Pre-replicative complex assembly with purified proteins

Mehanna, Amina; Diffley, John F.X.

doi:10.1016/j.ymeth.2012.06.008

Cited by 11 publications

(12 citation statements)

References 32 publications

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“…As negative controls, HA-hORC5 was not co-immunoprecipitated by anti-FLAG antibody from the extracts without hORC5-FLAG, and hORC5-FLAG was not coimmunoprecipitated by anti-HA antibody from extracts without HA-ORC5. A comparison of the pairwise interactions among the yeast and human ORC subunits observed in this study and by others, as cited below, indicate that the following positive interactions were common between the budding yeast and human proteins: ORC1 interacts with ORC2 [9,[33][34][35][36][37][38][39][40][41] and ORC4 [9,[36][37][38][39][42][43][44]; ORC2 interacts with ORC2 [37], ORC3 [9,34,35,37,40,[42][43][44][45][46][47][48], ORC4 [34,35,37,40,44,[47][48][49], ORC5 [9,34,35,37,40,[42][43]…”

Section: Pairwise Interactions Of the Yeast And Human Orc Subunitssupporting

confidence: 63%

The interaction networks of the budding yeast and human DNA replication-initiation proteins

Amin

Wang

et al. 2019

Cell Cycle

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DNA replication is a stringently regulated cellular process. In proliferating cells, DNA replicationinitiation proteins (RIPs) are sequentially loaded onto replication origins during the M-to-G 1 transition to form the pre-replicative complex (pre-RC), a process known as replication licensing. Subsequently, additional RIPs are recruited to form the pre-initiation complex (pre-IC). RIPs and their regulators ensure that chromosomal DNA is replicated exactly once per cell cycle. Origin recognition complex (ORC) binds to, and marks replication origins throughout the cell cycle and recruits other RIPs including Noc3p, Ipi1-3p, Cdt1p, Cdc6p and Mcm2-7p to form the pre-RC. The detailed mechanisms and regulation of the pre-RC and its exact architecture still remain unclear. In this study, pairwise protein-protein interactions among 23 budding yeast and 16 human RIPs were systematically and comprehensively examined by yeast two-hybrid analysis. This study tested 470 pairs of yeast and 196 pairs of human RIPs, from which 113 and 96 positive interactions, respectively, were identified. While many of these interactions were previously reported, some were novel, including various ORC and MCM subunit interactions, ORC self-interactions, and the interactions of IPI3 and NOC3 with several pre-RC and pre-IC proteins. Ten of the novel interactions were further confirmed by co-immunoprecipitation assays. Furthermore, we identified the conserved interaction networks between the yeast and human RIPs. This study provides a foundation and framework for further understanding the architectures, interactions and functions of the yeast and human pre-RC and pre-IC.

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Section: Pairwise Interactions Of the Yeast And Human Orc Subunitssupporting

confidence: 63%

The interaction networks of the budding yeast and human DNA replication-initiation proteins

Amin

Wang

et al. 2019

Cell Cycle

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“…Recent establishment of an in vitro helicase loading reaction with purified proteins and origin DNA has transformed these concepts into tangible biochemical processes that are amenable to mechanistic dissections (Evrin et al 2009;Mehanna and Diffley 2012). By taking advantage of the established in vitro pre-RC assembly process and the fact that the proteins form large complexes on DNA, we succeeded in capturing and visualizing a number of pre-RC assembly steps by EM, which, combined with image classification and detailed subunit mapping, provided mechanistic insights into key eukaryotic replication initiation events (Fig.…”

Section: Discussionmentioning

confidence: 99%

Structural and mechanistic insights into Mcm2–7 double-hexamer assembly and function

et al. 2014

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Eukaryotic cells license each DNA replication origin during G1 phase by assembling a prereplication complex that contains a Mcm2-7 (minichromosome maintenance proteins 2-7) double hexamer. During S phase, each Mcm2-7 hexamer forms the core of a replicative DNA helicase. However, the mechanisms of origin licensing and helicase activation are poorly understood. The helicase loaders ORC-Cdc6 function to recruit a single Cdt1-Mcm2-7 heptamer to replication origins prior to Cdt1 release and ORC-Cdc6-Mcm2-7 complex formation, but how the second Mcm2-7 hexamer is recruited to promote double-hexamer formation is not well understood. Here, structural evidence for intermediates consisting of an ORC-Cdc6-Mcm2-7 complex and an ORC-Cdc6-Mcm2-7-Mcm2-7 complex are reported, which together provide new insights into DNA licensing. Detailed structural analysis of the loaded Mcm2-7 double-hexamer complex demonstrates that the two hexamers are interlocked and misaligned along the DNA axis and lack ATP hydrolysis activity that is essential for DNA helicase activity. Moreover, we show that the head-to-head juxtaposition of the Mcm2-7 double hexamer generates a new protein interaction surface that creates a multisubunit-binding site for an S-phase protein kinase that is known to activate DNA replication. The data suggest how the double hexamer is assembled and how helicase activity is regulated during DNA licensing, with implications for cell cycle control of DNA replication and genome stability.

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“…However, their precise functions are still largely elusive. For characterization of their biochemical properties and ultimate reconstitution of the entire processes with purified factors, it would be crucial to purify each of these factors involved in a quantity sufficient for detailed enzymatic characterization [4][5][6]. Unfortunately, most of these factors are generally large in sizes and are sometimes present as a component of larger multi-factor complexes.…”

Section: Introductionmentioning

confidence: 99%

Purification of replication factors using insect and mammalian cell expression systems

Uno¹,

You²,

Masai³

2012

Methods

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Pre-replicative complex assembly with purified proteins

Cited by 11 publications

References 32 publications

The interaction networks of the budding yeast and human DNA replication-initiation proteins

The interaction networks of the budding yeast and human DNA replication-initiation proteins

Structural and mechanistic insights into Mcm2–7 double-hexamer assembly and function

Purification of replication factors using insect and mammalian cell expression systems

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