No abstract

The sixth transmembrane segment (TM6) of the CFTR chloride channel has been intensively investigated. The effects of amino acid substitutions and chemical modification of engineered cysteines (cysteine scanning) on channel properties strongly suggest that TM6 is a key component of the anion-conducting pore, but previous cysteine-scanning studies of TM6 have produced conflicting results. Our aim was to resolve these conflicts by combining a screening strategy based on multiple, thiol-directed probes with molecular modeling of the pore. CFTR constructs were screened for reactivity toward both channel-permeant and channel-impermeant thiol-directed reagents, and patterns of reactivity in TM6 were mapped onto two new, molecular models of the CFTR pore: one based on homology modeling using Sav1866 as the template and a second derived from the first by molecular dynamics simulation. Comparison of the pattern of cysteine reactivity with model predictions suggests that nonreactive sites are those where the TM6 side chains are occluded by other TMs. Reactive sites, in contrast, are generally situated such that the respective amino acid side chains either project into the predicted pore or lie within a predicted extracellular loop. Sites where engineered cysteines react with both channel-permeant and channel-impermeant probes occupy the outermost extent of TM6 or the predicted TM5−6 loop. Sites where cysteine reactivity is limited to channel-permeant probes occupy more cytoplasmic locations. The results provide an initial validation of two, new molecular models for CFTR and suggest that molecular dynamics simulation will be a useful tool for unraveling the structural basis of anion conduction by CFTR.

Deletion of Phe508 from CFTR results in a temperature-sensitive folding defect that impairs protein maturation and chloride channel function. Both of these adverse effects, however, can be mitigated to varying extents by second-site, suppressor mutations. To better understand the impact of second-site mutations on channel function, we compared the thermal sensitivity of CFTR channels in Xenopus oocytes. CFTR-mediated conductance of oocytes expressing wt or ΔF508 CFTR was stable at 22°C and increased at 28°C; a temperature permissive for ΔF508 CFTR expression in mammalian cells. At 37°C, however, CFTR-mediated conductance was further enhanced, whereas that due to ΔF508 CFTR channels decreased rapidly towards background, a phenomenon referred to here as “thermal inactivation.” Thermal inactivation of ΔF508 was mitigated by each of five suppressor mutations, I539T, R553M, G550E, R555K and R1070W; but each exerted unique effects on the severity of, and recovery from, thermal inactivation. Another mutation, K1250A, known to increase open probability (Po) of ΔF508 CFTR channels, exacerbated thermal inactivation. Application of potentiators known to increase Po of ΔF508 CFTR channels at room temperature failed to protect channels from inactivation at 37°C and one, PG-01, actually exacerbated thermal inactivation. Unstimulated ΔF508CFTR channels or those inhibited by CFTRinh-172, were partially protected from thermal inactivation, suggesting a possible inverse relationship between thermal stability and gating transitions. Thermal stability of channel function and temperature-sensitive maturation of the mutant protein appear to reflect related, but distinct facets of the ΔF508 CFTR conformational defect, both of which must be addressed by effective therapeutic modalities.

Following arrest by UV-induced DNA damage, replication is restored through a sequence of steps that involve partial resection of the nascent DNA by RecJ and RecQ, branch migration and processing of the fork DNA surrounding the lesion by RecA and RecF-O-R, and resumption of DNA synthesis once the blocking lesion has been repaired or bypassed. In vitro, the primosomal proteins (PriA, PriB, and PriC) and Rep are capable of initiating replication from synthetic DNA fork structures, and they have been proposed to catalyze these events when replication is disrupted by certain impediments in vivo. Here, we characterized the role that PriA, PriB, PriC, and Rep have in processing and restoring replication forks following arrest by UV-induced DNA damage. We show that the partial degradation and processing of the arrested replication fork occurs normally in both rep and primosome mutants. In each mutant, the nascent degradation ceases and DNA synthesis initially resumes in a timely manner, but the recovery then stalls in the absence of PriA, PriB, or Rep. The results demonstrate a role for the primosome and Rep helicase in overcoming replication forks arrested by UV-induced damage in vivo and suggest that these proteins are required for the stability and efficiency of the replisome when DNA synthesis resumes but not to initiate de novo replication downstream of the lesion. P riA, PriB, and PriC were originally identified as proteins required for replication of single-strand X174 phage DNA in vitro and in vivo (70,71). In vitro, the proteins function as a complex that is required for processive priming to occur behind the replicative helicase, DnaB (1, 2). PriA initially binds a hairpin structure on the X174 chromosome, followed by PriB, DnaT, and PriC. The resulting complex then recruits DnaC, which loads the DnaB helicase onto the chromosome. The DnaG primase is then able to associate with DnaB to synthesize RNA primers. While DnaG and DnaB are sufficient for primer synthesis on X174 DNA (1), specific and processive priming of singlestranded DNA binding protein-coated phage DNA requires PriA (2). In vivo, conversion of X174 from its plus-strand form to its minus-strand replication intermediate requires PriA and other Escherichia coli host proteins (40). E. coli strains lacking PriA have reduced viability, growth rates, and culture densities relative to wild-type cells (36). priA mutants are also constitutively induced for the SOS response, and cells lacking PriA produce filaments extensively (49). Taken together, these observations led early researchers to propose that the primosomal proteins promote efficient priming for Okazaki fragments during lagging-strand replication (35,38).rep was originally identified as a mutant that was unable to support replication of DNA of several double-stranded phage, including X174 (19,20,33,56,62). rep was subsequently shown to encode a DNA helicase that tracks on the leading-strand template and is essential to reconstitute replication of double-stranded phage in vitro (55). rep mutants ...