Abstract
DNA replication in eukaryotic cells initiates from replication origins that bind the Origin Recognition Complex (ORC). Origin establishment requires well-defined DNA sequence motifs in Saccharomyces cerevisiae and some other budding yeasts, but most eukaryotes lack sequence-specific origins. A 3.9 Å structure of S. cerevisiae ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) bound to origin DNA revealed that a loop within Orc2 inserts into a DNA minor groove and an α-helix within Orc4 inserts into a DNA major groove. Using a massively parallel origin selection assay coupled with a custom mutual-information-based modeling approach, and a separate analysis of whole-genome replication profiling, here we show that the Orc4 α-helix contributes to the DNA sequence-specificity of origins in S. cerevisiae and Orc4 α-helix mutations change genome-wide origin firing patterns. The DNA sequence specificity of replication origins, mediated by the Orc4 α-helix, has co-evolved with the gain of ORC-Sir4-mediated gene silencing and the loss of RNA interference.
Highlights
DNA replication in eukaryotic cells initiates from replication origins that bind the Origin Recognition Complex (ORC)
We demonstrate using two independent assays that the Orc[4] αhelix and Orc[2] loop are required for the initiation of DNA replication and that the Orc[4] α-helix contributes to determining DNA sequence-specific origins in the yeast S. cerevisiae
By examining ORC sequences in a wide variety of eukaryotes for the presence of these origin specification domains, we find that the specification of DNA replication origins by the Orc[4] αhelix and Orc[2] loop co-evolved with ORC-Sir4-dependent transcriptional gene silencing and the loss of RNA interference (RNAi)
Summary
DNA replication in eukaryotic cells initiates from replication origins that bind the Origin Recognition Complex (ORC). To better understand the effects of these mutations on origin activity and specificity, we performed two complementary, but independent deep-sequencing-based assays: massively parallel origin mutagenesis on plasmids and genome-wide DNA replication profiling.
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