Replication of the two template strands at eukaryotic cell DNA replication forks is a highly coordinated process that ensures accurate and efficient genome duplication. Biochemical studies, principally of plasmid DNAs containing the Simian Virus 40 origin of DNA replication, and yeast genetic studies have uncovered the fundamental mechanisms of replication fork progression. At least two different DNA polymerases, a single-stranded DNA-binding protein, a clamp-loading complex, and a polymerase clamp combine to replicate DNA. Okazaki fragment synthesis involves a DNA polymerase-switching mechanism, and maturation occurs by the recruitment of specific nucleases, a helicase, and a ligase. The process of DNA replication is also coupled to cell-cycle progression and to DNA repair to maintain genome integrity.

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The dna replication fork in eukaryotic cells

The Bloom’s Syndrome Helicase Unwinds G4 DNA

A DNA Helicase Activity Is Associated with an MCM4, -6, and -7 Protein Complex *

The RecQ helicases represent a subfamily of DNA helicases that are highly conserved in evolution. Loss of RecQ helicase function leads to a breakdown in the maintenance of genome integrity, in particular hyper-recombination. Germ-line defects in three of the five known human RecQ helicases give rise to defined genetic disorders associated with cancer predisposition and/or premature aging. These are Bloom's syndrome, Werner's syndrome and Rothmund-Thomson syndrome, which are caused by defects in the genes BLM, WRN and RECQ4 respectively. Here we review the properties of RecQ helicases in organisms from bacteria to humans, with an emphasis on the biochemical functions of these enzymes and the range of protein partners that they operate with. We will discuss models in which RecQ helicases are required to protect against replication fork demise, either through prevention of fork breakdown or restoration of productive DNA synthesis.

RecQ helicases: suppressors of tumorigenesis and premature aging.

http://depts.washington.edu/loeblabs/pdf/1999_Fry.pdf

Human Werner Syndrome DNA Helicase Unwinds Tetrahelical Structures of the Fragile X Syndrome Repeat Sequence d(CGG)n

Novel development of 5-aminolevurinic acid (ALA) in cancer diagnoses and therapy

The hammerhead ribozyme belongs to the class of molecules known as antisense RNAs However, because of short extra sequences that form the so-called catalytic loop, it can act as an enzyme Since the catalytic domain captures magnesium ions and magnesium ions can cleave phosphodiester bonds, hammerhead ribozymes are recognized as metalloenzymes In general, the cleavage of phosphodiester bonds involves acid/base catalysis, with proton transfer occurring in the transition state When the possibility of such a proton-transfer process was examined by measuring solvent isotope effects, it became apparent that no proton transfer occurs in the transition state during reactions catalyzed by a hammerhead ribozyme It is likely, therefore, that hammerhead ribozymes exploit the general double-metal-ion mechanism of catalysis, with Mg2+ ions coordinating directly with the attacking and leaving oxygen moieties Since the hammerhead ribozyme is one of the smallest RNA enzymes known and has potential as an antiviral agent, thus ribozyme has been extensively investigated for applications in vivo Ribozymes are described that have possible utility as agents against HIV-1

Ribozymes: from mechanistic studies to applications in vivo.

DNA-dependent ATPase activities in crude extracts prepared from HeLa cells were separated into five peaks designated Q1 to Q5 by FPLC Mono Q column chromatography. In our previous study, we observed that crude extracts prepared from xeroderma pigmentosum complementation group C (XP-C) cells contained no DNA-dependent ATPase activity at the peak position of Q1 and exhibited a broader peak with higher activity than normal Q2 at the peak position of Q2 [Yanagisawa, J., Seki, M., Ui, M., & Enomoto, T. (1992) J. Biol. Chem. 267, 3585-3588]. We have purified two DNA-dependent ATPases Q1 and Q2 from HeLa cells and characterized their properties in order to obtain a means to discriminate ATPase Q1 from Q2 in XP-C cells. The apparent molecular masses of Q1 and Q2 on SDS-polyacrylamide gel electrophoresis were 73 and 100 kDa, respectively. The two enzymes required a divalent cation for activity. DNA-dependent ATPase Q1 hydrolyzed ATP and dATP and Q2 hydrolyzed ATP preferentially among the nucleotides tested. Both enzymes preferred single-stranded DNA as a cofactor. The DNA-dependent ATPase activity of Q2 was inhibited by 90% in the presence of 200 mM NaCl, whereas that of Q1 was not affected by NaCl at concentrations up to 200 mM. Both enzymes had DNA helicase activity, that of Q1 being more resistant to NaCl than that of Q2. The DNA helicase activity of Q2 was about 150-fold higher than that of Q1, when compared with units of ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of Two DNA-Dependent Adenosinetriphosphatases Having DNA Helicase Activity from HeLa Cells and Comparison of the Properties of the Two Enzymes

5-aminolevulinic acid (5-ALA) has recently been employed for photodynamic diagnosis (ALA-PDD) and photodynamic therapy (ALA-PDT) of various types of cancer because hyperproliferating tumor cells do not utilize oxidative phosphorylation and do not efficiently produce heme; instead, they accumulate protoporphyrin IX (PpIX), which is a precursor of heme that is activated by violet light irradiation that results in the production of red fluorescence and singlet oxygen. The efficiencies of ALA-PDD and ALA-PDT depend on the efficient cellular uptake of 5-ALA and the inefficient excretion of PpIX. We employed the JFCR39 cell panel to determine whether tumor cells originating from different tissues can produce and accumulate PpIX. We also investigated cellular factors/molecules involved in PpIX excretion by tumor cells with the JFCR39 cell panel. Unexpectedly, the expression levels of ABCG2, which has been considered to play a major role in PpIX extracellular transport, did not show a strong correlation with PpIX excretion levels in the JFCR39 cell panel, although an ABCG2 inhibitor significantly increased intracellular PpIX accumulation in several tumor cell lines. In contrast, the expression levels of dynamin 2, which is a cell membrane-associated molecule involved in exocytosis, were correlated with the PpIX excretion levels. Moreover, inhibitors of dynamin significantly suppressed PpIX excretion and increased the intracellular levels of PpIX. This is the first report demonstrating the causal relationship between dynamin 2 expression and PpIX excretion in tumor cells.

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Mechanistic study of PpIX accumulation using the JFCR39 cell panel revealed a role for dynamin 2-mediated exocytosis.

A temperature-sensitive mutant defective in DNA replication, tsFT848, was isolated from the mouse mammary carcinoma cell line FM3A. In mutant cells, the DNA-dependent ATPase activity of DNA helicase B, which is a major DNA-dependent ATPase in wild-type cells, decreased at the nonpermissive temperature of 39 degrees C. DNA synthesis in tsFT848 cells at the nonpermissive temperature was analyzed in detail. DNA synthesis measured by incorporation of [3H]thymidine decreased to about 50% and less than 10% of the initial level at 8 and 12 h, respectively. The decrease in the level of thymidine incorporation correlated with a decrease in the number of silver grains in individual nuclei but not with the number of cells with labeled nuclei. DNA fiber autoradiography revealed that the DNA chain elongation rate did not decrease even after an incubation for 10 h at 39 degrees C, suggesting that initiation of DNA replication at the origin of replicons is impaired in the mutant cells. The decrease in DNA-synthesizing ability coincided with a decrease in the level of the DNA-dependent ATPase activity of DNA helicase B. Partially purified DNA helicase B from tsFT848 cells was more heat sensitive than that from wild-type cells. Inactivation of DNA-dependent ATPase activity of DNA helicase B from mutant cells was considerably reduced by adding DNA to the medium used for preincubation, indicating that the DNA helicase of mutant cells is stabilized by binding to DNA.

Characterization of DNA synthesis and DNA-dependent ATPase activity at a restrictive temperature in temperature-sensitive tsFT848 cells with thermolabile DNA helicase B.

Takeo Kohda

Papers

Novel development of 5-aminolevurinic acid (ALA) in cancer diagnoses and therapy

Ribozymes: from mechanistic studies to applications in vivo.

Purification of Two DNA-Dependent Adenosinetriphosphatases Having DNA Helicase Activity from HeLa Cells and Comparison of the Properties of the Two Enzymes

Mechanistic study of PpIX accumulation using the JFCR39 cell panel revealed a role for dynamin 2-mediated exocytosis.

Characterization of DNA synthesis and DNA-dependent ATPase activity at a restrictive temperature in temperature-sensitive tsFT848 cells with thermolabile DNA helicase B.