Characterization of Leishmania donovani MCM4: expression patterns and interaction with PCNA

Events leading to origin firing and fork elongation in eukaryotes involve several proteins which are mostly conserved across the various eukaryotic species. Nuclear DNA replication in trypanosomatids has thus far remained a largely uninvestigated area. While several eukaryotic replication protein orthologs have been annotated, many are missing, suggesting that novel replication mechanisms may apply in this group of organisms. Here, we characterize the expression of Leishmania donovani MCM4, and find that while it broadly resembles other eukaryotes, noteworthy differences exist. MCM4 is constitutively nuclear, signifying that, unlike what is seen in S.cerevisiae, varying subcellular localization of MCM4 is not a mode of replication regulation in Leishmania. Overexpression of MCM4 in Leishmania promastigotes causes progress through S phase faster than usual, implicating a role for MCM4 in the modulation of cell cycle progression. We find for the first time in eukaryotes, an interaction between any of the proteins of the MCM2-7 (MCM4) and PCNA. MCM4 colocalizes with PCNA in S phase cells, in keeping with the MCM2-7 complex being involved not only in replication initiation, but fork elongation as well. Analysis of a LdMCM4 mutant indicates that MCM4 interacts with PCNA via the PIP box motif of MCM4--perhaps as an integral component of the MCM2-7 complex, although we have no direct evidence that MCM4 harboring a PIP box mutation can still functionally associate with the other members of the MCM2-7 complex- and the PIP box motif is important for cell survival and viability. In Leishmania, MCM4 may possibly help in recruiting PCNA to chromatin, a role assigned to MCM10 in other eukaryotes.     

Two mcm3 mutations affect different steps in the initiation of DNA replication

Mcm3 is a subunit of the hexameric MCM2-7 complex required for the initiation and elongation of DNA replication in eukaryotes. We have characterized two mutant alleles, mcm3-1 and mcm3-10, in Saccharomyces cerevisiae and showed that they are defective at different steps of the replication initiation process. Mcm3-10 contains a P118L substitution that compromises its interaction with Mcm5 and the recruitment of Mcm3 and Mcm7 to a replication origin. P118 is conserved between Mcm3, Mcm4, Mcm5, and Mcm7. An identical substitution of this conserved residue in Mcm5 (P83L of mcm5-bob1) strengthens the interaction between Mcm3 and Mcm5 and allows cells to enter S phase independent of Cdc7-Dbf4 kinase (Hardy, C. F., Dryga, O., Pahl, P. M. B., and Sclafani, R. A. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3151-3155). Mcm3-1 contains a G246E mutation that diminishes the efficiency of replication initiation (Yan, H., Merchant, A. M., and Tye, B. K. (1993) Genes Dev. 7, 2149-2160) but not its interaction with Mcm5 or recruitment of the MCM2-7 complex to replication origin. These observations indicate that Mcm3-10 is defective in a step before, and Mcm3-1 is defective in a step after the recruitment of the MCM2-7 complex to replication origins.     

Fission yeast cdc21, a member of the MCM protein family, is required for onset of S phase and is located in the nucleus throughout the cell cycle.

The fission yeast cdc21 protein belongs to the MCM family, implicated in the once per cell cycle regulation of chromosome replication. In budding yeast, proteins in this family are eliminated from the nucleus during S phase, which has led to the suggestion that they may serve to distinguish unreplicated from replicated DNA, as in the licensing factor model. We show here that, in contrast to the situation in budding yeast, cdc21 remains in the nucleus after S phase, as is found for related proteins in mammalian cells. We suggest that regulation of nuclear import of these proteins may not be an essential aspect of their function in chromosome replication. To determine the function of cdc21+, we have analysed the phenotype of a gene deletion. cdc21+ is required for entry into S phase and, unexpectedly, a proportion of cells depleted of the gene product are able to enter mitosis in the absence of DNA replication. These results are consistent with the view that individual proteins in the MCM family are required for all initiation events, and defective initiation may impair the coordination between mitosis and S phase.     

The capacity of origins to load MCM establishes replication timing patterns

Loading of the MCM replicative helicase at origins of replication is a highly regulated process that precedes DNA replication in all eukaryotes. The stoichiometry of MCM loaded at origins has been proposed to be a key determinant of when those origins initiate replication during S phase. Nevertheless, the genome-wide regulation of MCM loading stoichiometry and its direct effect on replication timing remain unclear. In order to investigate why some origins load more MCM than others, we perturbed MCM levels in budding yeast cells and, for the first time, directly measured MCM levels and replication timing in the same experiment. Reduction of MCM levels through degradation of Mcm4, one of the six obligate components of the MCM complex, slowed progression through S phase and increased sensitivity to replication stress. Reduction of MCM levels also led to differential loading at origins during G1, revealing origins that are sensitive to reductions in MCM and others that are not. Sensitive origins loaded less MCM under normal conditions and correlated with a weak ability to recruit the origin recognition complex (ORC). Moreover, reduction of MCM loading at specific origins of replication led to a delay in their replication during S phase. In contrast, overexpression of MCM had no effects on cell cycle progression, relative MCM levels at origins, or replication timing, suggesting that, under optimal growth conditions, cellular MCM levels are not limiting for MCM loading. Our results support a model in which the loading capacity of origins is the primary determinant of MCM stoichiometry in wild-type cells, but that stoichiometry is controlled by origins’ ability to recruit ORC and compete for MCM when MCM becomes limiting.     

Chromatin loading of MCM hexamers is associated with di-/tri-methylation of histone H4K20 toward S phase entry

DNA replication is a key step in initiating cell proliferation. Loading hexameric complexes of minichromosome maintenance (MCM) helicase onto DNA replication origins during the G1 phase is essential for initiating DNA replication. Here, we examined MCM hexamer states during the cell cycle in human hTERT-RPE1 cells using multicolor immunofluorescence-based, single-cell plot analysis, and biochemical size fractionation. Experiments involving cell-cycle arrest at the G1 phase and release from the arrest revealed that a double MCM hexamer was formed via a single hexamer during G1 progression. A single MCM hexamer was recruited to chromatin in the early G1 phase. Another single hexamer was recruited to form a double hexamer in the late G1 phase. We further examined relationship between the MCM hexamer states and the methylation levels at lysine 20 of histone H4 (H4K20) and found that the double MCM hexamer state was correlated with di/trimethyl-H4K20 (H4K20me2/3). Inhibiting the conversion from monomethyl-H4K20 (H4K20me1) to H4K20me2/3 retained the cells in the single MCM hexamer state. Non-proliferative cells, including confluent cells or Cdk4/6 inhibitor-treated cells, also remained halted in the single MCM hexamer state. We propose that the single MCM hexamer state is a halting step in the determination of cell cycle progression.     

Visualization of the MCM DNA helicase at replication factories before the onset of DNA synthesis

In mammalian cells, DNA synthesis takes place at defined nuclear structures termed “replication foci” (RF) that follow the same order of activation in each cell cycle. Intriguingly, immunofluorescence studies have failed to visualize the DNA helicase minichromosome maintenance (MCM) at RF, raising doubts about its physical presence at the sites of DNA synthesis. We have revisited this paradox by pulse-labeling RF during the S phase and analyzing the localization of MCM at labeled DNA in the following cell cycle. Using high-throughput confocal microscopy, we provide direct evidence that MCM proteins concentrate in G1 at the chromosome structures bound to become RF in the S phase. Upon initiation of DNA synthesis, an active “MCM eviction” mechanism contributes to reduce the excess of DNA helicases at RF. Most MCM complexes are released from chromatin, except for a small but detectable fraction that remains at the forks during the S phase, as expected for a replicative helicase.     

SpSld3 is required for loading and maintenance of SpCdc45 on chromatin in DNA replication in fission yeast

Initiation of DNA replication in eukaryotic cells is regulated through the ordered assembly of replication complexes at origins of replication. Association of Cdc45 with the origins is a crucial step in assembly of the replication machinery, hence can be considered a target for the regulation of origin activation. To examine the process required for SpCdc45 loading, we isolated fission yeast SpSld3, a counterpart of budding yeast Sld3 that interacts with Cdc45. SpSld3 associates with the replication origin during G1-S phases and this association depends on Dbf4-dependent (DDK) kinase activity. In the corresponding period, SpSld3 interacts with minichromosome maintenance (MCM) proteins and then with SpCdc45. A temperature-sensitive sld3-10 mutation suppressed by the multicopy of the sna41+ encoding SpCdc45 impairs loading of SpCdc45 onto chromatin. In addition, this mutation leads to dissociation of preloaded Cdc45 from chromatin in the hydroxyurea-arrested S phase, and DNA replication upon removal of hydroxyurea is retarded. Thus, we conclude that SpSld3 is required for stable association of Cdc45 with chromatin both in initiation and elongation of DNA replication. The DDK-dependent origin association suggests that SpSld3 is involved in temporal regulation of origin firing.     

A requirement for MCM7 and Cdc45 in chromosome unwinding during eukaryotic DNA replication

In vertebrates, MCM2-7 and Cdc45 are required for DNA replication initiation, but it is unknown whether they are also required for elongation, as in yeast. Moreover, although MCM2-7 is a prime candidate for the eukaryotic replicative DNA helicase, a demonstration that MCM2-7 unwinds DNA during replication is lacking. Here, we use Xenopus egg extracts to investigate the roles of MCM7 and Cdc45 in DNA replication. A fragment of the retinoblastoma protein, Rb(1-400), was used to neutralize MCM7, and antibodies were used to neutralize Cdc45. When added immediately after origin unwinding, or after significant DNA synthesis, both inhibitors blocked further DNA replication, indicating that MCM7 and Cdc45 are required throughout replication elongation in vertebrates. We next exploited the fact that inhibition of DNA polymerase by aphidicolin causes extensive chromosome unwinding, likely due to uncoupling of the replicative DNA helicase. Strikingly, Rb(1-400) and Cdc45 antibodies both abolished unwinding by the uncoupled helicase. These results provide new support for the model that MCM2-7 is the replicative DNA helicase, and they indicate that Cdc45 functions as a helicase co-factor.     

Distinct patterns of MCM protein binding in nuclei of S phase and rereplicating SV40-infected monkey kidney cells.

BACKGROUND: Simian Virus 40 (SV40) infection of growth-arrested monkey kidney cells stimulates S phase entry and the continued synthesis of both viral and cellular DNA. Infected cells can attain total DNA contents as high as DNA Index, DI = 5.0-6.0 (10-12C), with host cell DNA representing 70-80% of the total. In this study, SV40-infected and uninfected control cells were compared to determine whether continued DNA replication beyond DI = 2.0 was associated with rebinding of the minichromosome maintenance (MCM) hexamer, the putative replicative helicase, to chromatin. METHOD: Laser scanning cytometry was used to measure the total expression per cell and the chromatin/matrix-association of two MCM subunits in relation to DNA content. RESULTS: MCM2 and MCM3 proteins that were associated with the chromatin/matrix fraction in G1 phase of both uninfected and SV40-infected cells were gradually released during progression through S phase. However, in SV40-infected cells that progressed beyond DI = 2.0, chromatin/matrix-associated MCM2 and MCM3 remained at the low levels observed at the end of S phase. Rereplication was not preceded by an obvious rebinding of MCM proteins to chromatin, as was observed in G1 phase. CONCLUSIONS: The rereplication of host cell DNA in the absence of the reassociation of MCM proteins with chromatin indicates that SV40 infection induces a novel mechanism of licensing cellular DNA replication.     

The GINS complex from Pyrococcus furiosus stimulates the MCM helicase activity

Pyrococcus furiosus, a hyperthermophilic Archaea, has homologs of the eukaryotic MCM (mini-chromosome maintenance) helicase and GINS complex. The MCM and GINS proteins are both essential factors to initiate DNA replication in eukaryotic cells. Many biochemical characterizations of the replication-related proteins have been reported, but it has not been proved that the homologs of each protein are also essential for replication in archaeal cells. Here, we demonstrated that the P. furiosus GINS complex interacts with P. furiosus MCM. A chromatin immunoprecipitation assay revealed that the GINS complex is detected preferentially at the oriC region on Pyrococcus chromosomal DNA during the exponential growth phase but not in the stationary phase. Furthermore, the GINS complex stimulates both the ATPase and DNA helicase activities of MCM in vitro. These results strongly suggest that the archaeal GINS is involved in both the initiation and elongation processes of DNA replication in P. furiosus, as observed in eukaryotic cells.     

Cryo-electron microscopy reveals a novel DNA-binding site on the MCM helicase

The eukaryotic MCM2-7 complex is recruited at origins of replication during the G1 phase and acts as the main helicase at the replication fork during the S phase of the cell cycle. To characterize the interplay between the MCM helicase and DNA prior to the melting of the double helix, we determined the structure of an archaeal MCM orthologue bound to a 5.6-kb double-stranded DNA segment, using cryo-electron microscopy. DNA wraps around the N-terminal face of a single hexameric ring. This interaction requires a conformational change within the outer belt of the MCM N-terminal domain, exposing a previously unrecognized helix-turn-helix DNA-binding motif. Our findings provide novel insights into the role of the MCM complex during the initiation step of DNA replication.     

Chromatin remodeler sucrose nonfermenting 2 homolog (SNF2H) is recruited onto DNA replication origins through interaction with Cdc10 protein-dependent transcript 1 (Cdt1) and promotes pre-replication complex formation

From late mitosis to the G(1) phase of the cell cycle, ORC, CDC6, and Cdt1 form the machinery necessary to load MCM2-7 complexes onto DNA. Here, we show that SNF2H, a member of the ATP-dependent chromatin-remodeling complex, is recruited onto DNA replication origins in human cells in a Cdt1-dependent manner and positively regulates MCM loading. SNF2H physically interacted with Cdt1. ChIP assays indicated that SNF2H associates with replication origins specifically during the G(1) phase. Binding of SNF2H at origins was decreased by Cdt1 silencing and, conversely, enhanced by Cdt1 overexpression. Furthermore, SNF2H silencing prevented MCM loading at origins and moderately inhibited S phase progression. Although neither SNF2H overexpression nor SNF2H silencing appeared to impact rereplication induced by Cdt1 overexpression, Cdt1-induced checkpoint activation was inhibited by SNF2H silencing. Collectively, these data suggest that SNF2H may promote MCM loading at DNA replication origins via interaction with Cdt1 in human cells. Because efficient loading of excess MCM complexes is thought to be required for cells to tolerate replication stress, Cdt1- and SNF2H-mediated promotion of MCM loading may be biologically relevant for the regulation of DNA replication.     

Distinct roles for Sld3 and GINS during establishment and progression of eukaryotic DNA replication forks

The Cdc45 protein is crucial for the initiation of chromosome replication in eukaryotic cells, as it allows the activation of prereplication complexes (pre-RCs) that contain the MCM helicase. This causes the unwinding of origins and the establishment of DNA replication forks. The incorporation of Cdc45 at nascent forks is a highly regulated and poorly understood process that requires, in budding yeast, the Sld3 protein and the GINS complex. Previous studies suggested that Sld3 is also important for the progression of DNA replication forks after the initiation step, as are Cdc45 and GINS. In contrast, we show here that Sld3 does not move with DNA replication forks and only associates with MCM in an unstable manner before initiation. After the establishment of DNA replication forks from early origins, Sld3 is no longer essential for the completion of chromosome replication. Unlike Sld3, GINS is not required for the initial recruitment of Cdc45 to origins and instead is necessary for stable engagement of Cdc45 with the nascent replisome. Like Cdc45, GINS then associates stably with MCM during S-phase.     

Is the MCM2-7 complex the eukaryotic DNA replication fork helicase?

The MCM2-7 complex is essential for both the initiation and elongation phases of eukaryotic chromosome replication. There is some evidence that MCM2-7 proteins may act as a DNA helicase; at the same time, a variety of other DNA helicases have also been implicated in the replication of eukaryotic chromosomes.     

Xenopus Cdc7 executes its essential function early in S phase and is counteracted by checkpoint-regulated protein phosphatase 1

The initiation of DNA replication requires two protein kinases: cyclin-dependent kinase (Cdk) and Cdc7. Although S phase Cdk activity has been intensively studied, relatively little is known about how Cdc7 regulates progression through S phase. We have used a Cdc7 inhibitor, PHA-767491, to dissect the role of Cdc7 in Xenopus egg extracts. We show that hyperphosphorylation of mini-chromosome maintenance (MCM) proteins by Cdc7 is required for the initiation, but not for the elongation, of replication forks. Unlike Cdks, we demonstrate that Cdc7 executes its essential functions by phosphorylating MCM proteins at virtually all replication origins early in S phase and is not limiting for progression through the Xenopus replication timing programme. We demonstrate that protein phosphatase 1 (PP1) is recruited to chromatin and rapidly reverses Cdc7-mediated MCM hyperphosphorylation. Checkpoint kinases induced by DNA damage or replication inhibition promote the association of PP1 with chromatin and increase the rate of MCM dephosphorylation, thereby counteracting the previously completed Cdc7 functions and inhibiting replication initiation. This novel mechanism for regulating Cdc7 function provides an explanation for previous contradictory results concerning the control of Cdc7 by checkpoint kinases and has implications for the use of Cdc7 inhibitors as anti-cancer agents.