Colocalization of FFA-1 with RPA and Replication Foci
We first used indirect immunofluorescence staining to establish the relationship among FFA-1, RPA, and sites of DNA replication in nuclei reconstituted in Xenopus egg extracts. To do this, nuclei were reconstituted by mixing demembranated sperm chromatin and egg extracts. After various lengths of time they were fixed and then stained for FFA-1, RPA, and DNA synthesis. Replication began asynchronously, but generally occurred between 45 and 85 min in most nuclei. It was monitored by the incorporation of biotin-dCTP which was added to the reaction 5 min before fixation. As shown in , FFA-1 and RPA displayed an almost identical spatial distribution throughout DNA replication. During the early stage (, A–D; 40–45 min), they formed discrete foci on chromatin. Replication was not detectable in most nuclei, which is consistent with the observation that focus structure is formed before DNA synthesis. But when replication was detected, it occurred at a subset of FFA-1 foci (, A–C), suggesting that FFA-1 foci are indeed where replication is initiated. During the middle stage, both FFA-1 and RPA showed a more diffuse staining colocalizing with chromatin (, E–H; 45–85 min). Replication proceeded rapidly and was also detected throughout chromatin (, D–F). The diffuse FFA-1 and RPA staining subsided by the end of this stage and FFA-1 and RPA could again be detected as foci which often showed incorporation of biotin-dCTP (data not shown). During the late stage (after 85 min), replication was no longer detectable (data not shown), the diffuse staining of FFA-1 and RPA further decreased, but more discrete foci containing FFA-1 and RPA appeared again (, I–L) and persisted up to at least 150 min (the maximum time assayed).
Figure 1 Colocalization of FFA-1 and RPA in nuclei reconstituted around sperm chromatin in the interphase egg extract. Representative nuclei from early (40–45 min; A–D), middle (45–85 min; E–H), and late (85–150 (more ...)
Figure 2 Colocalization of FFA-1 and sites of DNA synthesis. (A–F) In normal reconstituted nuclei. Biotin-dCTP was added 5 min before fixation. (A–C) 45 min. (D–F) 60 min. (G–L) In reconstituted nuclei formed in the presence of (more ...)
The more diffuse staining of FFA-1 and RPA was always observed in the nuclei that displayed extensive DNA synthesis, suggesting that the two phenomena might be mechanistically linked. A reasonable explanation is that during DNA synthesis more single-stranded DNA is generated, which in turn attracts more RPA and FFA-1 and the foci consequently appear expanded in size. To test this hypothesis, we blocked DNA synthesis at the priming step with aphidicolin, which inhibits DNA polymerases α and δ (Melendy and Stillman 1991
). As shown in , aphidicolin completely blocked the incorporation of biotin-dCTP, yet FFA-1 foci still formed and their number increased to >1,000 after 80 min of incubation. To determine whether these foci were where replication would have occurred, we designed a replication run-on experiment based on the observation that replication elongation resumes synchronously after removal of aphidicolin (Strausfeld et al. 1994
). In brief, the aphidicolin-arrested nuclei were spun onto a coverslip through a 1 M sucrose cushion. The nuclei were then incubated with a mixture of dATP, dGTP, TTP, and biotin-dCTP to allow replication elongation to resume for a short period of time (usually 20 s). As shown in , the sites of biotin-dCTP incorporation overlapped with FFA-1 foci, suggesting that FFA-1 foci are indeed sites for DNA replication even in the middle stage nuclei.
Immunodepletion of FFA-1 on DNA Replication
We then directly tested the role of FFA-1 in DNA replication by immunodepleting it from egg extracts. As shown in A, the anti–FFA-1 antibody but not the control antibody removed FFA-1 to a level below detection (>98% depletion). Yet when assayed for DNA replication in the nuclei reconstituted with the depleted extracts, no significant difference was observed between the two, suggesting that FFA-1 is not essential for DNA replication ( B). However, we also failed to notice a significant reduction in the ability of the FFA-1–depleted extract to form RPA foci ( and ). Although it remains formally possible that our depletion may have been incomplete, in the context that neither human WRN nor its mouse homologue is an essential gene, this experiment suggests that there is at least one more focus-forming activity in the egg extract. It should be mentioned that this result does not contradict our previous report that depletion of FFA-1 blocks RPA focus formation (Yan et al. 1998
). In those experiments we used partially purified column fractions of RPA and FFA-1 as the starting material for depletion and presumably the redundant activity had already been removed.
Figure 3 The effect of FFA-1 depletion on DNA replication and RPA focus formation. (A) Western blot for FFA-1 in the extracts depleted with either anti–FFA-1 antibody or control antibody. Arrowhead indicates the position of FFA-1. (B) DNA synthesis in (more ...)
Inhibitory Effect of Dominant Negative FFA-1 Mutants on DNA Replication in Reconstituted Nuclei
Therefore, we tried an alternative approach by using dominant negative mutants. FFA-1's involvement in focus formation suggests that it should interact with itself and/or other proteins. Therefore, it should be possible to make mutants that are functionally deficient, but still capable of protein–protein interaction. Adding such mutant proteins to the extract may interfere with the function of not only the endogenous FFA-1 protein but also the redundant protein.
We tested several GST fusion proteins containing various regions of FFA-1 ( and ) for inhibitory effects on DNA replication. Nuclei were reconstituted in the extract in the presence of the fusion proteins and [32P]dATP for various lengths of time. The incorporation of [32P]dATP into DNA was assayed by running the replication products on agarose gels. As shown in A, GST-Xho (containing FFA-1 amino acids 1–587, just upstream of the helicase domain) efficiently inhibited DNA synthesis (5–10-fold) ( A). GST alone did not exert any effect on DNA synthesis. The inhibition was most effective when GST-Xho was added at the beginning of the reaction and was leaky as DNA synthesis did occur later. The minimal concentration of GST-Xho to inhibit DNA synthesis was ~400 nM (data not shown), about 80 times that of the endogenous FFA-1 (estimated at 5 nM in the reaction). This is most likely an overestimate, because not all the fusion protein is in active conformation and the concentration of the putative redundant protein is not taken into account.
Figure 4 Effect of GST–FFA-1 fusion protein on DNA replication. (A) Replication of nuclei reconstituted around sperm chromatin in the presence of various fusion proteins (500 nM final concentration for GST-Xho and GST-Stu/Xho; 1 μM for (more ...)
Although these characteristics are consistent with a dominant negative effect, there are two potentially trivial explanations. The first is that GST-Xho nonspecifically inactivates the egg extract. This is not the case, however, since nuclear reformation was not significantly affected (see below; , A–F). The second is that since GST-Xho contains the putative 3′→5′ exonuclease domain, it may inhibit replication simply by degrading DNA. To rule out the this possibility, we expressed and tested a smaller fusion protein, GST-Stu (containing FFA-1 amino acids 1–296), which still contains the nuclease domain. In vitro assays indicated that it possessed good 3′→5′ exonuclease activity (data not shown). However, it did not exert any significant inhibitory effect on DNA replication in reconstituted nuclei even at twice the molar concentration of GST-Xho ( A). This result suggests that the inhibitory domain is located in the region spanning amino acids 297–587, which lies between the nuclease domain and the helicase domain. When the GST fusion protein containing this region (GST-Stu/Xho) was expressed and tested, it indeed effectively inhibited DNA replication at about the same concentration as that of GST-Xho ( A). Together these experiments, in combination with the immunofluorescence studies, strongly suggest that FFA-1 participates in DNA replication in reconstituted nuclei.
Figure 5 Formation of hybrid foci. (A–F) Nuclei were reconstituted around sperm chromatin for 52 min in the presence of GST-Xho and stained with the affinity-purified anti-GST (A and D), RPA (B), or FFA-1 (E). (C) Merge of GST staining and RPA staining. (more ...)
Formation of Hybrid Replication Foci
The presence of FFA-1 at replication foci raises the question of whether the inhibitory fusion proteins block DNA replication by interfering with the assembly, or the activity of replication foci. To address this issue, we determined the localization of the GST-Xho fusion protein by immunofluorescence staining. Interestingly, GST-Xho was itself incorporated into 300–500 discrete foci (, A–F). The GST-Xho foci were mostly hybrid in nature because they also contained RPA ( B) and the endogenous FFA-1 ( E). They were well formed by 52 min, but did not show incorporation of biotin-dCTP, whereas extensive incorporation of biotin-dCTP had already occurred by this time in the control nuclei. In addition, whereas normal foci tend to expand in size during DNA synthesis, the GST foci changed very little in size upon further incubation (data not shown).
In support of the correlation between the formation of “hybrid foci” and the inhibitory effect on replication, we found that the number of foci containing GST-Xho per nucleus decreased dramatically from 300–500 at 400 nM down to <50 at 100 nM, which correlates well with the precipitous drop in replication inhibition by GST-Xho at the lower concentration (data not shown). Moreover, we found that GST-Stu/Xho, which can inhibit DNA replication, also induced the formation of hybrid foci within reconstituted nuclei (, G–I). In contrast, GST and GST-Stu, which cannot inhibit DNA replication, could not form any foci detectable by the anti-GST antibody (, J–O). Together, these observations strongly suggest that the Stu/Xho domain inhibits DNA replication by forming “hybrid foci” that are incapable of executing DNA synthesis.
Physical Interaction between FFA-1 and RPA
We then studied the biochemical mechanism by which the dominant negative fusion proteins inhibit DNA synthesis at “hybrid foci.” The simplest explanation is that the fusion proteins displace the endogenous FFA-1 and the redundant protein from “hybrid foci.” However, since the endogenous FFA-1 is still present at “hybrid foci,” this explanation is unlikely to be the case. The second explanation is that, since helicases usually act as oligomers, the dominant negative fusion proteins, which do not contain the helicase domain, might interact with the endogenous FFA-1, leading to the formation of inactive helicase oligomers. We have tested this idea, but found no interaction between the fusion proteins and FFA-1 (data not shown); as such it is also unlikely to be true. The third explanation is based on the report that the helicase activity of human WRN can be stimulated by the human RPA protein, probably by protein–protein interaction (Shen et al. 1998b
; Brosh et al. 1999
). Conceivably, the helicase activity of FFA-1 may also be stimulated by RPA through protein–protein interaction. If the dominant negative fusion proteins can also interact with RPA, they may then interfere with this stimulation. To test this idea, we first determined whether FFA-1 could indeed interact with RPA by coimmunoprecipitation. Protein A beads were coated with different antibodies and then incubated with the cytosol. The proteins bound to the beads were then separated on SDS-PAGE, transferred to PVDF membranes, and probed with either anti–FFA-1 or anti-RPA antibodies. As shown in A, the anti–FFA-1 antibody brought down not only FFA-1, but also a small amount of RPA. Conversely, the anti-RPA antibody brought down RPA and a small amount of FFA-1. In neither case did the control antibody bring down FFA-1 and RPA. This experiment suggests that FFA-1 and RPA can physically interact with each other.
Figure 6 Interaction between FFA-1 and RPA. (A) Coimmunoprecipitation of FFA-1 and RPA. Western blot analysis of the proteins brought down from the cytosol by the Affi-gel protein A beads precoated with the indicated antibodies. Blots were probed with the rabbit (more ...)
We then determined which region of FFA-1 mediates its interaction with RPA. Various GST–FFA-1 fusion proteins were incubated in the cytosol and then brought down by glutathione-agarose beads. Proteins bound to the beads were analyzed by Western blot for the presence of RPA. As shown in B, RPA was efficiently brought down with GST-Xho and GST-Stu/Xho, but not with GST or GST-Stu. Two other fusions containing the middle and COOH-terminal regions of FFA-1 also failed to bring down RPA (data not shown). Therefore, the Stu/Xho domain, which can inhibit DNA replication and induce the formation of “hybrid foci,” also mediates FFA-1's interaction with RPA. Further characterization revealed that the interaction can still be observed between the fusion proteins and the purified RPA, and in the presence of DNase I ( C), suggesting that it is by direct contact, rather than indirectly mediated by other proteins or DNA. These results are consistent with the colocalization of FFA-1, RPA, and the GST fusions containing the Stu/Xho domain at “hybrid foci.”
The direct interaction between the Stu/Xho domain and RPA raised the possibility that replication inhibition is due to the titration of RPA, rather than the interference with FFA-1 activity. To test this possibility, we determined whether RPA was completely bound to the GST-Stu/Xho fusion protein in the pull-down assay. As shown in D, at the concentration effective for replication inhibition, the fusion protein bound to only a small fraction of RPA (~10%). This result suggests that FFA-1 interacts with only a subset of RPA and the dominant negative effect is not due to the titration of RPA.
Functional Effect of FFA-1–RPA Interaction on FFA-1 Helicase Activity
We then determined whether the interaction between FFA-1 and RPA could augment the DNA helicase activity of FFA-1. FFA-1 was incubated with a radioactively labeled DNA substrate and various amounts of RPA in the presence of ATP. The substrate was made by extending a primer hybridized to single-stranded m13 DNA in the presence of [32P]dATP, dNTPs, and dilute ddTTP. The rationale for using this type of substrate is that RPA may preferentially stimulate the unwinding of long DNA strands. After incubation, the reactions were terminated and separated on 8% polyacrylamide gels. The undissociated fragments could not enter the gel due to the large size of m13 DNA, whereas the dissociated fragments would run as a series of bands of different sizes. As shown in A, FFA-1 alone showed very weak unwinding activity at the concentration used in this experiment (~2 nM). However, the unwinding of both short and long fragments was greatly stimulated by RPA at a concentration as low as 60 nM. RPA alone, even at the highest concentration (120 nM), did not cause any significant dissociation of the substrate.
Figure 7 Effect of RPA and gp32 on the helicase activity of FFA-1. (A) Helicase reactions containing the indicated amounts of FFA-1 and RPA (in nM). (B) Helicase reactions containing the indicated amounts of FFA-1 (in nM) and gp32 (in μM). In both A and (more ...)
There are two likely mechanisms to explain how RPA might stimulate FFA-1 helicase activity. The more trivial explanation is that RPA simply prevents the dissociated DNA from reassociation. Alternatively, the effect may be due to the specific interaction between the two proteins. To differentiate between these two possibilities, we tested the effect on FFA-1 helicase activity by another single-stranded DNA binding protein, the gene 32 protein of E. coli
phage T4 (gp32). As shown in B, this protein had no significant stimulatory effect on the unwinding reaction over a wide range of concentrations (from 37.5 nM to 3 μM). The size of binding site of gp32 is seven nucleotides (Ferrari et al. 1994
), so 3 μM of protein is in ~10-fold excess over the number of binding sites. In contrast, assuming that the size of binding site of Xenopus
RPA is similar to that of human RPA (30 nucleotides; Wold 1997
), 60 nM of RPA occupies at most 75% of the binding sites. These results suggest that the stimulation on FFA-1 helicase activity by RPA depends on the direct interaction between the two proteins.
Effect of GST–FFA-1 Fusion Proteins on FFA-1/RPA Helicase Activity
Since GST-Xho and GST-Stu/Xho can interact with RPA, we then addressed whether they can inhibit the stimulation of FFA-1 helicase activity by RPA. To do this, various amounts of the fusion proteins were added to the helicase reaction containing FFA-1 and RPA. As shown in A, GST-Xho and GST-Stu/Xho inhibited the unwinding reaction in a dose-dependent manner. In contrast, GST-Stu had no effect on unwinding, even at the highest concentration (2.4 μM). In the absence of RPA, FFA-1 had a low intrinsic helicase activity, particularly on small fragments. But as shown in B, this activity was not significantly affected by the fusion proteins. In addition, the affinity of RPA for single-stranded DNA was not affected by the fusion proteins (data not shown). Collectively, these results support the interpretation that RPA stimulates FFA-1 through direct protein–protein interaction and this stimulation is blocked by the two fusion proteins containing the Stu/Xho domain.
Figure 8 Effect of GST–FFA-1 fusion proteins on FFA-1 helicase activity. (A) In the presence of RPA. Lanes 1–10 contain 2 nM FFA-1 and 60 nM RPA, but lanes 1–9 also contain the various fusion proteins at the indicated final concentrations (more ...)
It is worth mentioning that the concentrations of the proteins in the helicase inhibition experiment agree reasonably well with those in the replication inhibition experiment. In the helicase experiment, the concentrations for FFA-1, RPA, and the two dominant negative fusion proteins are 2, 60, and 300 nM (minimum), respectively. In the replication experiment, the corresponding concentrations are 5, 33, and 400 nM (minimum). Therefore, it is reasonable to believe that the FFA-1 helicase activity is also stimulated by RPA during normal DNA replication and that this stimulation is abolished by the two dominant negative fusion proteins at “hybrid foci.”