Rate for nucleotide release from tubulin.

The lower limit for the first order rate constant for dissociation of GDP from the tubulin E-site has been determined to be 0.14 s-1; this corresponds to a reaction with a half-life of 5 s. Using this rate constant and the previously determined equilibrium constant for GDP dissociation, equal to 6.1 X 10(-8) M (Zeeberg, B., and Caplow, M. (1979) Biochemistry 18, 3880-3886), the calculated association rate constant is 2.2 X 10(6) M-1 s-1. The tubulin E-site is highly reactive and it is calculated that: the half-life is 5 s for quantitative displacement of E-site bound radioactive GDP, by added excess nonradioactive GDP; the half-life is about 260 ms for isotopic equilibration when a trace amount of radioactive GDP is added to 20 microM tubulin-GDP; the half-life is about 850 ms for re-establishing the equilibrium for GDP binding, when 20 microM tubulin is diluted 20-fold. Thus, tubulin-GDP nucleotide exchange is rapid, so that added radioactive guanine nucleotides can be used in studies of relatively rapid reactions involving the tubulin subunit

Submodular maximization over multiple matroids via generalized exchange properties

In this paper, we consider the problem of maximizing a non-negative submodular function f, defined on a (finite) ground set N, subject to matroid constraints. A function f:2NR is submodular if for all S, T ⊆ N, f(S ∪ T) + f(S ∩ T) ≤ f(S) + f(T). Furthermore, all submodular functions that we deal with are assumed to be non-negative. Throughout, we assume that our submodular function f is given by a value oracle; i.e., for a given set S ⊆ N, an algorithm can query an oracle to find the value f(S). Without loss of generality, we take the ground set N to be [n] = {1,2,...,n}

Retinoblastoma Tumor Suppressor Protein Signals through Inhibition of Cyclin-Dependent Kinase 2 Activity To Disrupt PCNA Function in S Phase

The retinoblastoma tumor suppressor protein (RB) is a negative regulator of the cell cycle that inhibits both G(1) and S-phase progression. While RB-mediated G(1) inhibition has been extensively studied, the mechanism utilized for S-phase inhibition is unknown. To delineate the mechanism through which RB inhibits DNA replication, we generated cells which inducibly express a constitutively active allele of RB (PSM-RB). We show that RB-mediated S-phase inhibition does not inhibit the chromatin binding function of MCM2 or RPA, suggesting that RB does not regulate the prereplication complex or disrupt early initiation events. However, activation of RB in S-phase cells disrupts the chromatin tethering of PCNA, a requisite component of the DNA replication machinery. The action of RB was S phase specific and did not inhibit the DNA damage-mediated association of PCNA with chromatin. We also show that RB-mediated PCNA inhibition was dependent on downregulation of CDK2 activity, which was achieved through the downregulation of cyclin A. Importantly, restoration of cyclin-dependent kinase 2 (CDK2)–cyclin A and thus PCNA activity partially restored S-phase progression in the presence of active RB. Therefore, the data presented identify RB-mediated regulation of PCNA activity via CDK2 attenuation as a mechanism through which RB regulates S-phase progression. Together, these findings identify a novel pathway of RB-mediated replication inhibition