A Conserved Hsp10-like Domain in Mcm10 Is Required to Stabilize the Catalytic Subunit of DNA Polymerase-α in Budding Yeast

Ricke, Robin M.; Bielinsky, Anja‐Katrin

doi:10.1074/jbc.m513551200

Cited by 51 publications

(91 citation statements)

References 65 publications

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“…This core has been shown to interact with single-stranded DNA, DNA polymerase a, and PCNA Chattopadhyay and Bielinsky 2007;Robertson et al 2008;Warren et al 2008Warren et al , 2009). Genetic studies also support the assertion that this central core supports the essential function(s) of Mcm10 as mutations in this region of Mcm10 affect viability of cells and affect DNA replication (Merchant et al 1997;Homesley et al 2000;Ricke and Bielinsky 2006). However, mutations in this region have also revealed a role for Mcm10 in heterochromatic silencing (Liachko and Tye 2005).…”

Section: Discussionmentioning

confidence: 59%

Multiple Functions for Drosophila Mcm10 Suggested Through Analysis of Two Mcm10 Mutant Alleles

et al. 2010

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DNA replication and the correct packaging of DNA into different states of chromatin are both essential processes in all eukaryotic cells. High-fidelity replication of DNA is essential for the transmission of genetic material to cells. Likewise the maintenance of the epigenetic chromatin states is essential to the faithful reproduction of the transcriptional state of the cell. It is becoming more apparent that these two processes are linked through interactions between DNA replication proteins and chromatin-associated proteins. In addition, more proteins are being discovered that have dual roles in both DNA replication and the maintenance of epigenetic states. We present an analysis of two Drosophila mutants in the conserved DNA replication protein Mcm10. A hypomorphic mutant demonstrates that Mcm10 has a role in heterochromatic silencing and chromosome condensation, while the analysis of a novel C-terminal truncation allele of Mcm10 suggests that an interaction with Mcm2 is not required for chromosome condensation and heterochromatic silencing but is important for DNA replication.T HE essential process of DNA replication does not occur in a vacuum; rather, it takes place within the context of the cell. More specifically, DNA replication occurs within the context of chromatin: an integrated network of DNA-associated proteins that have roles in packaging DNA, controlling transcription, and maintaining genome integrity. The maintenance and manipulation of these chromatin proteins are, like DNA replication, an essential process. The packaging of DNA has significant consequences for the transcriptional state of the underlying DNA. Repression or activation of different regions of the genome through packaging as open euchromatin or as repressive heterochromatin is cell type specific (Fraser et al. 2009;Minard et al. 2009). Moreover, these transcriptional states must be maintained and passed on to daughter cells during mitosis. If not passed on faithfully, genome instability and/or transcriptional misregulation can occur, both of which may lead to defects in cell proliferation, cancer, and other disease states ( Jones et al. 2007;Hirst and Marra 2009).By necessity, the process of DNA replication requires unencumbered access to the nitrogenous bases that make up the DNA strand. As a result, chromatin proteins must be removed. In the wake of the DNA replication fork this nascent DNA must be repackaged to recapitulate the previous chromatin state. While DNA replication benefits from complementary base pairing to build a DNA molecule through semiconservative replication, the reestablishment of epigenetic states occurs through more subtle and varied mechanisms (Groth et al. 2007). One central question in reconciling the processes of DNA replication and the establishment and/or maintenance of chromatin states is how are these processes linked? One model suggests that DNA replication proteins interact with separate chromatin establishment factors, thereby spatially linking the two processes. Supporting this model has ...

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Section: Discussionmentioning

confidence: 59%

Multiple Functions for Drosophila Mcm10 Suggested Through Analysis of Two Mcm10 Mutant Alleles

et al. 2010

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“…This follows a common strategy for numerous modular proteins involved in DNA processing; there is a significant kinetic advantage to deconstructing protein interactions into two or more weak binding sites (68). The recruitment of pol ␣ to origins of replication by Mcm10 would be a significant step to signal nascent DNA synthesis and contribute to fork stability (6,12,16,24,26,27,29,71). Indeed, Mcm10 has been shown to be necessary for pol ␣ loading onto chromatin (4).…”

Section: A Molecular Mechanism For Mcm10mentioning

confidence: 99%

“…Importantly, Mcm10 physically interacts with and stabilizes pol ␣ and helps to maintain its association with chromatin (16,26,27). This is a critical interaction during replication because pol ␣ is the only enzyme in eukaryotic cells that is capable of initiating DNA synthesis de novo.…”

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Physical Interactions between Mcm10, DNA, and DNA Polymerase α

Warren

Huang²,

Fanning³

et al. 2009

Journal of Biological Chemistry

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Mcm10 is an essential eukaryotic protein required for the initiation and elongation phases of chromosomal replication. Specifically, Mcm10 is required for the association of several replication proteins, including DNA polymerase ␣ (pol ␣), with chromatin. We showed previously that the internal (ID) and C-terminal (CTD) domains of Mcm10 physically interact with both single-stranded (ss) DNA and the catalytic p180 subunit of pol ␣. However, the mechanism by which Mcm10 interacts with pol ␣ on and off DNA is unclear. As a first step toward understanding the structural details for these critical intermolecular interactions, x-ray crystallography and NMR spectroscopy were used to map the binary interfaces between Mcm10-ID, ssDNA, and p180. The crystal structure of an Mcm10-ID⅐ssDNA complex confirmed and extended our previous evidence that ssDNA binds within the oligonucleotide/oligosaccharide binding-fold cleft of Mcm10-ID. We show using NMR chemical shift perturbation and fluorescence spectroscopy that p180 also binds to the OB-fold and that ssDNA and p180 compete for binding to this motif. In addition, we map a minimal Mcm10 binding site on p180 to a small region within the p180 N-terminal domain (residues 286 -310). These findings, together with data for DNA and p180 binding to an Mcm10 construct that contains both the ID and CTD, provide the first mechanistic insight into how Mcm10 might use a handoff mechanism to load and stabilize pol ␣ within the replication fork.

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“…Besides its role in DNA replication, Mcm10 has also been implicated in transcriptional silencing (Liachko and Tye, 2005). It is important to note that the general protein domain structure of Mcm10 is not unique in Saccharomyces cerevisiae, but it is highly conserved among eukaryotic species (Izumi et al, 2000;Ricke and Bielinsky, 2006). Mcm10 has a central oligonucleotide/ oligosaccharide binding (OB)-fold , a signature motif of RNA-and single-stranded (ss) DNA binding proteins.…”

Section: Introductionmentioning

confidence: 99%

Human Mcm10 Regulates the Catalytic Subunit of DNA Polymerase-α and Prevents DNA Damage during Replication

Chattopadhyay

Bielinsky

2007

MBoC

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In Saccharomyces cerevisiae, minichromosome maintenance protein (Mcm) 10 interacts with DNA polymerase (pol)-␣ and functions as a nuclear chaperone for the catalytic subunit, which is rapidly degraded in the absence of Mcm10. We report here that the interaction between Mcm10 and pol-␣ is conserved in human cells. We used a small interfering RNA-based approach to deplete Mcm10 in HeLa cells, and we observed that the catalytic subunit of pol-␣, p180, was degraded with similar kinetics as Mcm10, whereas the regulatory pol-␣ subunit, p68, remained unaffected. Simultaneous loss of Mcm10 and p180 inhibited S phase entry and led to an accumulation of already replicating cells in late S/G2 as a result of DNA damage, which triggered apoptosis in a subpopulation of cells. These phenotypes differed considerably from analogous studies in Drosophila embryo cells that did not exhibit a similar arrest. To further dissect the roles of Mcm10 and p180 in human cells, we depleted p180 alone and observed a significant delay in S phase entry and fork progression but little effect on cell viability. These results argue that cells can tolerate low levels of p180 as long as Mcm10 is present to "recycle" it. Thus, human Mcm10 regulates both replication initiation and elongation and maintains genome integrity. INTRODUCTIONDNA replication is a highly regulated process in which multiprotein complexes generate an exact copy of a cell's DNA. These multiprotein complexes are assembled into replication forks. Fork assembly takes place at replication origins that are spread throughout the genome in all eukaryotic cells. The first step is the formation of a prereplicative complex (pre-RC) (Diffley et al., 1994), which is subsequently transformed into a preinitiation complex, and finally into a pair of functional replication forks that have the ability to unwind parental DNA and synthesize new daughter strands (Mendez and Stillman, 2003). DNA unwinding is likely catalyzed by the minichromosome maintenance (Mcm) 2-7 complex (Aparicio et al., 1997;Labib et al., 2000;Shechter et al., 2004) in conjunction with two coactivators, Cdc45 and GINS (Pacek and Walter, 2004;Gambus et al., 2006;Moyer et al., 2006;Pacek et al., 2006). The association of Cdc45 and GINS with DNA is interdependent (Kubota et al., 2003;Takayama et al., 2003), and it requires yet another factor, Mcm10 (Wohlschlegel et al., 2002;Gregan et al., 2003;Sawyer et al., 2004). Despite its name, Mcm10 is not a member of the MCM protein family, although it was isolated in the same genetic screen in budding yeast that identified the MCM2-7 genes (Solomon et al., 1992;Merchant et al., 1997).Mcm10 is a constitutively nuclear DNA binding protein (Merchant et al., 1997;Burich and Lei, 2003) that is essential in yeast (Burich and Lei, 2003). Besides its role in DNA replication, Mcm10 has also been implicated in transcriptional silencing (Liachko and Tye, 2005). It is important to note that the general protein domain structure of Mcm10 is not unique in Saccharomyces cerevisiae, but it is highly con...

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A Conserved Hsp10-like Domain in Mcm10 Is Required to Stabilize the Catalytic Subunit of DNA Polymerase-α in Budding Yeast

Cited by 51 publications

References 65 publications

Multiple Functions for Drosophila Mcm10 Suggested Through Analysis of Two Mcm10 Mutant Alleles

Multiple Functions for Drosophila Mcm10 Suggested Through Analysis of Two Mcm10 Mutant Alleles

Physical Interactions between Mcm10, DNA, and DNA Polymerase α

Human Mcm10 Regulates the Catalytic Subunit of DNA Polymerase-α and Prevents DNA Damage during Replication

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