Isolation of human Dna2 endonuclease and characterization of its enzymatic properties.

In eukaryotes, the creation of ligatable nicks in DNA from flap structures generated by DNA polymerase delta-catalyzed displacement DNA synthesis during Okazaki fragment processing depends on the combined action of Fen1 and Dna2. These two enzymes contain partially overlapping but distinct endonuclease activities. Dna2 is well-suited to process long flaps, which are converted to nicks by the subsequent action of Fen1. In this report, we purified human Dna2 as a recombinant protein from human cells transfected with the cDNA of the human homologue of Saccharomyces cerevisiae Dna2. We demonstrated that the purified human Dna2 enzyme contains intrinsic endonuclease and DNA-dependent ATPase activities, but is devoid of detectable DNA helicase activity. We determined a number of enzymatic properties of human Dna2 including its substrate specificity. When both 5' and 3' tailed ssDNAs were present in a substrate, such as a forked-structured one, both single-stranded regions were cleaved by human Dna2 (hDna2) with equal efficiency. Based on this and other properties of hDna2, it is likely that this enzyme facilitates the removal of 5' and 3' regions in equilibrating flaps that are likely to arise during the processing of Okazaki fragments in human cells.

WITHDRAWN: The MCM2p-binding is crucial for the function of CDC7-DBF4 protein kinase during the initiation of chromosomal DNA replication in budding yeast.

This manuscript was withdrawn at the request of the authors.

X-ray structure of prephenate dehydratase from Streptococcus mutans.

Prephenate dehydratase is a key enzyme of the biosynthesis of L-phenylalanine in the organisms that utilize shikimate pathway. Since this enzymatic pathway does not exist in mammals, prephenate dehydratase can provide a new drug targets for antibiotics or herbicide. Prephenate dehydratase is an allosteric enzyme regulated by its end product. The enzyme composed of two domains, catalytic PDT domain located near the N-terminal and regulatory ACT domain located near the C-terminal. The allosteric enzyme is suggested to have two different conformations. When the regulatory molecule, phenylalanine, is not bound to its ACT domain, the catalytic site of PDT domain maintain open (active) state conformation as Sa-PDT structure. And the open state of its catalytic site become closed (allosterically inhibited) state if the regulatory molecule is bound to its ACT domain as Ct-PDT structure. However, the X-ray structure of prephenate dehydratase from Streptococcus mutans (Sm-PDT) shows that the catalytic site of Sm-PDT has closed state conformation without phenylalanine molecule bound to its regulatory site. The structure suggests a possibility that the binding of phenylalanine in its regulatory site may not be the only prerequisite for the closed state conformation of Sm-PDT.

Leukocyte common antigen-related (LAR) tyrosine phosphatase positively regulates osteoblast differentiation by modulating extracellular signal-regulated kinase (ERK) activation.

Protein tyrosine phosphatases (PTPs) are pivotal regulators of key cellular functions, including cell growth, differentiation, and adhesion. Previously, we reported that leukocyte common antigen-related (LAR) tyrosine phosphatase promotes osteoblast differentiation in MC3T3-E1 preosteoblast cells. In the present study, the mechanism of the regulatory action of LAR on osteoblast differentiation was investigated. The mineralization of extracellular matrix and calcium accumulation in MC3T3-E1 cells were markedly enhanced by LAR overexpression, and these effects were further increased by treatment with a MEK inhibitor. In addition, LAR overexpression dramatically reduced extracellular signal-regulated kinase (Erk) activation during osteoblast differentiation. In contrast, a marginal effect of the inactive LAR mutant on Erk activation was detected. Expression of osteoblast-related genes such as ALP, BSP, DLX5, OCN, and RUNX2, was increased by LAR overexpression during osteoblast differentiation. On the basis of these results, we propose that LAR functions as a positive regulator of osteoblast differentiation by modulating ERK activation. Therefore, LAR phosphatase could be used as a novel regulatory target protein in many bone-associated diseases, including osteoporosis.

Reconstitution of Saccharomyces cerevisiae prereplicative complex assembly in vitro.

The assembly of the prereplicative complex (pre-RC) at the origin of replication in eukaryotes is a highly regulated and highly conserved process that plays a critical role in preventing multiple rounds of DNA replication per cell division cycle. This study analyzes the molecular dynamics of the assembly of Saccharomyces cerevisiae pre-RC in vitro using ARS1 plasmid DNA and yeast whole cell extracts. In addition, pre-RC assembly was reconstituted in vitro using ARS1 DNA and purified origin-recognition complex (ORC), Cdc6p and Cdt1p-Mcm2-7p. The results reveal sequential recruitment of ORC, Cdc6p, Cdt1p and Mcm2-7p on to ARS1 DNA. When Mcm2-7p is maximally loaded, Cdc6p and Cdt1p are released, suggesting that these two proteins are co-ordinately regulated during pre-RC assembly. In extracts from sid2-21 mutant cells that are deficient in CDT1, ORC and Cdc6p bind to ARS1 but Cdt1p and Mcm2-7p do not. However, Mcm2-7p does bind in the presence of exogenous Cdt1p or Cdt1p-Mcm2-7p complex. Cdt1p-Mcm2-7p complex, which was purified from G1-, early S or G2/M-arrested cells, exhibits structure-specific DNA binding, interacting only with bubble- or Y-shape-DNA, but the biological significance of this observation is not yet known.

Coupling of DNA helicase and endonuclease activities of yeast Dna2 facilitates Okazaki fragment processing.

Saccharomyces cerevisiae Dna2 possesses both helicase and endonuclease activities. Its endonuclease activity is essential and well suited to remove RNA-DNA primers of Okazaki fragments. In contrast, its helicase activity, although required for optimal growth, is not essential when the rate of cell growth is reduced. These findings suggest that DNA unwinding activity of Dna2 plays an auxiliary role in Okazaki fragment processing. To address this issue, we examined whether the Dna2 helicase activity influenced its intrinsic endonuclease activity using two mutant proteins, Dna2D657A and Dna2K1080E, which contain only helicase or endonuclease activity, respectively. Experiments performed with a mixture of Dna2D657A and Dna2K1080E enzymes revealed that cleavage of a single-stranded DNA by endonuclease activity of Dna2 occurs while the enzyme translocates along the substrate. In addition, DNA unwinding activity efficiently removed the secondary structure formed in the flap structure, which was further aided by replication protein A. Our results suggest that the Dna2 unwinding activity plays a role in facilitating the removal of the flap DNA by its intrinsic endonuclease activity.