High-resolution analysis of four efficient yeast replication origins reveals new insights into the ORC and putative MCM binding elements

Abstract

In budding yeast, the eukaryotic initiator protein ORC (origin recognition complex) binds to a bipartite sequence consisting of an 11 bp ACS element and an adjacent B1 element. However, the genome contains many more matches to this consensus than actually bind ORC or function as origins in vivo. Although ORC-dependent loading of the replicative MCM helicase at origins is enhanced by a distal B2 element, less is known about this element. Here, we analyzed four highly active origins (ARS309, ARS319, ARS606 and ARS607) by linker scanning mutagenesis and found that sequences adjacent to the ACS contributed substantially to origin activity and ORC binding. Using the sequences of four additional B2 elements we generated a B2 multiple sequence alignment and identified a shared, degenerate 8 bp sequence that was enriched within 228 known origins. In addition, our high-resolution analysis revealed that not all origins exist within nucleosome free regions: a class of Sir2-regulated origins has a stably positioned nucleosome overlapping or near B2. This study illustrates the conserved yet flexible nature of yeast origin architecture to promote ORC binding and origin activity, and helps explain why a strong match to the ORC binding site is insufficient to identify origins within the genome.

Maintaining replication origins in the face of genomic change

“…We do note that the few L. waltii ARSs that fail to function in S. cerevisiae are less well matched with the S. cerevisiae ACS at the B1 element than at the A element. This observation is in line with what has been previously reported for S. cerevisiae: Deviations in sequences outside of the ACS, especially the B1 element, can affect the function of the ARS (Chang et al 2011).…”

“…In addition to the A and B1 elements, S. cerevisiae origins are also comprised of additional A-rich B elements lacking a defined consensus sequence (Eaton et al 2010;Chang et al 2011). These sequences are located between 50 and 100 bp downstream from the T-rich ACS and as a result create an apparent A-T asymmetry at the ACS (Breier et al 2004;Eaton et al 2010).…”

A Link between ORC-Origin Binding Mechanisms and Origin Activation Time Revealed in Budding Yeast

“…Specifically, ORC binds to a bipartite ∼35 bp element consisting of a 17-bp EACS-element ( e xtended A RS c onsensus s equence) and a less conserved B1-element that contains a common 3 bp WTW motif [48]–[50]. However, recent work has shown that local chromatin structure may also contribute to origin selection by ORC [43], [44].…”

“…Origins lacking a predicted ACS were omitted from these analyses. All nucleosome traces were centered on the start of the EACS (Expanded ACS) on the T rich strand [49], [50]. For origins containing an ORCACS match, the first nucleotide of the ORCACS on the T rich strand marked the start of the EACS.…”

“…The sub-clones of ARS1529.5 in Figure S7 were generated using sequence from the KANMX coding sequence as substitutes for regions of ARS1529.5 as indicated in Figure S7 and Table S3. Plasmids were transformed into wild-type W303 yeast using standard techniques, and ARS assays were performed with four independent cultures per plasmid as in [50].…”