The human F-Box DNA helicase FBH1 faces Saccharomyces cerevisiae Srs2 and postreplication repair pathway roles

Abstract

The Saccharomyces cerevisiae Srs2 UvrD DNA helicase controls genome integrity by preventing unscheduled recombination events. While Srs2 orthologues have been identified in prokaryotic and lower eukaryotic organisms, human orthologues of Srs2 have not been described so far. We found that the human F-box DNA helicase hFBH1 suppresses specific recombination defects of S. cerevisiae srs2 mutants, consistent with the finding that the helicase domain of hFBH1 is highly conserved with that of Srs2. Surprisingly, hFBH1 in the absence of SRS2 also suppresses the DNA damage sensitivity caused by inactivation of postreplication repair-dependent functions leading to PCNA ubiquitylation. The F-box domain of hFBH1, which is not present in Srs2, is crucial for hFBH1 functions in substituting for Srs2 and postreplication repair factors. Furthermore, our findings indicate that an intact F-box domain, acting as an SCF ubiquitin ligase, is required for the DNA damage-induced degradation of hFBH1 itself. Overall, our findings suggest that the hFBH1 helicase is a functional human orthologue of budding yeast Srs2 that also possesses self-regulation properties necessary to execute its recombination functions.

FIG. 1.

The UvrD helicase family. (A) Amino acid sequence alignments, performed with CLUSTALW, among the seven helicase motifs found in E. coli (ec) UvrD, S. cerevisiae (sc) Srs2, S. pombe (sp) Srs2, S. pombe Fbh1, mouse (m) Fbox18, and hFBH1. Identical and conserved amino acids are indicated within gray and white boxes, respectively. (B) Schematic representation of UvrD helicases and conserved motifs.

FIG. 2.

hFBH1 suppresses srs2 recombination defects arising from spontaneous DNA lesions. (A) Gene conversion rates were determined for wt, srs2Δ, and srs2::hFBH1 strains. (B) Tetrads obtained from sporulation of diploids heterozygous for rad54Δ and srs2Δ (left) or rad54Δ and srs2::hFBH1 (right). (C) The indicated strains, containing SRS2 cloned into a URA3⁺ vector, were grown at an equal cellular concentration and sequentially diluted 1:6 before being spotted onto plates without treatment (UNT) or with 5-fluoroorotic acid (5-FOA) to counterselect the plasmid. Cellular growth was evaluated after incubation at 28°C for 3 to 5 days. (D) Tetrads obtained from sporulation of diploids heterozygous for rad27Δ and srs2Δ (left) or rad27Δ and srs2::hFBH1 (right).

FIG. 3.

hFBH1 suppresses the hypersensitivity of srs2 mutants to mutagens. (A) The indicated strains were grown at an equal cellular concentration, sequentially diluted 1:6, and spotted onto plates containing MMS, HU, or 4-NQO at the indicated concentrations. Cellular growth was evaluated after incubation at 28°C for 3 to 5 days. (B) An appropriate number of log-phase cells of wt, srs2Δ, and srs2::hFBH1 yeast strains were plated and exposed to different doses of UV light. Cell survival was then evaluated compared to that of an untreated control. Log-phase cultures of the same strains were incubated with different doses of zeocin for 1 h, and cell survival was then calculated as the plating efficiency with respect to untreated cells. (C) Schematic view of hFBH1, Srs2, and mutants used in this study. Crude protein extracts were prepared from the indicated Myc-tagged strains grown under unperturbed conditions (−) or in the presence of 0.02% MMS for 3 h (+) and were analyzed by Western blotting using antibodies against Myc or α-tubulin as a loading control.

FIG. 4.

hFBH1 and the hybrid F-Srs2 protein suppress the DNA damage sensitivity of PRR mutants. (A and B) The indicated strains were grown at equal cellular concentrations, sequentially diluted 1:6, and spotted onto plates containing MMS at the indicated concentrations. Cellular growth was evaluated after incubation at 28°C for 3 to 5 days.

FIG. 5.

hFBH1 protein turnover is stimulated by DNA damage and depends on a functional F-box domain and yeast SCF complex. (A) Different cell cultures of an hFBH1 Myc-tagged yeast strain were treated for 3 h at the indicated MMS concentrations. Cycloheximide (chx) was then added to each culture to prevent further protein synthesis. At the indicated times, crude protein extracts were prepared and analyzed by Western blotting using anti-Myc or anti-tubulin antibody as a loading control. Quantification analysis of hFBH1 protein levels is also shown. (B) Log-phase cells of the indicated yeast strains were presynchronized by nocodazole treatment, released at 25°C into 0.02% MMS for 2 h, and then incubated at 37°C for 1.5 h. Cycloheximide was subsequently added, and protein samples were analyzed at the indicated time points as described for panel A. hFBH1 and F-box-truncated mutant proteins are indicated by asterisks. Quantification analysis of hFBH1 protein levels is also shown.

FIG. 6.

Model for SCF-mediated regulation of hFBH1 and Srs2 turnover in response to DNA damage. The F-box domain of hFBH1 forms an SCF ubiquitin ligase that controls hFBH1 DNA damage-induced turnover. In budding yeast under unperturbed conditions, Srs2 is recruited by sumoylated PCNA, and in response to DNA damage, the conserved Rad6/Rad18/Rad5/Ubc13 PRR pathway triggers K63 ubiquitylation of PCNA. PCNA ubiquitylation stimulates the recruitment of a putative SCF complex that, as in the case of the hFBH1 F-box domain, enhances Srs2 turnover. In addition to its role in monoubiquitylating PCNA, Rad6, by promoting the modification of another unknown target(s), aids hFBH1 and Srs2 recombination functions. Increased hFBH1 and Srs2 turnover might lead to the stimulation of recombinational repair of DNA lesions.