Origins of DNA replication in eukaryotes

Abstract

Errors occurring during DNA replication can result in inaccurate replication, incomplete replication, or re-replication, resulting in genome instability that can lead to diseases such as cancer or disorders such as autism. A great deal of progress has been made toward understanding the entire process of DNA replication in eukaryotes, including the mechanism of initiation and its control. This review focuses on the current understanding of how the origin recognition complex (ORC) contributes to determining the location of replication initiation in the multiple chromosomes within eukaryotic cells, as well as methods for mapping the location and temporal patterning of DNA replication. Origin specification and configuration vary substantially between eukaryotic species and in some cases co-evolved with gene-silencing mechanisms. We discuss the possibility that centromeres and origins of DNA replication were originally derived from a common element and later separated during evolution.

Keywords: DNA replication; epigenetic inheritance; evolution; origin recognition complex.

Figure 1 |. Replication Timing.

a shows the DNA replication process with various replication timing domains. Early-firing replication origins are indicated as E. Mid-firing replication origins are indicated as M. Late-firing replication origins are indicated as L. Dormant replication origins are indicated as D. Replication bubbles are indicated in green color. b shows the replication profile correspond to a measured in a population of cells with 1C and 2C genome copy number indicated. C equals to the genome copy number.

Figure 2 |. Pre-Replicative Complex Assembly Model.

[Adapted from with image credit: molecular structures taken and adapted from the RCSB protein database, deposits 3JA8, 5ZR1 5BK4, 5V8F, 5XF8, 6RQC, 6F0L, 6WGG and 6WGF and 7MCA.) a shows the replication origin DNA (+ strand in cantaloupe color, - strand in lavender color), which in S. cerevisiae contains four elements (indicated as black segments) with A and B2 elements binding ORC in opposite orientations. b shows that ORC (in teal color) first binds to the A and B2 elements. c shows that ORC recruits Cdc6 (in orchid color). d shows that Cdt1-Mcm2-7 complex in open ring conformation (Cdt1 in Mocha color, Mcm2-7 in Asparagus color, a channel between Mcm2 and Mcm5 subunits is indicated) is recruited by ORC-Cdc6. DNA is aligned to the channel in the Mcm2-7 hexamer. The Mcm2-7 complex is oriented as the hexamer C-terminus binding to ORC-Cdc6. e shows the intermediate known as OCCM with the double stranded DNA inserted into the channel between Mcm2 and Mcm5 subunits in the Mcm2-7 hexamer. The hexamer is partially closed. f shows that the ATP hydrolysis by the Mcm2-7 expels the first Cdc6 and then Cdt1, creating the OM complex. g shows that ORC flips over to the N-terminus side of Mcm2-7 and presumably binds to the B2 element on DNA, creating the MO complex. The structure of the MO complex was modeled by real-space-refining docked coordinates of MCM (PDB 6EYC), ORC (PDB 5ZR1) and an N-terminal Orc6 homology. h shows that ORC can now recruit a second Cdc6, creating a binding site for a second Cdt1-Mcm2-7 complex that can be loaded in an opposite orientation to the first Mcm2-7. The Mcm2-7 double hexamer, possibly with ORC still bound to the DNA, is then ready to be activated and can unwind the double stranded DNA when entering S phase.

Figure 3 |. Waves of cyclins and DNA replication proteins in human and budding yeast during cell cycle progression.

Human and budding yeast replication proteins ORC1/Orc1 and CDT1/Cdt1 (in blue) and CDC6/Cdc6 (in pink) proteins levels as well as Mcm2-7/Mcm2-7 single hexamer (in lime green) and Mcm2-7/Mcm2-7 double hexamer (in dark green) loading levels are shown as lines. Pre-RC assembly corresponds to Mcm2-7/Mcm2-7 double hexamer formation. Cyclin-dependent kinases activities are shown as solid areas with Cyclin E-CDK2 in human and Cln-Cdc28 in budding yeast are shown in yellow, while Cyclin A-CDK2 in human and Clb5-Cdc28 and Clb6-Cdc28 in budding yeast are shown in dark blue. G1, S, G2, M phases in cell cycle are indicated.

Figure 4 |. Co-evolution of gene silencing mechanisms, centromeres and replication origin sequence specificity.

[Adapted from and]. The phylogenetic tree is not drawn to scale. Most eukaryotes (font in black), including basal branching yeasts (font in green), have complete RNAi machinery or full complements of heterochromatin. Intermediate branching yeasts (font in purple) harbor partial components of RNAi or heterochromatin machinery, whereas the Saccharomycetaceae yeast family, the most recently branching yeasts, (font in blue) have completely lost RNAi/heterochromatin machinery and acquired ORC-Sir4 mediated gene silencing. Meanwhile, the selfishly propagating 2-micron plasmids exist in Saccharomycetaceae lineage, where the strains have DNA sequence-defined point centromeres as well as sequence specific replication origins. Y. lipolytica lacks RNAi as well as the SIR proteins, so it is not clear what gene silencing mechanism it uses.