatin assembly factor 1 (CAF-1) and the posttranslationally modified histones H3 and H4 is discovered and characterized. A cDNA for p48, the small subunit of CAF-1 and a member of a highly conserved subfamily of WD-repeat proteins, is cloned. Experiments show that the CAC plays a key role in the de novo nucleosome assembly pathway and that the p48 subunit participates in other aspects of histone metabolism.

A model system is designed with the Xenopus egg extracts and human cell extracts to examine DNA repair and chromatin assembly. In the former, repair of lesions occurs on an ultraviolet-treated plasmid together with chromatin assembly. A complementation assay is also demonstrated with human cell extracts that support DNA repair, with the incapacity for chromatin assembly remedied by Xenopus extract. The human chromatin assembly factor 1, which is responsible for the specificity of replicating DNA assembly, also supports chromatin assembly of repaired DNA.

The origin recognition complex 1 protein (Orc1p) of Saccharomyces cervisiae is structurally similar to Cdc6p, Cdc18p, and Sir3p. The yeast proteins Cdc6p and Cdc18p control DNA replication. The region of Orc1p that are similar to Cdc6p probably signals the initiation and completion of transcription. Sir3p controls transcription silencing of the mating-type loci. The N-terminal domain regions, that bind to histones and the regions required for protein localization, are similar in Orc1p and Sir3p.

The human chromatin assembly factor I(CAF I), is a protein complex having three subunits, p150, p60 and p50, which bind newly synthesized histones onto DNA replication sites indicating that these complexes are intermediates between histone synthesis and assembly onto replicating DNA. In eukaryotic organisms, the DNA is packed in the nucleus with a number of proteins to form a nucleoprotein structure, the chromatin. Of the three subunits in the CAF I protein, p150 and p60 are essential for chromatin assembly activity whereas p50 is uncertain.

Byline: Paul D. Kaufman, Ryuji Kobayashi, Naama Kessler, Bruce Stillman Abstract: Chromatin assembly factor I (CAF-I) from human cellnuclei is a three-subunit protein complex that assembles histone octamers onto replicating DNA in a cell-free system. Sequences of cDNAs encoding the two largest CAF-I subunits reveal that the p150 protein contains large clusters of charged residues, whereas p60 contains WD repeats. p150 and p60 directly interact and are both required for DNA replication-dependent assembly of nucleosomes. Deletion of the p60-binding domain from the p150 protein prevents chromatin assembly. p150 and p60 form complexes with newly synthesized histories H3 and acetylated H4 in human cell extracts, suggesting that such complexes are intermediates between histone synthesis and assembly onto replicating DNA. Author Affiliation: Cold Spring Harbor LaboratoryCold Spring Harbor, New York 11724, USA

A study of DNA replication in origin recognition complex (ORC) mutants binding to Saccharomyces cerevisiae under specific temperature conditions revealed that temperature influences the rate of replication in wild-type cells, orc2-1 ad orc5-1, with rate of replication decreasing as temperature increases. Use of genetic screening indicated CDC6 as the multicopy suppressor of orc5-1, 2-D gel and biochemical analyses suggested an interaction between Cdc6p and ORC.

P21, an inhibitor of cyclin-dependent kinases, does not inhibit proliferating-cell nuclear antigen (PCNA)-dependent nucleotide-excision repair. P21 limitedly inhibits the short gap-filling DNA proliferation by PCNA-dependent DNA polymerases delta and epsilon more than the long primer-extension proliferation. P21 inhibition of PCNA's role in DNA duplication but not in DNA repair provides reason for the theory that genetic damage inactivates chromosomal replication but permits damage-induced repair.

Identification and characterization of the proteins that replicate genomes of bacteriophage T4 and Escherichia coli reveal that the protein apparatus is capable of monitoring progress in different molecular processes and detecting abnormalities. The functions of these proteins are conserved even after replication in both prokaryotes and eukaryotes.

Simian virus 40 DNA duplication indicates that p21 controls the proliferating cell nuclear antigen (PCNA)-based DNA duplications without a cyclin-dependent protein kinase. p21 prevents PCNA stimulation of DNA polymerase delta, the main replicative DNA polymerase which is due to a reaction between p21 and PCNA. p21 and PCNA are vital for cell-cycle development, DNA duplication and restructuring impaired DNA during p53-mediated restriction of cell proliferation.

Studies on a cell-free system developed to replicate DNA from SV40 established the identity of eukaryotic cell DNA replication machinery and the role of all the replication proteins. Biochemical fractionation of human cell extracts identified the cellular replication components that combine with SV40 large T antigen to replicate SV40 containing DNA. Now highly purified proteins replicate SV40 containing plasmid DNA including the two DNA polymerases alpha and delta.

DNA replication origins serve as sites of replicative helicase loading. In all eukaryotes, the six-subunit origin recognition complex (Orc1-6; ORC) recognizes the replication origin. During late M-phase of the cell-cycle, Cdc6 binds to ORC and the ORC–Cdc6 complex loads in a multistep reaction and, with the help of Cdt1, the core Mcm2-7 helicase onto DNA. A key intermediate is the ORC–Cdc6–Cdt1–Mcm2-7 (OCCM) complex in which DNA has been already inserted into the central channel of Mcm2-7. Until now, it has been unclear how the origin DNA is guided by ORC–Cdc6 and inserted into the Mcm2-7 hexamer. Here, we truncated the C-terminal winged-helix-domain (WHD) of Mcm6 to slow down the loading reaction, thereby capturing two loading intermediates prior to DNA insertion in budding yeast. In "semi-attached OCCM," the Mcm3 and Mcm7 WHDs latch onto ORC–Cdc6 while the main body of the Mcm2-7 hexamer is not connected. In "pre-insertion OCCM," the main body of Mcm2-7 docks onto ORC–Cdc6, and the origin DNA is bent and positioned adjacent to the open DNA entry gate, poised for insertion, at the Mcm2–Mcm5 interface. We used molecular simulations to reveal the dynamic transition from preloading conformers to the loaded conformers in which the loading of Mcm2-7 on DNA is complete and the DNA entry gate is fully closed. Our work provides multiple molecular insights into a key event of eukaryotic DNA replication. [ABSTRACT FROM AUTHOR]