Oligomer formation and G-quadruplex binding by purified murine Rif1 protein, a key organizer of higher-order chromatin architecture

Kenji Moriyama,Naoko Yoshizawa-Sugata,Hisao Masai

doi:10.1074/jbc.ra117.000446

Kenji Moriyama, Naoko Yoshizawa-Sugata + Show 1 more

Open Access

https://doi.org/10.1074/jbc.ra117.000446

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Abstract

Rap1-interacting protein 1 (Rif1) regulates telomere length in budding yeast. We previously reported that, in metazoans and fission yeast, Rif1 also plays pivotal roles in controlling genome-wide DNA replication timing. We proposed that Rif1 may assemble chromatin compartments that contain specific replication-timing domains by promoting chromatin loop formation. Rif1 also is involved in DNA lesion repair, restart after replication fork collapse, anti-apoptosis activities, replicative senescence, and transcriptional regulation. Although multiple physiological functions of Rif1 have been characterized, biochemical and structural information on mammalian Rif1 is limited, mainly because of difficulties in purifying the full-length protein. Here, we expressed and purified the 2418-amino-acid-long, full-length murine Rif1 as well as its partially truncated variants in human 293T cells. Hydrodynamic analyses indicated that Rif1 forms elongated or extended homo-oligomers in solution, consistent with the presence of a HEAT-type helical repeat segment known to adopt an elongated shape. We also observed that the purified murine Rif1 bound G-quadruplex (G4) DNA with high specificity and affinity, as was previously shown for Rif1 from fission yeast. Both the N-terminal (HEAT-repeat) and C-terminal segments were involved in oligomer formation and specifically bound G4 DNA, and the central intrinsically disordered polypeptide segment increased the affinity for G4. Of note, pulldown assays revealed that Rif1 simultaneously binds multiple G4 molecules. Our findings support a model in which Rif1 modulates chromatin loop structures through binding to multiple G4 assemblies and by holding chromatin fibers together.

Highlights

Rap1-interacting protein 1 (Rif1) regulates telomere length in budding yeast
Because the addition of a small tag at the N or C terminus did not affect the functions of SpRif1, we assumed that addition of these tags to mouse Rif1 (muRif1) would not affect its functions
Because a C-terminal peptide derived from budding yeast Rif1 (ScRif1) was shown to form a tetramer [27], we first observed hydrodynamic behaviors of the purified muRif1 to analyze its oligomer formation

Summary

Edited by Joel Gottesfeld

Rap1-interacting protein 1 (Rif1) regulates telomere length in budding yeast. Vertebrate Rif contains a long intrinsically disordered polypeptide (more than 1000 amino acids long), which is located between the N- and C-terminal segments and encoded by a single exon Another conserved feature of Rif is the presence of a phosphoprotein phosphatase 1 (PP1)-binding motif, which was shown to be important for suppression of late-firing origins through counteracting Cdc7mediated phosphorylation events [11, 13, 28]. We reported that SpRif binds to G-quadruplex (G4) structures and regulates replication timing over a long distance [6] It has not been known whether G4-binding activity is conserved in Rif from other species. We set out to purify and characterize the full-length and various domains of murine Rif protein to clarify the mechanisms by which Rif regulates chromatin architecture

Results

Discussion

Experimental procedures

Recombinant plasmids

Oligonucleotide name

Preparation of structured oligonucleotides