Laminin 511 partners with laminin 332 to mediate directional migration of Madin-Darby canine kidney epithelial cells.

Sustained directional migration of epithelial cells is essential for regeneration of injured epithelia. Front-rear polarity of migrating cells is determined by local activation of a signaling network involving Cdc42 and other factors in response to spatial cues from the environment, the nature of which are obscure. We examined the roles of laminin (LM)-511 and LM-332, two structurally different laminin isoforms, in the migration of Madin-Darby canine kidney cells by suppressing expression of their α subunits using RNA interference. We determined that knockdown of LM-511 inhibits directional migration and destabilizes cell-cell contacts, in part by disturbing the localization and activity of the polarization machinery. Suppression of integrin α3, a laminin receptor subunit, in cells synthesizing normal amounts of both laminins has a similar effect as knockdown of LM-511. Surprisingly, simultaneous suppression of both laminin α5 and laminin α3 restores directional migration and cell-cell contact stability, suggesting that cells recognize a haptotactic gradient formed by a combination of laminins.

Kinetics of mismatch formation opposite lesions by the replicative DNA polymerase from bacteriophage RB69.

The fidelity of DNA replication is under constant threat from the formation of lesions within the genome. Oxidation of DNA bases leads to the formation of altered DNA bases such as 8-oxo-7,8-dihydroguanine, commonly called 8-oxoG, and 2-hydroxyadenine, or 2-OHA. In this work we have examined the incorporation kinetics opposite these two oxidatively derived lesions as well as an abasic site analogue by the replicative DNA polymerase from bacteriophage RB69. We compared the kinetic parameters for both wild type and the low fidelity L561A variant. While nucleotide incorporation rates (k(pol)) were generally higher for the variant, the presence of a lesion in the templating position reduced the ability of both the wild-type and variant DNA polymerases to form ternary enzyme-DNA-dNTP complexes. Thus, the L561A substitution does not significantly affect the ability of the RB69 DNA polymerase to recognize damaged DNA; instead, the mutation increases the probability that nucleotide incorporation will occur. We have also solved the crystal structure of the L561A variant forming an 8-oxoG.dATP mispair and show that the propensity for forming this mispair depends on an enlarged polymerase active site.