In this report, we have described the isolation of the recombinant SCFhFBH1 complex and demonstrated that hFBH1 forms a functional complex with SKP1, CUL1 and ROC1. In the presence of E1, E2 and monomeric ubiquitin, the recombinant complex catalyzed formation of a polyubiquitin chain. Polyubiquitin chain formation by the recombinant SCFhFBH1 complex was unaffected by ssDNA or dsDNA, that markedly stimulated ATP hydrolysis by the complex (Fig. B). These results indicate that the E3 ligase activity of the complex is not changed when it interacts with DNA and that the recombinant SCFhFBH1 complex interacts with the ubiquitination machinery.
We also showed that the helicase and DNA-dependent ATPase activities of SCFhFBH1 are nearly identical to those of hFBH1 in every aspect we examined. For example, both enzymes acted in a distributive manner in the unwinding reaction (Fig. ), and their unwinding efficiencies were unaffected by the presence of fork structures in the substrate (Fig. ). DNA helicase substrates of different duplex lengths were unwound with comparable efficiencies by both enzymes (Fig. ). The SCFhFBH1 complex is distributive and incapable of unwinding long duplex DNA and, thus, it is inferred that the complex is most likely to act in a catalytic fashion and may be involved in a DNA transaction that requires unwinding of short stretches of DNA such as DNA repair or recombination (see also below).
The properties of the helicase and ATPase of hFBH1 are similar to those of SpFBH1, the S.pombe homolog. We noted two differences between these two enzymes; one is that hFBH1 is neither stimulated nor inhibited by hRPA, whereas SpFHB1 is stimulated by its cognate RPA particularly at low ATP concentrations. However, we found that RPA and hFBH1 interact in the yeast two-hybrid assays (data not shown). At present, the significance of this interaction is not clear. The other difference noted is that hFBH1 can utilize ADP and AppNp as an energy source, although the efficiencies are relatively poor (25 and 45%, respectively) compared with ATP. SpFHB1, however, used neither ADP nor AppNP as energy source (J.Kim and Y.S.Seo, unpublished observation). Our findings suggest that both hFBH1 and SpHBH1 can act as a DNA helicase even when the ATP supply is inadequate. The activity of SpFBH1 is stimulated by RPA, while hFBH1 utilizes ADP when the level of ATP is low.
Since hFBH1 contains an F-box motif critical for its association with SKP1, we tested whether deletion of the F-box had any effect on its helicase activity. The F-box-deleted mutant protein (ΔF-hFBH1), which was purified as a soluble protein, contained substantially lower ATPase and helicase activities (data not shown). Both activities of ΔF-hFBH1 were reduced >95%. It is believed that the deletion of the F-box motif impaired the integrity of the enzyme required for the catalytic function of enzyme, since deletion did not include any part of the helicase motifs. The fact that the efficiency of the SCFhFBH1 complex formation in vitro is very low suggests that the in vivo assembly of SCFhFBH1 may be a regulated process, requiring an additional factor(s) such as a chaperone. If this were the case, the assembly step could constitute a key step controlling the level of the complex in cells.
At present, it is unclear whether cells contain free hFBH1, a mixture of hFBH1 and SCFhFBH1
or the SCFhFBH1
complex only. We previously isolated SpFBH1 as a single polypeptide during its purification (1
), and recently we found that SpFBH1 was co-immunoprecipiated from crude extracts prepared from S.pombe
cells (H.Y.Kang and Y.S.Seo, unpublished observation). These findings suggest that in S.pombe
, SpFBH1 and SCFSpFBH1
are present within the same cell. However, we cannot rule out the possibility that the SCFSpFBH1
complex is dissociated during the purification procedure. We speculate that this is unlikely since the SCFhFBH1
complex is very stable and does not dissociate even in the presence of 1 M NaCl (data not shown). Since both enzymes are almost identical in their ability to unwind duplex DNA, it is possible that both hFBH1 and SCFhFBH1
, although differing in their architecture, can function similarly as a DNA helicase. However, SCFhFBH1
may have additional roles in vivo
. We currently do not understand what DNA transactions require hFBH1 and SCFhFBH1
, making it difficult to assign the role(s) of hFBH1 and hence the SCFhFBH1
complex in reactions involving DNA. Identification of the protein(s) targeted by the SCFhFBH1
complex may contribute to this solution. Since the two different activities, DNA unwinding and ubiquitin ligase, of SCFhFBH1
reside in one complex, these two activities may act in a coupled manner rather than acting in two seemingly unrelated functions. One scenario is that SCFhFBH1
can catalyze polyubiquitination of a DNA-bound substrate protein that it encounters while it unwinds duplex DNA. This possibility is consistent with our finding that unwinding occurs when ssDNA is available for binding, which suggests that the natural target is pre-existing ssDNA. This possibility is consistent with our finding that the enzyme did not unwind blunt-ended duplex DNA despite its ability to stimulate ATP hydrolysis (data not shown). The other scenario is that the energy derived from ATP hydrolysis may allow the complex to translocate along the duplex DNA rather than unwinding the duplex, since dsDNA efficiently stimulated hydrolysis of ATP by SCFhFBH1
(Table ). The chromatin-remodeling factor SWI2/SNF2 that is devoid of helicase activity despite the presence of helicase motifs, hydrolyzes ATP in a dsDNA-dependent manner (22
). ATP hydrolysis may provide the energy required for SWI2/SNF2 to translocate along chromatin DNA and/or alter protein–DNA structure when necessary, rather than directly altering the structure of duplex DNAs. If this is true for SCFhFBH1
, the helicase activity associated with the SCFhFBH1
complex may help the enzyme move along the chromatin DNA, resulting in its interaction with proteins on chromatin, which are ubiquitinated and targeted for degradation by 26S proteasome.
Recently, Shinagawa and his colleagues discovered that the S.pombe homolog of hFBH1 plays a role in post-replicational recombination events in their genetic studies (personal communication). This result is in keeping with the helicase properties we showed, since this process does not require extensive unwinding of duplex DNA and processive helicase activity of the SCFhFBH1 complex. The strategy above used by SCFhFBH1 may be essential to remove proteins associated with DNA and/or alter nucleoprotein structures, which can then facilitate subsequent DNA transactions such as post-replication recombination. For a clearer definition of a function(s) of SCFhFBH1 in vivo, we are currently attempting to identify a substrate protein that is specifically ubiquitinated by the complex.