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

Abstract

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 ...

MCM10: One tool for all—Integrity, maintenance and damage control

“…Co-immunoprecipitation studies in human cells and Xenopus egg extracts have demonstrated that Mcm10, Pol-α and Ctf4 are in a common complex and postulated that Mcm10 and Ctf4 connect the lagging strand polymerase, Pol-α, with the CMG helicase [38]. This notion is consistent with the finding that genetic disruption of Mcm10 and knockdown of Ctf4 result in very similar cellular phenotypes in Drosophila melanogaster [42, 43]. Moreover, depletion of either Ctf4 or Mcm10 has been implicated in regulating the turnover of Pol-α [5, 38, 44, 45].…”

From structure to mechanism—understanding initiation of DNA replication

“…Therefore, the two proteins might affect Mcm2 in alternative ways, with Cdc45 closing the gate to support helicase activity, and Cdt1 allowing opening and closing of the gate during MCM2-7 loading. Moreover, Mcm10 might affect these surfaces as well, as it contacts the Mcm2 NTD (Apger et al 2010;Lee et al 2010;Looke et al 2017) in order to stabilize the CMG complex (Looke et al 2017). At an in vitro assembled replication fork, ssDNA appears from the N-terminal MCM2-7 face during DNA unwinding (Georgescu et al 2017).…”

Structural Biology of Replication Initiation Factor Mcm10

“…Furthermore, spMcm10 has been reported to stimulate DDK phosphorylation of Mcm2-7 (Lee et al 2003), and is thus believed to play a role in helicase activation. Studies in S. cerevisiae have shown that scMcm10 facilitates assembly of the Cdc45/Mcm2-7/GINS helicase complex (Gambus et al 2009), and physical interactions have been observed with Mcm2, Mcm4, Mcm5, Mcm6, and Mcm7 subunits (Apger et al 2010; Hart et al 2002; Homesley et al 2000; Merchant et al 1997). Recent work suggested that scMcm10 serves as a functional linker between the MCM helicase and pol α by coordinating their activities and ensuring their physical stability and integrity at the replication fork (Lee et al 2010), a role also identified for Ctf4 (Gambus et al 2009).…”