Multiple regulation of Rad51-mediated homologous recombination by fission yeast Fbh1.

Fbh1, an F-box helicase related to bacterial UvrD, has been proposed to modulate homologous recombination in fission yeast. We provide several lines of evidence for such modulation. Fbh1, but not the related helicases Srs2 and Rqh1, suppressed the formation of crossover recombinants from single HO-induced DNA double-strand breaks. Purified Fbh1 in complex with Skp1 (Fbh1-Skp1 complex) inhibited Rad51-driven DNA strand exchange by disrupting Rad51 nucleoprotein filaments in an ATP-dependent manner; this disruption was alleviated by the Swi5-Sfr1 complex, an auxiliary activator of Rad51. In addition, the reconstituted SCFFbh1 complex, composed of purified Fbh1-Skp1 and Pcu1-Rbx1, displayed ubiquitin-ligase E3 activity toward Rad51. Furthermore, Fbh1 reduced the protein level of Rad51 in stationary phase in an F-box-dependent, but not in a helicase domain-independent manner. These results suggest that Fbh1 negatively regulates Rad51-mediated homologous recombination via its two putative, unrelated activities, namely DNA unwinding/translocation and ubiquitin ligation. In addition to its anti-recombinase activity, we tentatively suggest that Fbh1 might also have a pro-recombination role in vivo, because the Fbh1-Skp1 complex stimulated Rad51-mediated strand exchange in vitro after strand exchange had been initiated.

Drug-sensitive DNA polymerase δ reveals a role for mismatch repair in checkpoint activation in yeast.

We have used a novel method to activate the DNA damage S-phase checkpoint response in Saccharomyces cerevisiae to slow lagging-strand DNA replication by exposing cells expressing a drug-sensitive DNA polymerase δ (L612M-DNA pol δ) to the inhibitory drug phosphonoacetic acid (PAA). PAA-treated pol3-L612M cells arrest as large-budded cells with a single nucleus in the bud neck. This arrest requires all of the components of the S-phase DNA damage checkpoint: Mec1, Rad9, the DNA damage clamp Ddc1-Rad17-Mec3, and the Rad24-dependent clamp loader, but does not depend on Mrc1, which acts as the signaling adapter for the replication checkpoint. In addition to the above components, a fully functional mismatch repair system, including Exo1, is required to activate the S-phase damage checkpoint and for cells to survive drug exposure. We propose that mismatch repair activity produces persisting single-stranded DNA gaps in PAA-treated pol3-L612M cells that are required to increase DNA damage above the threshold needed for checkpoint activation. Our studies have important implications for understanding how cells avoid inappropriate checkpoint activation because of normal discontinuities in lagging-strand replication and identify a role for mismatch repair in checkpoint activation that is needed to maintain genome integrity.

Fission yeast Swi5/Sfr1 and Rhp55/Rhp57 differentially regulate Rhp51-dependent recombination outcomes.

Several accessory proteins referred to as mediators are required for the full activity of the Rad51 (Rhp51 in fission yeast) recombinase. In this study, we analyzed in vivo functions of the recently discovered Swi5/Sfr1 complex from fission yeast. In normally growing cells, the Swi5-GFP protein localizes to the nucleus, where it forms a diffuse nuclear staining pattern with a few distinct foci. These spontaneous foci do not form in swi2Delta mutants. Upon UV irradiation, Swi5 focus formation is induced in swi2Delta mutants, a response that depends on Sfr1 function, and Sfr1 also forms foci that colocalize with damage-induced Rhp51 foci. The number of UV-induced Rhp51 foci is partially reduced in swi5Delta and rhp57Delta mutants and completely abolished in an swi5Delta rhp57Delta double mutant. An assay for products generated by HO endonuclease-induced DNA double-strand breaks (DSBs) reveals that Rhp51 and Rhp57, but not Swi5/Sfr1, are essential for crossover production. These results suggest that Swi5/Sfr1 functions as an Rhp51 mediator but processes DSBs in a manner different from that of the Rhp55/57 mediator.

Cooperative structural change of actin filaments interacting with activated myosin motor domain, detected with copolymers of pyrene-labeled actin and acto-S1 chimera protein.

Acto-S1 chimera proteins CP24 and CP18 carry the entire actin sequence, inserted in loop 2 of the motor domain of Dictyostelium myosin II, and have MgATPase activity close to that of natural Dictyostelium actomyosin [M.S.P. Siddique, T. Miyazaki, E. Katayama, T.Q.P. Uyeda, M. Suzuki, Evidence against essential roles of subdomain 1 of actin in actomyosin sliding movements, Biochem. Biophys. Res. Commun. 332 (2005) 474-481]. Here, we examined and detected cooperative structural change of actin filaments accompanying interaction with myosin motor domain in the presence of ATP using copolymer filaments consisting of pyrene-labeled skeletal actin (SA) and either CP24 or CP18. Upon addition of ATP, the fluorescence intensity increased over the range from 380 to 480nm using 365-nm excitation. The relative increases of fluorescence intensity at 390nm were 14%, 46%, and 77% for the copolymer filaments with the CP24 to actin molar ratios of 0.0625, 0.143, and 0.333, respectively, and demonstrated a sigmoid behavior. Stoichiometric analysis indicates that each CP24 molecule appears to affect four actin molecules, on average, in SA-CP24 copolymers, and each CP18 molecule appears to affect three actin molecules in SA-CP18 copolymers.

Evidence against essential roles for subdomain 1 of actin in actomyosin sliding movements.

We have engineered acto-S1chimera proteins carrying the entire actin inserted in loop 2 of the motor domain of Dictyostelium myosin II with 24 or 18 residue-linkers (CP24 and CP18, respectively). These proteins were capable of self-polymerization as well as copolymerization with skeletal actin and exhibited rigor-like structures. The MgATPase rate of CP24-skeletal actin copolymer was 1.06 s(-1), which is slightly less than the V(max) of Dictyostelium S1. Homopolymer filaments of skeletal actin, CP24, and CP18 moved at 4.7+/-0.6, 2.9+/-0.6, and 4.1+/-0.8 microm/s (mean+/-SD), respectively, on coverslips coated with skeletal myosin at 27 degrees C. Statistically thermodynamic considerations suggest that the S1 portion of chimera protein mostly resides on subdomain 1 (SD-1) of the actin portion even in the presence of ATP. This and the fact that filaments of CP18 with shorter linkers moved faster than CP24 filaments suggest that SD-1 might not be as essential as conventionally presumed for actomyosin sliding interactions.