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The Ras proteins are aberrantly activated in a wide range of human cancers, often endowing tumors with aggressive properties and resistance to therapy. Decades of effort to develop direct Ras inhibitors for clinical use have thus far failed, largely because of a lack of adequate small-molecule– binding pockets on the Ras surface. Here, we report the discovery of Ras-binding miniproteins from a naïve library and their evolution to afford versions with midpicomolar affinity to Ras. A series of biochemical experiments indicated that these miniproteins bind to the Ras effector domain as dimers, and midpicomolar crystal structures revealed that these miniprotein dimers bind Ras in an unprecedented mode in which the Ras effector domain is remodeled to expose an extended pocket that connects two isolated pockets previously found to engage small-molecule ligands. We also report a Ras point mutant that stabilizes the protein in the open conformation trapped by these miniproteins. These findings provide new tools for studying Ras structure and function and present opportunities for the development of both miniprotein and small-molecule inhibitors that directly target the Ras proteins. [ABSTRACT FROM AUTHOR]

In computer graphics applications, the standard diffusion model (SDA) is used to represent translucent materials. However, the SDA is not suitable for the representation of the translucent materials with weakly scattering properties. We represent the translucent materials with the weakly scattering properties by using the P3 approximation, since the P3 approximation can more accurately represent such translucent materials than the SDA. We also present an efficient and stable image-based bidirectional subsurface scattering reflectance distribution function measurement system that can measure the reflectance property of homogeneous translucent materials. From the high-dynamic-range image of a translucent material acquired from the proposed image-based measurement systems, the reduced scattering and the absorption coefficients of the material are estimated to represent its translucent property in computer graphics applications. We demonstrate the strength of the P3 approximation for representing the translucent materials using various examples with realistic illumination based on an environmental map. [ABSTRACT FROM AUTHOR]

T7 gene 5 protein (gp5) is distributive DNA polymerase. When bound to its processivity factor, Escherichia coli thioredoxin (trx), its processivity is increased from few nucleotides to few hundreds of nucleotides. In the presence of the T7 gene 4 helicase (gp4), the processivity of gp5/trx on duplex DNA increases to thousands of nucleotides. During such processive DNA synthesis on duplex DNA we find that DNA polymerase in solution can exchange with the active DNA polymerase via a mechanism that does not affect either the rate or processivity of replisome. In these studies we have made use of a T7 gp5 in which tyrosine 526 is replaced with phenylalanine (gp5-Y526F). The altered polymerase discriminates against the chain terminating dideoxynucleotides thus providing a probe for the exchange reaction. Using a genetically altered gp5/trx that can not interact with the C-terminus of the helicase we show that the exchange of polymerase is mediated by the interaction of polymerases for solution with gp4. We postulate that the hexameric gp4 not only stabilizes the polymerase at the replication fork but that it also serves as a reservoir for DNA polymerases in the event that the functioning polymerase dissociates. [ABSTRACT FROM AUTHOR]

The formation of a replication loop on the lagging strand facilitates coordinated synthesis of the leading- and lagging- DNAstrands and provides a mechanism for recycling of the lagging- strand DNA polymerase. As an Okazaki fragment is completed, the loop is released, and a new loop is formed as the synthesis of a new Okazaki fragment is initiated. Loop release requires the dissociation of the complex formed by the interactions among helicase, DNA polymerase, and DNA. The completion of the Okazaki fragment may result in either a nick or a single-stranded DNA region. In the replication system of bacteriophage T7, the dissociation of the polymerase from either DNA region is faster than that observed for the dissociation of the helicase from DNA polymerase, implying that the replication loop is released more likely through the dissociation of the lagging-strandDNAfrom polymerase, retaining the polymerase at replication fork. Both dissociation of DNA polymerase from DNA and that of helicase from a DNA polymeraseDNA complex are much faster at a nick DNA region than the release from a ssDNA region. These results suggest that the replication loop is released as a result of the nick formed when the lagging-strand DNA polymerase encounters the previously synthesized Okazaki fragment, releasing lagging-strand DNA and retaining DNApolymerase at the replication fork for the synthesis of next Okazaki fragment. [ABSTRACT FROM AUTHOR]

Flap endonucleases remove flap structures generated during DNA replication. Gene 6 protein of bacteriophage T7 is a 5'-3'-exonuclease specific for dsDNA. Here we show that gene 6 protein also possesses a structure-specific endonuclease activity similar to known flap endonucleases. The flap endonuclease activity is less active relative to its exonuclease activity. The major cleavage by the endonuclease activity occurs at a position one nucleotide into the duplex region adjacent to a dsDNA-ssDNA junction. The efficiency of cleavage of the flap decreases with increasing length of the 5'-overhang. A 3'-single-stranded tail arising from the same end of the duplex as the 5-tail inhibits gene 6 protein flap endonuclease activity. The released flap is not degraded further, but the exonuclease activity then proceeds to hydrolyze the 5'-terminal strand of the duplex. T7 gene 2.5 single-stranded DNA-binding protein stimulates the exonuclease and also the endonuclease activity. This stimulation is attributed to a specific interaction between the two proteins because Escherichia coli single-stranded DNA binding protein does not produce this stimulatory effect. The ability of gene 6 protein to remove 5'-terminal overhangs as well as to remove nucleotides from the 5'-termini enables it to effectively process the 5'-termini of Okazaki fragments before they are ligated. [ABSTRACT FROM AUTHOR]

Synthesis of the leading DNA strand requires the coordinated activity of DNA polymerase and DNA helicase, whereas synthesis of the lagging strand involves interactions of these proteins with DNA primase. We present the first structural model of a bacteriophage T7 DNA helicase-DNA polymerase complex using a combination of small angle x-ray scattering, single-molecule, and biochemical methods. We propose that the proteinprotein interface stabilizing the leading strand synthesis involves two distinct interactions: a stable binding of the helicase to the palm domain of the polymerase and an electrostatic binding of the carboxyl-terminal tail of the helicase to a basic patch on the polymerase. DNA primase facilitates binding of DNA helicase to ssDNA and contributes to formation of the DNA helicase-DNA polymerase complex by stabilizing DNA helicase. [ABSTRACT FROM AUTHOR]