Decreased MCM2-6 in Drosophila S2 Cells Does Not Generate Significant DNA Damage or Cause a Marked Increase in Sensitivity to Replication Interference

A reduction in the level of some MCM proteins in human cancer cells (MCM5 in U20S cells or MCM3 in Hela cells) causes a rapid increase in the level of DNA damage under normal conditions of cell proliferation and a loss of viability when the cells are subjected to replication interference. Here we show that Drosophila S2 cells do not appear to show the same degree of sensitivity to MCM2-6 reduction. Under normal cell growth conditions a reduction of >95% in the levels of MCM3, 5, and 6 causes no significant short term alteration in the parameters of DNA replication or increase in DNA damage. MCM depleted cells challenged with HU do show a decrease in the density of replication forks compared to cells with normal levels of MCM proteins, but this produces no consistent change in the levels of DNA damage observed. In contrast a comparable reduction of MCM7 levels has marked effects on viability, replication parameters and DNA damage in the absence of HU treatment.

[1]  A. Davies,et al.  Ubiquitin-dependent DNA damage bypass is separable from genome replication , 2010, Nature.

[2]  Jonathan R. Hall,et al.  Analysis of re-replication from deregulated origin licensing by DNA fiber spreading , 2008, Nucleic acids research.

[3]  E. Schwob,et al.  Excess MCM proteins protect human cells from replicative stress by licensing backup origins of replication , 2008, Proceedings of the National Academy of Sciences.

[4]  Xin Quan Ge,et al.  Dormant origins licensed by excess Mcm2-7 are required for human cells to survive replicative stress. , 2007, Genes & development.

[5]  M. Yamaguchi,et al.  Differential Requirements for MCM Proteins in DNA Replication in Drosophila S2 Cells , 2007, PloS one.

[6]  A. Gartner,et al.  Excess Mcm2–7 license dormant origins of replication that can be used under conditions of replicative stress , 2006, The Journal of cell biology.

[7]  D. Wigley,et al.  Pumps, paradoxes and ploughshares: mechanism of the MCM2-7 DNA helicase. , 2005, Trends in biochemical sciences.

[8]  S. Cotterill,et al.  The Drosophila Cdc6/18 protein has functions in both early and late S phase in S2 cells , 2005, Journal of Cell Science.

[9]  R. Abraham,et al.  Interaction between human MCM7 and Rad17 proteins is required for replication checkpoint signaling , 2004, The EMBO journal.

[10]  M. Pacek,et al.  A requirement for MCM7 and Cdc45 in chromosome unwinding during eukaryotic DNA replication , 2004, The EMBO journal.

[11]  S. Elledge,et al.  Minichromosome maintenance proteins are direct targets of the ATM and ATR checkpoint kinases. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[12]  J. Blow,et al.  The role of Cdc6 in ensuring complete genome licensing and S phase checkpoint activation , 2004, The Journal of cell biology.

[13]  Susan L. Forsburg,et al.  Eukaryotic MCM Proteins: Beyond Replication Initiation , 2004, Microbiology and Molecular Biology Reviews.

[14]  H. Kimura,et al.  Enhanced expression of Mcm proteins in cancer cells derived from uterine cervix. , 2003, European journal of biochemistry.

[15]  R. Laskey,et al.  A rotary pumping model for helicase function of MCM proteins at a distance from replication forks , 2003, EMBO reports.

[16]  T. Prokhorova,et al.  MCM2–7 Complexes Bind Chromatin in a Distributed Pattern Surrounding the Origin Recognition Complex inXenopus Egg Extracts* , 2002, The Journal of Biological Chemistry.

[17]  M. Yamaguchi,et al.  Nearest neighbour analysis of MCM protein complexes in Drosophila melanogaster. , 2001, Nucleic acids research.

[18]  S Povey,et al.  Dynamic molecular combing: stretching the whole human genome for high-resolution studies. , 1997, Science.

[19]  L. Drury,et al.  Cdc6p-dependent loading of Mcm proteins onto pre-replicative chromatin in budding yeast. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[20]  J. Blow,et al.  Cell Cycle Regulation of the Replication Licensing System: Involvement of a Cdk-dependent Inhibitor , 1997, The Journal of cell biology.

[21]  M. Lei,et al.  Physical interactions among Mcm proteins and effects of Mcm dosage on DNA replication in Saccharomyces cerevisiae , 1996, Molecular and cellular biology.

[22]  R. Knippers,et al.  Interactions of human nuclear proteins P1Mcm3 and P1Cdc46. , 1995, European journal of biochemistry.

[23]  P. Thömmes,et al.  Purification and characterisation of dRP‐A: a single‐stranded DNA binding protein from Drosophila melanogaster , 1994, FEBS letters.