Atomic force microscopic analysis of the binding of the Schizosaccharomyces pombe origin recognition complex and the spOrc4 protein with origin DNA

Abstract

In eukaryotes, the initiation of DNA replication requires the interaction between origin sequences and the origin recognition complex (ORC), which is highly conserved. In this report, atomic force microscopy (AFM) was used to examine the binding of Schizosaccharomyces pombe (sp) ORC and the spOrc4 protein with the sp autonomously replicating sequence 1 (ars1). AFM imaging revealed that spORC binding to ars1 occurred solely through spOrc4p and depended on the N-terminal AT-hook domains present in spOrc4p. At high molar ratios of spORC (or spOrc4p alone) to DNA (6:1), all of the input ars1 was bound in a one protein complex to one plasmid manner. Restriction digestion and AFM analysis of protein-DNA fragments revealed the presence of two binding sites in ars1. One site mapped to a region centered at nucleotide 838 of ars1 previously detected by DNase I protection that was reported to be essential for the autonomously replicating sequence activity of ars1. The second site mapped to a previously uncharacterized region centered at nucleotide 1148. AFM showed that the length of the DNA fragment complexed with either spORC or spOrc4p was shortened by Ϸ140 bp, suggesting the wrapping of two turns of the DNA around the spOrc4p alone as well as the spOrc4p in spORC. We also show that treatment of the spORC (spOrc4p)-ars1 complex with topoisomerase I induced a negative shift in the topoisomer distribution. These findings suggest that the binding of spORC to origin DNA alters the structure of the DNA. Thus, in the case of spORC, due to its unusual spOrc4p, at least two factors are likely to influence ars1 activation. These include the selective binding of the complex to A-and T-rich regions and the alteration of the DNA structure due to its wrapping around spOrc4p.atomic force microscopy ͉ topoisomers ͉ replication C hromosomal replication in eukaryotes requires the origin recognition complex (ORC), a heteromeric six-subunit complex that acts as the initiator for the assembly of other key initiator factors such as Cdc18 (Cdc6), Cdt1, and the MCM2-7 complex (the probable replicative helicase) (1). The ORC was isolated initially from Saccharomyces cerevisiae (Sc) based on its origin binding activity (2). Replication origins of Sc are relatively short AT-rich sequences (Ϸ150 bp) that include an essential autonomously replicating sequence (ARS) with an 11-bp consensus sequence (ACS) (3-5) that acts as the key binding site for ScORC in a reaction requiring ATP (2). Additional sequences in Sc ARSs, referred to as B domains, enhance the efficiency of origin activity (3, 6).The structure and initiator role of ORC is conserved in all eukaryotes, and six-subunit complexes have been purified from species including Schizosaccharomyces pombe (sp), Drosophila melanogaster (Dm), Xenopus laevis, and humans (7-11). In vitro and in vivo studies indicate that although ORC is essential for DNA replication in these species (1), the sequences recognized by these ORC complexes differ. In metazoans, initiation of replication occurs at multiple sit...

New insights into replication origin characteristics in metazoans

“…30 Atomic force microscopy studies of the S. pombe ORC have suggested that approximately 150 bp of DNA are wrapped around the protein complex. 31 DNA wrapping of Ori binding proteins has been observed also with the Drosphila DmORC, 32 and this could explain the distance of 160 bp that (Fig. 5E).…”

Effect of minichromosome maintenance protein 2 deficiency on the locations of DNA replication origins

“…One explanation for this discrepancy is that an additional, as yet unidentified, protein interacts at this location (Cayrou et al 2011). Alternatively, it has been shown for S. pombe (Gaczynska et al 2004) and Drosophila (Remus et al 2004) that following the initial binding, DNA is wrapped around the ORC complex, which could result in a distance of ∼160-190 bp between an initial ORC binding site and the location at which the CDT1-MCM2-7 complex is bound. Alternatively, MCM2-7 complex loading has been studied only on naked DNA substrates, and it is unclear whether these studies accurately reflect loading to chromatin.…”

Mechanisms for initiating cellular DNA replication

“…Many of the AAA+ domains within ORC and Cdc6 are linked to other modules, in particular a predicted variant of an HTH fold termed a winged-helix domain (WHD), which may be involved in DNA binding (4,84,93). The N terminus of S. pombe Orc4 is further decorated with several AT-hook repeats that promote interactions between the initiator complex and origin DNA (40,41,94), whereas the N terminus of Orc1 universally bears a conserved bromo-adjacent homology (BAH) domain implicated in binding multiple factors (50,95,96), including transcriptional silencing factors such as Sir1 (in yeast) and HP1 (in human and Drosophila) (97)(98)(99). The Orc1 BAH domain binds to nucleosomes, specifically recognizing dimethylated histone H4 tails (100).…”