Abstract
A central event in the initiation of DNA replication in eukaryotes is the assembly of pre-replicative complex (pre-RC) on specific chromatin sites known as DNA replication origins. The pre-RC assembly process differs between budding and fission yeasts. In fission yeast, Sap1 directly participates in pre-RC assembly, together with the four initiation factors: ORC, Cdc18/Cdc6, Cdt1, and MCM. In metazoans, the nature of DNA replication origins is not defined and the mechanism of pre-RC assembly remains incompletely known. In this study, Girdin was identified as an essential replication initiation factor in human cells. Similar to the activity of Sap1, human Girdin binds to DNA origins, interacts with ORC, and is required for pre-RC assembly due to its essential role in recruitment of Cdc6 to DNA origins. Thus, DNA origins in human or metazoans are defined as including two elements, one bound by ORC and the other bound by Girdin.
Introduction
In eukaryotes, the biochemical process of DNA replication initiation is divided into two steps. In the first step, a pre-replication complex (pre-RC) is assembled on specific chromatin sites or DNA replication origins at late M and G1 phases of the cell division cycle. In the second step, when cells are at the transition of G1 to S phase, the pre-RCs are activated by CDK and DDK kinases (1), triggering the recruitment of Cdc45 and GINS for the formation of CMG (Cdc45-MCM-GINS) replicative helicase, and the recruitment of other replication factors that act at replication forks, such as RPA, DNA pol α-primase, DNA pol δ, and DNA pol ε. Once these factors are assembled, DNA replication starts.
In budding yeast, pre-RC is assembled by four protein factors—ORC (origin recognition complex), Cdc6, Cdt1, and MCM (mini-chromosome maintenance). In the early 1970s, Cdc6 gene was first identified by Hartwell and his colleagues in a screen of temperature-sensitive mutants defective in gene functions needed at specific stages of the cell division cycles (2). Their studies suggested that Cdc6 may specify a function necessary for the proper initiation of DNA replication, but its exact function was unknown at that time (3). Over the next twenty years, studies carried out in several labs finally revealed the direct participation of Cdc6 in pre-RC assembly, with Cdc6 essential for the recruitment of MCM to DNA origins (4–12). In a similar genetic screen, several temperature-sensitive mutants, named cdc21, nda1, and nda4 in the fission yeast S. pombe and CDC54, CDC46, and CDC47 in the budding yeast S. cerevisiae, were isolated in the early 1980s (13–18). These mutants were later determined to harbor mutations in Mcm2, Mcm4, Mcm5, and Mcm7 gene, respectively. Just one or two years later, mutants defective in maintaining circular minichromosomes (Mcm−) were also isolated in S. cerevisiae (19,20). MCM was later identified to function in fungi to homo sapiens as a complex composed of six distinct and highly conserved subunits (21–27). ORC was first isolated in the budding yeast S. cerevisiae in 1992 (28). ORC is a six-subunit complex that remains bound to DNA origins throughout the cell cycle. Cdt1 was first identified in the fission yeast S. pombe (29). The cdt1 gene is essential in fission yeast; cells with a null allele of cdt1 arrest in the G1 phase, suggesting that Cdt1 function is related to DNA replication with either a direct role as a replication factor or an indirect regulator, such as a transcription factor that could promote entry into S phase (29). Cdt1 was subsequently found to be required for loading MCM to DNA origins for pre-RC assembly (30,31). Using recombinant ORC, Cdc6, Cdt1, and MCM, pre-RC could be reconstituted in vitro in the budding yeast S. cerevisiae, suggesting that these four proteins are sufficient for the assembly of pre-RC in budding yeast (32–34).
As defined, pre-RC assembles on DNA replication origins. However, the structures of DNA origins are remarkably different from budding yeast to fission yeast and metazoans. In the budding yeast S. cerevisiae, DNA origins are approximately 100 bp in size. Each origin contains a consensus sequence of 11 bp termed an “A” element that is essential for origin activity and is recognized and bound by ORC (35–38). In the fission yeast S. pombe, DNA origins range from 500-1500 bp in size (39–42), making them five to ten times larger than DNA origins in S. cerevisiae. The S. pombe DNA origins are large because they possess two essential elements that are recognized and bound by two origin recognition proteins, ORC and Sap1 (43,44). The average distance between ORC- and Sap1-bound elements ranges from ~300 to ~700 bp, and this distance combined with the size of the two origin elements explains the large size of DNA origins in S. pombe. Sap1, as an origin recognition factor, exhibits the same functions as ORC (44). Sap1 binds to DNA origins throughout the cell division cycle, Sap1 interacts with ORC to form a complex, and Sap1 is required for pre-RC assembly because it recruits Cdc18 (the homologue of Cdc6 in fission yeast) to DNA origins (44). Therefore, DNA origins in S. cerevisiae contain a single element that is bound by ORC, but those in S. pombe possess two discrete elements and two origin recognition factors, ORC and Sap1.
Similar to what occurs in budding and fission yeasts, the initiation of DNA replication in metazoans also occurs at specific chromosome sites, however, the nature of metazoan DNA origins remains elusive (45–54). DNA origins in metazoans are large (55–58), contain AT-rich sequences important for origin activity (42,59–61), and lack an easily recognizable consensus sequence. These three characteristics also generally describe S. pombe DNA origins, suggesting the possibility that metazoan and S. pombe DNA origins may have a similar structure. Initially, it was though that S. pombe DNA origins are AT-rich but lack a consensus sequence. Later, it was found that the S. pombe ORC binds to asymmetric AT-rich sequences with A in one strand and T in the other strand, in a process that depends on its 9-AT hook motifs (42,43,62,63). And Sap1 binds to a DNA sequence of 5’-(A/T)n(C/G)(A/T)9–10(G/C)(A/T)n-3’ (n ≧ 1) (44). Thus, based on the recent findings DNA origins in fission yeast do exhibit a certain level of sequence specificity. However, it remains to be determined if DNA origins in metazoans are similar to those in S. cerevisiae or S. pombe.
In this study, Girdin was identified in human cells as the homologue of the pre-RC component Sap1. Girdin is a highly conserved protein in metazoans, and sequence alignment indicates that hGirdin contains a region of ~200 amino acids that is highly homologous (~41% amino acid identity) to the highly conserved middle region of Sap1 protein in fission yeast. Sap1 has 254 amino acids in total. Like Sap1, hGirdin binds to DNA origins, physically interacts with ORC, and is required for loading Cdc6 to DNA origins for pre-RC assembly. The Sap1-homologous region in hGirdin can partially complement the function of Sap1 in fission yeast, further suggesting that Girdin is the homologue of Sap1 in metazoans. Thus, like those in fission yeast, DNA origins in human/metazoan cells possess two essential elements, with one bound by ORC and the other by Girdin. The assembly of pre-RC in metazoans requires the five components of ORC, Girdin, Cdc6, Cdt1, and MCM.
Results
Identification of the Sap1 homologue in metazoans
The similarity of DNA replication origins between fission yeast and metazoans suggests the likelihood of a Sap1 homologue in metazoans. Database-searching and immunoprecipitation against human Cdc6 identified the human protein Girdin. Girdin contains a region (~200 amino acids) that is highly homologous to Sap1 (Fig. 1A, B), with ~41% amino acids identity (with D/E, I/L, and R/K considered identical amino acids). The homologous region in Sap1 consists of amino acids 8 to 210, and this region is the most conserved in Sap1 proteins in fission yeast. Girdin is a very conserved protein present from C. elegans to human. Comparison of Girdin proteins from different organisms reveals that the Sap1-homologous region is the most conserved region, suggesting this region is probably the most important region for Girdin function. Girdin is expressed in all examined tissues and cell lines (64,65).
Girdin is an essential protein for cell growth
To investigate whether Girdin, like Sap1, is essential in pre-RC assembly, we first examined its cellular distribution. Fluorescent analysis of GFP-tagged Girdin revealed that the majority of Girdin was in the cytoplasm, but a weak fluorescent signal was also detected in nuclei (Fig. 1C). To determine whether Girdin in the nucleus associates with chromatin, the fractions of cytoplasm, nucleoplasm, and chromatin were carefully separated and the amount of Girdin in each fraction was quantified by Western blotting. As shown in Fig. 1D, ~95% of Girdin was found in the cytoplasm and the remained (~5-10%) was in the nuclear fraction, where the Girdin was associated with chromatin.
Next, we examined whether Girdin is essential for cell growth. To do this, 293T cells were infected with lentiviral particles harboring either Girdin shRNA or control shRNA. After eight to nine days of incubation, the amount of Girdin was reduced by more than 90% in Girdin shRNA-introduced cells but the levels of the other initiation proteins, Orc2, Cdc6, Cdt1, and Mcm2, remained the same (Fig. 1E). At this point, Girdin shRNA-treated cells grew more slowly compared to the control cells. Beginning at the tenth or eleventh day, ~30-40% of Girdin shRNA-treated cells were dying or dead, but the control cells still grew well at the twentieth day or beyond (Fig. 1F, G). A similar result was obtained with HeLa cells or using a different shRNA. Efforts to knock out the gene of hGirdin by CRISPA-Cas9 were not successful. Together, these results indicate that Girdin is an essential protein for cell growth.
The S phase progression of the cell cycle is affected when Girdin is reduced
Slower cell growth was observed in the Girdin siRNA- or shRNA-treated cells. To examine if Girdin affects the progression of S phase, the percentage of S phase cells was measured in the Girdin siRNA- or control siRNA-treated cells by BrdU incorporation and subsequent florescent assay and FACS analysis. Based on the percentage of cells that incorporated BrdU, ~53% of the Girdin siRNA-transfected cells were in S phase when Girdin was reduced by ~90%, while only ~41% of the control cells were in the S phase (Fig. 2A, B). The FACS analysis also clearly shows more cells in the S phase when Girdin was reduced (Fig. 2C). This result is consistent with that of a previous study showing that the depletion of another replication initiation protein Treslin from human cells increases the number of cells in S phase (66). Consistent with slower progression of S phase, DNA synthesis was inhibited by ~20-35% in the Girdin siRNA-treated cells compared to the control cells (Fig. 2D).
Girdin and ORC bind to the same regions on genomic DNA and they physically interact with each other
To determine whether Girdin and ORC bind to the same regions on genomic DNA, their genomic binding sites were identified with ChlP-seq assays. As shown in Fig. 3A, the genomic binding sites of Girdin and ORC are the same. The completely overlapping binding sites suggests that Girdin and ORC are under physical interaction on chromatin DNA. To confirm that, reciprocal IPs were performed. The results in Fig. 3B show that IP against Girdin or ORC brought down ORC (Orc2 subunit) or Girdin, respectively, indicating that like Sap1 and ORC in S. pombe, human Girdin and ORC physically interact with each other. The association of Girdin with DNA was also examined during the cell cycle. The result shown in Fig. 3C indicates that Girdin, like ORC, binds to DNA origins throughout the cell cycle.
Girdin is required to recruit and load Cdc6 onto DNA for pre-RC assembly
The interaction between Girdin and Cdc6 was further confirmed by performing reciprocal IPs with cell extracts containing a physiological level of Cdc6 (Fig. 4A). Next, we examined the requirement of Girdin for Cdc6 loading onto DNA for pre-RC assembly. ORC- and MCM-dependent S. cerevisiae or human cell-free systems for pre-RC assembly and DNA replication were previously reported (32–34,67–69). In this assay, nuclear extracts prepared from 293T cells in late G1 phase were used. During IP depletion experiments, we identified ORC, Girdin, Cdc6, Cdt1, and MCM in sub-complexes even in nuclear extracts free of genomic DNA. Thus, immuno-depletion of Girdin or Orc2 was carried out in the presence of 0.6 M KCl. For removal of ~95-98% of ORC or Girdin from the extracts, immuno-depletion of Girdin or Orc2 was routinely conducted two or three times. As shown in Fig. 4B, this method allowed effective removal of Girdin or Orc2 subunit, but not the other pre-RC components. Assembly of pre-RC was performed on salmon sperm DNA at a salt concentration of 0.1 to 0.15 M (see Methods). The result presented in Fig. 4C shows that Girdin was required to load Cdc6 and, subsequently, MCM to DNA, but was not required for Cdt1 loading. Additionally, the binding of ORC to DNA was not affected in the absence of Girdin, and that ORC, as shown previously, was required to load Cdc6 and Cdt1 and then MCM to DNA. In a control reaction, Cdc6, Cdt1, and MCM were loaded onto DNA when both Girdin and ORC were present. To verify that the absence of Girdin was responsible for the observed disruption of the pre-RC assembly (Fig. 4C), recombinant Girdin, which was overexpressed and subsequently purified from S. pombe cells (Fig. 4D), was added back to the Girdin-depleted extract and then the assembly of pre-RC was restored (Fig. 4 E).
Girdin complements the function of Sap1 in fission yeast S. pombe
To confirm that Girdin is functionally related to Sap1, we asked if Girdin could complement the function of Sap1 in the fission yeast. A plasmid that expresses the Sap1-homologous region of Girdin under the control of nmt41 promoter was transformed into the fission yeast Sp-dk1-sap1ts5 cells and the transformed cells were examined for their ability to grow at the restrictive temperature of 32°C or 34°C. The results in Fig. 5A, B show that the expression of the Sap1-homologous region of Girdin could enhance the survival rate of Sp-dk1-sap1ts5 cells at 32°C or 34°C by ~5 to 25 folds. This result indicates that the Sap1-homologous region of Girdin can partially complement the function of Sap1 in the fission yeast, consistent with the conservation of biological function from Sap1 to Girdin.
Discussion
There is significant experimental evidence that DNA replication initiates at specific sites in chromosomes in metazoans. However, the molecular mechanism for metazoan cells to choose replication initiation sites remains unclear. In particular, the nature of metazoan DNA replication origins and how DNA replication is initiated by assembly of pre-RC have been unresolved for more than two decades, significantly hampering further elucidation of the mechanism of replication initiation in metazoans.
The identification of a second DNA origin recognition factor, Sap1, in S. pombe suggests that a Sap1 homologue may also exist in metazoans, since S. pombe and metazoans appear to have a similar origin structure. By searching for proteins that interact with Cdc6 in human cells, we identified Girdin, which contains a region highly homologous to a region of ~200 amino acids in Sap1. This homologous region is also highly conserved among Sap1 proteins in the fission yeast species whose genomic sequences are available. However, the N-terminal ~10 amino acids and C-terminal ~40-50 amino acids in Sap1 are not very conserved. The nearly 41% identity of amino acids in the homologous region between Sap1 and hGirdin suggests that Girdin is a homologue of Sap1 in metazoans, and this was subsequently demonstrated by several lines of experimental evidence. First, like Sap1 and other replication proteins acting either in replication initiation or at replication forks, a reduced level of hGirdin slowed S phase progression (Fig. 2A-D). Second, like Sap1, hGirdin binds to DNA regions that are bound by ORC, and hGirdin and ORC physically interact as a complex on DNA origins. This hGirdin-ORC complex provides the biochemical basis for their collective action in recruiting Cdc6 to replication origins (Fig. 3A, B). Third, like Sap1, hGirdin is required to recruit Cdc6 to DNA origins (Fig. 4). Finally, a functional complementation test revealed that the homologous region in hGirdin partially complemented Sap1 function (Fig. 5), suggesting that hGirdin possesses a function that Sap1 also utilizes. Taken together, the experimental evidence presented in this study indicates that hGirdin is a genuine homologue of Sap1 and functions in pre-RC assembly and replication initiation.
Why do human/metazoan cells, like the fission yeast S. pombe, require a factor in addition to ORC for their pre-RC assembly? The answer to this question is likely related to the nature of the ORC complex. There are some obvious differences among ORC complexes in the budding yeast S. cerevisiae, the fission yeast S. pombe, and metazoans. First, the Orc6 subunits (~28-30 kDa) in fission yeast and metazoans differ from S. cerevisiae Orc6 (~50 kDa) in size and amino acid sequence. Second, S. pombe Orc4 contains an additional domain (~60 kDa) that contains 9 AT-hook motifs at its N-terminus. This AT-hook domain is not present in S. cerevisiae ORC and is solely responsible for binding of S. pombe ORC to AT-rich sequences in DNA origins(42,43,62,63). Third, a mutant S. pombe ORC that lacks the 9 AT-hook domain shows random, low affinity binding to DNA (62). Fourth, human ORC, similar to S. pombe ORC lacking the 9 AT-hook motifs, appears to have a sequence-independent DNA binding property (70). Fifth, S. cerevisiae ORC interacts with DNA using its five subunits (71), which is significantly different from S. pombe ORC which uses only one domain in Orc4 (the 9 AT-hook motifs) to interact with DNA. Whether human or metazoan ORC utilizes an accessory subunit similar to the 9 AT-hook motifs in S. pombe ORC remains to be determined. It is possible that such a subunit exists and promotes human/metazoan ORC binding to DNA. The difference in ORC and ORC-DNA interaction may explain the requirement of Sap1/Girdin for pre-RC assembly in fission yeast and metazoans.
The identification of replication initiation factor Girdin provides insight into the structure of DNA origins in metazoans. Like DNA origins in fission yeast, DNA origins in metazoans possess two origin elements, one that is recognized and bound by ORC and one that is recognized and bound by Girdin. Thus, metazoan DNA origins are defined as possessing two discrete elements that are separately bound by two origin recognition factors. Future investigation of metazoan DNA origins should determine how Girdin interacts with DNA and whether metazoan ORC associates with an accessory factor that is analogous to the AT-hooks domain in S. pombe Orc4 subunit.
Experimental procedures
Purification of hGirdin and preparation of polyclonal antibodies against human Orc2, Mcm2, Cdc6, Cdt1, and the Sap1-homologous region of hGirdin
The tagged hGirdin was overexpressed as 6His-hGirdin in S. pombe cells. All expressed 6His-Girdin was in the nuclei and the majority bound to chromatin. The 6His-Girdin in the chromatin extract was first precipitated by 40% ammonium sulfate. The pellet containing 6His-Girdin was then resuspended in buffer A (50 mM HEPES-HCl [pH 7.5], 100 mM KCl, 5 mM magnesium acetate, 5 mM dithiothreitol, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 0.04% NP-40, and protease inhibitors (Sigma)) and applied to Sephacryl S-300 column for filtration chromatography. The fractions containing 6His-Girdin were then applied to Q-sepharose column and 6His-Girdin was eluted at 0.5 to 0.6 M KCl. Then, the eluted 6His-Girdin was further purified with Ni2+-column and eluted at 250 mM imidazole. The purified 6His-Girdin was dialyzed against buffer A and then stored in −80°C freezer.
A polyclonal antibody against the Sap1-homologous region of hGirdin was obtained by immunizing rabbits with recombinant protein of the Sap1-homologous region of Girdin that was overexpressed in E. coli and purified. Polyclonal antibodies against human Orc2, Mcm2, Cdc6, Cdt1 were similarly obtained.
ChIP-seq assays to determine the binding sites of human ORC and hGirdin on genomic DNA
To determine human ORC and hGirdin binding sites, ChIP-seq assay was carried out as described (Euskirchen et al., Genome Res 17, 898-909; Johnson et al., Science 316, 1497-1502) with some modifications. First, 293T cells were grown to the confluence of 70% in DMEM containing 10% fetal bovine serum, and then arrested at G2/M phase after treatment of the culture with nocodazole (50 μg/ml) for 12 hrs. The chromatin-bound proteins were cross-linked to DNA by treating the cells with 1% formaldehyde for 10 min. The formaldehyde-treated cells were collected, washed with PBS twice, and then homogenized to obtain nuclei. The nuclei were lysed with RIPA Buffer (1XPBS / 1% NP-40 / 0.5% sodium deoxycholate / 0.1% SDS) and the chromatin was sheared to an average of 250 bp with sonication. The ChIP was then carried out to obtain ORC or Girdin-bound chromatin fragments using polyclonal antibody against Orc2 or hGirdin.
RNA interference
In total, five sets of siRNA sequences were tested. Three of them effectively mediated silencing of Girdin expression. One sequence is 5’-GCAATTAGAGAGTGAACTA-3’ (nucleotide 2340-2358 in hGirdin cDNA gene, only sense sequence is shown). This sequence was purchased as a 21 nucleotide synthetic duplex. 293T cells were transfected with the siRNA or a 21-nucleotide non-related RNA as a control by using lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. For shRNA-mediated knockdown of hGirdin, the targeted sequence is 5’-GAAGGAGAGGCAACTGGAT-3’ (nucleotides 4166-4184, only the sense sequence is shown). A set of single-stranded oligonucleotides encoding the Girdin target shRNA and its complement was synthesized, annealed, and inserted into lentiviral shRNA expression vector pLKO.1 (forward oligo, 5’-CCGGGAAGGAGAGGCAACTGGATCTCGAGATCCAGTTGCCTCTCCTTCTTTTTG-3’; reverse oligo, 5’-AATTCAAAAAGAAGGAGAGGCAACTGGATCTCGAGATCCAGTTGCCTCTCCTTC-3’). The production of lentiviral particles and lentiviral infection were performed according to manufacturer’s instructions.
Preparation of nuclear extract from 293T cells at late G1 phase and immune-depletion of Girdin or Orc2
The 293T cells were incubated in DMEM medium containing 10% fetal bovine serum, were first arrested at G1/S phase in the presence of thymidine (2 mM) for ~20 hrs, and were then released to G2/M phase in the presence of nocodazole (50 μg/ml) for 14 hrs. The cells were then released from nocodazole arrest and grew into late G1 phase by incubation in fresh medium for 6.5 hrs. The nuclei obtained with hypotonic buffer treatment and homogenization were frozen once and then crushed at high speed (37000g) to obtain the nucleoplasm supernatant. The pellet containing chromatin was sequentially extracted with buffer A containing 0.3 and then buffer A containing 0.6 M KCl. These extracts were mixed with the nucleoplasm to obtain whole nuclear extract at a salt concentration of ~0.2 M. To specifically remove ORC or Girdin protein from the nuclear extract, the salt concentration in the nuclear extract was first adjusted to 0.6 M to prevent removal of ORC or Girdin-interacting proteins such as Cdc6 during immunodepletion. The immunodepletion of Orc2 or Girdin was performed two or three times, allowing removal of more than 95% to 98% of Orc2 or Girdin from the extract. As a positive control in the subsequent pre-RC assembly reaction, whole nuclear extract samples were also mock-depleted with rabbit IgG.
Assembly of pre-RC on salmon sperm DNA-cellulose with human 293T nuclear extracts
The mock-, Orc2-, or hGirdin-depleted nuclear extracts were dialyzed against buffer A to adjust the salt concentration to 0.15 M. In a standard pre-RC assembly reaction, ~0.5 ml of nuclear extract from 293T cells at late G1 phase was mixed with 50 μl of DNA-cellulose in the presence of 5 mM ATP, 3 mM DTT, 10 mM of creatine phosphate, 20 units of creatine phosphokinase/ml, and proteinase inhibitors. The reactions were incubated at 4°C for 30 min, and then spun and the supernatant was removed. The pellet was washed with one ml of buffer A four times and then washed quickly twice with 0.5 ml of buffer A with salt (0.3 M KCl) to remove any proteins that were non-specifically bound to the salmon sperm DNA or cellulose. The pre-RC components assembled onto the DNA-cellulose were examined by SDS-PAGE electrophoresis and Western blotting assay.
Acknowledgements
We thank the members of the Kong Lab for their support during the course of this project. This work was supported by grants from the Ministry of Science and Technology of China (2013CB911000 and 2016YFA0500301), the National Natural Science Foundation of China (no. 31661143032, no. 31230021, and no. 31730022), the Peking-Tsinghua Center for Life Sciences, and the National Key Laboratory of Protein and Plant Gene Research.