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Arachidonic acid stimulates cell adhesion by activating α2β1 integrins in a process that depends on protein kinases, including p38 mitogen activated protein kinase. Here, we describe the interaction of cytoskeletal components with key signaling molecules that contribute to the spreading of, and morphological changes in, arachidonic acid-treated MDA-MB-435 human breast carcinoma cells. Arachidonic acid-treated cells showed increased attachment and spreading on collagen type IV, as measured by electric cell-substrate impedance sensing. Fatty acid-treated cells displayed short cortical actin filaments associated with an increased number of f β1 integrin-containing pseudopodia, whereas untreated cells displayed elongated stress fibers and fewer clusters of β1 integrins. Confocal microscopy of arachidonic acid- treated cells showed that vinculin and phospho-p38 both appeared enriched in pseudopodia and at the tips of actin filaments, and fluorescence ratio imaging indicated the increase was specific for the phospho-(active) form of p38. Immunoprecipitates of phospho-p38 from extracts of arachidonic acid-treated cells contained vinculin, and GST-vinculin fusion proteins carrying the central region of vinculin bound phospho-p38, whereas fusion proteins expressing the terminal portions of vinculin did not. These data suggest that phospho-p38 associates with particular domains on critical focal adhesion proteins that are involved in tumor cell adhesion and spreading, and that this association can be regulated by factors in the tumor microenvironment. Key words: p38 MAP kinase, vinculin, signal transduction, arachidonic acid, cell adhesion. L'acide arachidonique stimule l'adhesion cellulaire en activant les integrines al&1 dans un processus qui depend de proteines kinases, notamment la MAP kinase p38. Nous decrivons ici l'interaction des composantes du cytosquelette avec des molecules de signalisation importantes qui contribuent a l'etalement et aux changements morphologiques des cellules de cancer du sein MBA-MB-435 traitees a l'acide arachidonique. Les cellules traitees a l'acide arachidonique presentaient un attachement et un etalement accrus sur le collagene de type IV, tel que mesure par la detection par impedance electrique des substrats cellulaires. Les cellules traitees a l'acide gras presentaient de courts filaments d'actine corticaux associes a un nombre accru de pseudopodes contenant de la β1-integrine, alors que les cellules non traitees presentaient des fibres de stress allongees et moins d'amas de β1-integrines. L'examen en microscopie confocale des cellules traitees a l'acide arachidonique montrait que la vinculine et la p38 semblaient enrichies dans les pseudopodes et au bout des filaments d'actine, et l'examen en imagerie par ratio de fluorescence indiquait que l'accroissement etait specifique a la forme phosphorylee (active) de p38. Les immunoprecipites de p38 phosphorylee provenant d'extraits de cellules traitees a l'acide arachidonique contenaient de la vinculine, et des proteines de fusion GST-vinculine comportant la region centrale de la vinculine liaient la p38 phosphorylee, contrairement aux proteines de fusion qui exprimaient les portions terminales de la vinculine. Ces donnees suggerent que la p38 phosphorylee s'associe a des domaines particuliers de proteines d'adhesion focale importantes qui sont impliquees dans l'adhesion et l'etalement des cellules tumorales, et que cette association peut etre regulee par des facteurs du microenvironnement de la tumeur. [Traduit par la Redaction] Mots-cles: MAP kinase p38, vinculine, transduction de signal, acide arachidonique, adhesion cellulaire., Introduction The adhesion of tumor cells to the extracellular matrix (ECM) is a critical step in the process of cancer metastasis (Evans 1991; Weinberg 2007). Components of the tumor microenvironment [...]

Mismatch repair (MMR) of replication errors requires DNA ends that can direct repair to the newly synthesized strand containing the error. For all but those organisms that use adenine methylation to generate nicks, the source of these ends in vivo is unknown. One possibility is that MMR may have a 'special relation to the replication complex' [Wagner R, Jr., Meselson M (1976) Proc Natl Acad Sci USA 73:4135-4139], perhaps one that allows 5' or 3' DNA ends associated with replication to act as strand discrimination signals. Here we examine this hypothesis, based on the logic that errors made by yeast DNA polymerase [alpha] (Pol [alpha]), which initiates Okazaki fragments during lagging-strand replication, will always be closer to a 5' end than will be more internal errors generated by DNA polymerase [delta] (Pol [delta]), which takes over for Pol [alpha] to complete lagging-strand replication. When we compared MMR efficiency for errors made by variant forms of these two polymerases, Msh2-dependent repair efficiencies for mismatches made by Pol [alpha] were consistently higher than for those same mismatches when made by Pol [delta]. Thus, one special relationship between MMR and replication is that MMR is more efficient for the least accurate of the major replicative polymerases, exonuclease-deficient Pol [alpha]. This observation is consistent with the close proximity and possible use of 5' ends of Okazaki fragments for strand discrimination, which could increase the probability of Msh2-dependent MMR by 5' excision, by a Msh2-dependent strand displacement mechanism, or both. replication fork | DNA repair | infidelity | mutagenesis | base substitutions doi/ 10.1073/pnas.1013048107

Selection of spontaneous, loss-of-function mutations at two chromosomal loci (pyrF and pyrE) enabled the first molecular-level analysis of replication fidelity in the extremely thermophilic bacterium Thermus thermophilus. Two different methods yielded similar mutation rates, and mutational spectra determined by sequencing of independent mutants revealed a variety of replication errors distributed throughout the target genes. The genomic mutation rate estimated from these targets, 0.00097 [+ or -] 0.00052 per replication, was lower than corresponding estimates from mesophilic microorganisms, primarily because of a low rate of base substitution. However, both the rate and spectrum of spontaneous mutations in T. thermophilus resembled those of the thermoacidophilic archaeon Sulfolobus acidocaldarius, despite important molecular differences between these two thermophiles and their genomes.

Numerous studies of the impact of accessory proteins upon the fidelity of DNA synthesis have provided a complex and sometimes discordant picture. We previously described such an analysis conducted in vitro using various bacteriophage RB69 gp43 mutator DNA polymerases with or without the accessory proteins gp32 (which binds single-stranded DNA) plus gp45/44/62 (processivity clamp and its loaders). Mutations were scored at many sites in the lacZ[alpha] mutation reporter sequence. Unexpectedly, the accessory proteins sometimes decreased and sometimes increased fidelity at a handful of specific sites. Here, we enlarge our analysis with one particular mutator polymerase compromised in both insertion accuracy and proofreading and also extend the analysis to reactions supplemented only with gp32 or only with gp45/44/62. An overall 1.56-fold increase in mutation frequencies was produced by adding single or multiple accessory proteins and was driven mainly by increased [T.sup.template]*[G.sub.primer] mispairs. Evidence was found for many additional sites where the accessory proteins influence fidelity, indicating the generality of the effect. Thus, accessory proteins contribute to the site-specific variability in mutation rates characteristically seen in mutational spectra.