Identification of Elg1 interaction partners and effects on post-replication chromatin re-formation

Abstract

Elg1, the major subunit of a Replication Factor C-like complex, is critical to ensure genomic stability during DNA replication, and is implicated in controlling chromatin structure. We investigated the consequences of Elg1 loss for the dynamics of chromatin re-formation following DNA replication. Measurement of Okazaki fragment length and the micrococcal nuclease sensitivity of newly replicated DNA revealed a defect in nucleosome organization in the absence of Elg1. Using a proteomic approach to identify Elg1 binding partners, we discovered that Elg1 interacts with Rtt106, a histone chaperone implicated in replication-coupled nucleosome assembly that also regulates transcription. A central role for Elg1 is the unloading of PCNA from chromatin following DNA replication, so we examined the relative importance of Rtt106 and PCNA unloading for chromatin reassembly following DNA replication. We find that the major cause of the chromatin organization defects of an ELG1 mutant is PCNA retention on DNA following replication, with Rtt106-Elg1 interaction potentially playing a contributory role.

Fig 1. An elg1Δ mutant shows extended Okazaki fragments after DNA ligase depletion, suggesting a nucleosome organization defect.

A. Outline of the experiment for detection of Okazaki fragment length. B. Flow-cytometry profiles of indicated strains showing progression through S phase. C. Autoradiograph of Okazaki fragments in the strains indicated. Okazaki fragments show extended length in elg1Δ, similar to cac2Δ and unlike ctf18Δ. Dotted lines show Okazaki fragments corresponding to mono- and di-nucleosome sizes. Trace of signal intensity for each lane is shown.

Fig 2. Sensitivity of nascent chromatin in elg1Δ to micrococcal nuclease digestion reveals defective nucleosome assembly.

A. Outline of experiment. Thick grey line indicates the presence of BrdU in the culture medium. B. Budding index (% of budded cells) in WT and elg1Δ indicating synchronous progression through S phase. C. Micrococcal nuclease digestion of chromatin from WT and elg1Δ strains at indicated times after release into S phase. Upper panel: total DNA on agarose gel detected by Ethidium bromide staining. Lower panel: nascent DNA on membrane probed with anti-BrdU antibody. Micrococcal nuclease (MNase) concentrations: 200 and 600 gel units. D. Signal traces of 45 min, 600 gel units MNase concentration lanes from WT and elg1Δ. Blue and green arrows indicate di- and mono-nucleosomal peaks.

Fig 3. Genome-wide MNase-seq analysis of EdU labelled nascent DNA shows defective nucleosome organization in elg1Δ.

A. Outline of the MNase-seq experiment. Thick grey line indicates the presence of EdU in culture medium. B & C. Plots showing protection from MNase of EdU-labelled nascent DNA, aligned to origins of replication (ARS sites) in elg1Δ (B) and cac1Δ (C) compared to WT. Plots in (B) are mean of two biological replicates shown individually in S5 Fig. G1 samples show MNase-digested total DNA. 27.5–60 min samples show MNase-digested nascent DNA recovered by EdU pull down.

Fig 4. Rtt106 is identified as Elg1-binding protein by SILAC-IP.

A. Untagged or Elg1-3FLAG tagged strains were differentially labelled with light or heavy lysine and arginine respectively. Following immunoprecipitation with anti-FLAG antibody, IP samples were analysed by SDS-PAGE followed by SYPRO Ruby staining. B & C. Isotope ratios (Elg1-IP/mock IP) and peptide intensities of the proteins identified by SILAC-IP. Rtt106 is enriched at levels similar to those for the RFC2-5 subunits of Elg1 complex.

Fig 5. Rtt106 interacts with Elg1 but not other RFC like complexes.

A. Confirmation of the interaction between Elg1 and Rtt106 by FLAG-IP and Western blot analysis. Asterisk denotes a degradation product. B. Co-immunoprecipitation experiments showing Rtt106 interacts with Elg1 but not the major subunits of other RFC-like complexes, RFC1 and CTF18. C. Co-immunoprecipitation under different salt concentrations (potassium acetate as indicated) shows interaction of Elg1 with Rtt106 is not mediated by PCNA (also, see S7 Fig). Asterisk denotes degradation product.

Fig 6. Extended Okazaki fragments in the elg1Δ mutant are rescued by disassembly-prone mutants of PCNA.

A. Okazaki fragments are extended less in rtt106Δ mutant than in elg1Δ, suggesting that Elg1 affects Okazaki fragment length independent of Rtt106. B. Disassembly-prone mutant of PCNA (pol30-R14E or pol30-D150E) rescue the Okazaki fragment length extension observed in elg1Δ. Dotted lines show Okazaki fragments corresponding to mono- and di-nucleosome sizes. Trace of signal intensity for each lane is shown.

Fig 7. Model for the role of Elg1 in chromatin re-organization in lagging strand replication.