ORC binds and remodels nucleosomes to specify MCM loading onto DNA

Saccharomyces cerevisiae has been a faithful guide for study of eukaryotic DNA replication, as the numerous initiation and elongation proteins are conserved from yeast to human. However, there is a gap in our knowledge of why yeast uses a consensus DNA sequence at replication origins, while higher eukaryotes do not. The current study closes this gap. By direct single-molecule visualization, we show that the Origin Recognition Complex (ORC) searches for and stably binds nucleosomes, and that nucleosomes funtionalize ORC to load MCM helicase onto DNA, regardless of DNA sequence. Furthermore, we discover that ORC can remodel nucleosomes and expel H2A-H2B histone dimers, a heretofore unexpected function. Thus ORC helps create a chromatin environment permissive to origin function. The finding that ORC binding to nucleosomes leads to MCM loading at any DNA sequence is likely to generalize, and that higher eukaryotes follow this same paradigm for origin selection INTRODUCTION Complete and accurate duplication of the genome is critical for the proliferation of all organisms (1). The basic mechanism for the initiation of DNA replication is shared by all three domains of cellular life (2). An “initiator” first binds to a genomic site known as the origin of replication, then recruits the replicative helicase that is responsible for unwinding parental DNA duplex and creating templates for daughter strand synthesis. In eukaryotes, multiple origins across the genome are licensed for firing once, and only once, per cell cycle and their firing follows a temporally controlled program (3). The eukaryotic initiator is known as the origin recognition complex (ORC), which consists of six highly conserved subunits Orc1-6 (4, 5). ORC works in concert with Cdc6 and Cdt1 to coordinate the loading of two Mcm2-7 helicase complexes onto the origin (6, 7), forming the pre-replication complex (pre-RC) that potentially becomes activated during the subsequent S phase to produce bidirectional replication forks. In the model organism Saccharomyces cerevisiae (budding yeast), origins are located at a set of “replicator” positions known as autonomously replicating sequence (ARS) elements, each containing an AT-rich ARS consensus sequence (ACS) and other less conserved “B elements” (1). However, consensus ARS elements have yet to be found in higher eukaryotes including human (8). In fact, even in S. cerevisiae, the ARS and its internal ACS and B elements are not absolutely required for ORC binding, Mcm2-7 loading, or origin firing in vitro and in vivo (7, 9, 10). These observations indicate that there exist yet other chromosomal features that enable licensing and firing of eukaryotic replication origins (8, 11). One distinct challenge for ORC is the need to navigate through chromosomes predominantly packaged into nucleosomes (12), which influence multiple aspects of eukaryotic replication (13-15). Although still an area of active research, it is generally presumed that the nucleosome presents a barrier to ORC binding and origin activity, which must be overcome by DNA sequence or chromainremodeling enzymes. Consistent with the inhibitory effect of nucleosomes, it was shown that a nucleosome-free region (NFR) flanked by regularly positioned nucleosomes is a pronounced feature of ARS origin sites (16, 17). Notably, ORC binding and pre-RC assembly also play a direct role in shaping a local chromatin state permissive to replication initiation, often widening the NFR (18, 19). These studies suggest possible active participation of ORC in the formation of an adequate NFR for replication initiation. The interplay between ORC and nucleosomes is clearly a critical determinant for origin selection and function, but the molecular underpinning of this interplay remains incompletely understood. Single-molecule studies using reconstituted replication proteins have greatly aided our understanding of origin recognition and pre-RC formation (20-22). However, these studies have not examined the effect of nucleosomes on ORC activity. In this work, we employ correlative singlemolecule fluorescence and force microscopy, complemented with biochemical assays and genomic analysis, to interrogate the dynamic behavior of ORC on nucleosomal DNA. We find that, contrary to expectation, ORC searches for and actively remodels nucleosomes in a chromatinzed environment, and that nucleosomes are the primary determinant of origin recognition and licensing, regardless of the ARS DNA sequence. This finding of ARS-independent, nucleosome-dependent origin selection makes the yeast system much more like that expected for higher eukaryotes, and expands the role of ORC to one that can also help form the chromatin environment for initiation. RESULTS Direct observation of ORC interaction with nucleosomal DNA To perform the single-molecule studies we engineered a native S. cerevisiae origin sequence ARS1 (23) into the λ phage genome (Supplementary Fig. 1A), generating a DNA template (λARS1) that is biotinylated on both ends (Fig. 1A). We also purified the yeast Orc1-6 and Mcm2-7 complexes (referred to as ORC and MCM hereafter) (Supplementary Fig. 1B), as well as Cdc6 and Cdt1. To generate fluorescently labeled ORC for single-molecule visualization, we site-specifically attached a Cy3 fluorophore to the N terminus of the Orc1 subunit via a 12-residue peptide tag (S6), much smaller than the Halo-tag used in a recent single-molecule study (22). A single λARS1 molecule was tethered between a pair of streptavidin-coated beads in the microfluidic chamber of a dual-trap optical tweezers instrument also equipped with multicolor confocal fluorescence microscopy (24, 25) (Supplementary Fig. 1C). Upon moving the tethered DNA into a channel containing Cy3-ORC, Cdc6 and ATP, we observed ORC binding to DNA in real time (Fig. 1B). Consistent with previous results (21, 22), ORC-Cdc6 displayed diffusive behavior at non-ARS1 sites until it dissociated or encountered the ARS1 site, where the rapid diffusion behavior halted and, instead, ORC-Cdc6 resided stably (Fig. 1B). The distinctive behavior of ORC-Cdc6 at the engineered ARS1 site versus all other sites indicates that the λ genome does not contain a strong ACS motif, consistent with sequence analysis using a published algorithm (26) (Supplementary Fig. 2). Next, we set out to examine the behavior of ORC on nucleosomal DNA. We juxtaposed a Widom601 nucleosome positioning sequence next to the ARS1 site and generated the λ601ARS1 DNA template (Fig. 1C). We then loaded tethered λ601ARS1 with histone octamers in situ by the histone chaperone Nap1. This method has been previously used to form nucleosomes (14, 27) and also confirmed by us via single-molecule pulling experiments (Supplementary Fig. 3). The octamers were fluorescently labeled with Cy5 for direct visualization. We observed nucleosomes formed both at the 601 site next to ARS1 and at other locations (Fig. 1D), which allowed us to compare ORC-nucleosome interaction at ARS1 versus non-ARS1 sites. Surprisingly, we found that in the vast majority of cases ORC stably associated with the nucleosome, regardless of whether it was located adjacent to the ARS1 site or was at a non-ARS1 site (Fig. 1D). This is in contrast to the discrimination by ORC between ARS1 and non-ARS1 sites on non-nucleosomal DNA (Fig. 1E, F). Nucleosome targeting by ORC can conceivably be achieved by either a three-dimensional (3D) search (direct binding from solution) or a one-dimensional (1D) search (sliding along the DNA from a non-nucleosomal site). Indeed, we observed both modes in our data (Fig. 1D), with 3D search being the more dominant mode under our experimental conditions (Fig. 1G). These results suggest that ORC preferentially binds to nucleosomes when in a chromatinized