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. 2019 Nov 25;15(11):e1008426.
doi: 10.1371/journal.pgen.1008426. eCollection 2019 Nov.

Sumoylation of the DNA polymerase ε by the Smc5/6 complex contributes to DNA replication

Xiangzhou Meng  1 , Lei Wei  1 , Xiao P Peng  1   2 , Xiaolan Zhao  1
Affiliations

Sumoylation of the DNA polymerase ε by the Smc5/6 complex contributes to DNA replication

Xiangzhou Meng et al. PLoS Genet. .

Abstract

DNA polymerase epsilon (Pol ε) is critical for genome duplication, but little is known about how post-translational modification regulates its function. Here we report that the Pol ε catalytic subunit Pol2 in yeast is sumoylated at a single lysine within a catalytic domain insertion uniquely possessed by Pol2 family members. We found that Pol2 sumoylation occurs specifically in S phase and is increased under conditions of replication fork blockade. Analyses of the genetic requirements of this modification indicate that Pol2 sumoylation is associated with replication fork progression and dependent on the Smc5/6 SUMO ligase known to promote DNA synthesis. Consistently, the pol2 sumoylation mutant phenotype suggests impaired replication progression and increased levels of gross chromosomal rearrangements. Our findings thus indicate a direct role for SUMO in Pol2-mediated DNA synthesis and a molecular basis for Smc5/6-mediated regulation of genome stability.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Pol2 is sumoylated at a lysine residue within an insertion in its catalytic domain.
(A) Schematics of Pol2 protein domains and candidate sumoylation sites. The Pol2 domains depicted include its N-terminal domain (NTD), exonuclease domain (EXO), catalytic domain, and C-terminal structural domain. Eight lysine residues fitting within the sumoylation consensus motif are labeled. (B) Sumoylation of wild-type and mutant Pol2 proteins. TAF-tagged Pol2 was immunoprecipitated and examined by Western blotting using anti-SUMO antibody. In wild-type (WT) cells, the Pol2 sumoylated form (Pol2-S) was detected as a band migrating above the unmodified form (Pol2). Detection of the unmodified form arises from the interaction of the nonspecific region of the antibody with the Protein A (ProA) portion of TAF, as elucidated previously [8]. In each of the pol2 mutant constructs, dots indicate the lysine residues mutated to arginine. (C) Sumoylation of Pol2 is largely abolished by mutating lysine 571, but not lysine 575. Experiments were done as in panel (A), except that HA-tagged Pol2 was examined and immunoblots were first probed with an anti-HA antibody to detect the unmodified form of Pol2. Due to the low level of Pol2 sumoylation, the sumoylated Pol2 form was not visible at the exposure shown when probing with anti-HA antibody but was detectable using anti-SUMO antibody. In all figure panels when proteins were examined, representative Western blots of two or more biological replicates are shown. (D) The pol2 sumoylation mutant does not affect protein levels. Total protein extracts were examined during normal growth (-MMS) and after MMS treatment (+MMS). Stain indicates equal loading levels. (E) Overlay of the catalytic domain structures from the budding yeast Pol2 and Pol3. The crystal structure of Pol2 catalytic domain (cornflower blue) (PDB: 4M8O) is superimposed upon that of Pol3 (light grey) (PDB: 3IAY). DNA is indicated in gold. The insertion containing the Pol2 sumoylation site is colored green and K571 is colored pink. (F) Sequence alignments of the insertion containing the Pol2 sumoylation and adjacent regions among replicative polymerases. These regions from Pol2 (ScPol2) and human POLE (HsPOLE) are boxed blue and absent in the catalytic subunits of DNA polymerase α (ScPol1 and HsPolA) and δ (ScPol3 and HsPolD). Adjacent regions share homology amongst all polymerases. Asterisks and dots label conserved and similar residues, respectively.
Fig 2
Fig 2. Pol2 interacts with the Smc5/6 complex, and its sumoylation is dependent on this complex and Mec1 under genotoxic conditions.
(A) Pol2 sumoylation is induced by HU treatment. Cells were treated with HU (+HU) or without HU (-HU) and examined for Pol2 sumoylation as in Fig 1C. In both situations, pol2-K571R abolished Pol2 sumoylation. (B)-(C) Pol2 sumoylation is abolished by mutation of the Mms21 but not Siz1/2 SUMO E3s. Experiments were performed as described for panel (A). (D) Smc5 and Pol2 associate with each other in vivo. Immunoprecipitation of HA-tagged Pol2 co-purifies Flag-tagged Smc5 in both HU treated and non-treated cells. The control (–) shows that Smc5 exhibits a low level of bead-binding when Pol2 is untagged. (E) Mec1 is required for Pol2 sumoylation under HU and MMS conditions. Experiments were performed as described in panel (A) for HU conditions and in Fig 1C for MMS conditions. The viability of mec1Δ cells was maintained by sml1Δ, which does not affect Mec1-mediated checkpoint functions [27]. (F) A major population of sumoylated Pol2 is associated with chromatin. Left top: Whole cell extract (WCE), chromatin fraction (Chr), and soluble fraction (Sup) were examined by Western blotting. H3 and Pgk1 were used as markers for the chromatin and non-chromatin fractions, respectively. Left bottom: HA-tagged Pol2 was immunoprecipitated from chromatin-bound and soluble fractions and examined as in panel (A). Right: The relative levels of sumoylated vs. unmodified Pol2 in both fractions were plotted based on experiments using two different spore clones for each genotype.
Fig 3
Fig 3. S phase-specific Pol2 sumoylation and its genetic determinants.
(A) The temporal pattern of Pol2 sumoylation throughout the cell cycle. Wild-type cells containing Pol2-HA were arrested in G1 and then released into the cell cycle. Pol2 sumoylation was examined at the timepoints indicated, as in Fig 2A. Cell cycle progression was monitored by flow cytometry (Bottom). (B)-(C) Pol2 sumoylation in normal S phase requires Mms21, but not Mec1. As in panel (A), cells were examined after G1 cells had progressed into S phase. Note that mec1Δ cells contain sml1Δ to sustain viability. (D) Pol2 sumoylation requires the replication initiation factor Dpb11. Cells containing iAID-degron-tagged Dpb11 were analyzed as described for panel (A). As shown previously [56], cell became defective in replication initiation upon the addition of doxycycline (Dox), which turns off Dpb11-iAID expression, and IAA, which degrades Dpb11-iAID fusion proteins. (E) Pol2 sumoylation increases in cells lacking Dpb4, Rrm3, or Mrc1. Experiments were done, and data is presented as described for panel (B). The reduction of Pol2 sumoylation levels was based on experiments using two different spore clones for each genotype.
Fig 4
Fig 4. The pol2-KR sensitization phenotype reflects a defect in replication fork progression.
(A) pol2-KR cells exhibit no overt growth defects or genotoxic sensitivities. Two spore clones of each genotype were examined; cells were spotted in 10-fold serial dilutions on plates containing no drug (YPD) or indicated concentration of drugs. (B) pol2-KR is synthetically sick with dpb2-1. As in panel (A), results of a set of representative spore clones are shown. (C) Examination of Chr XII replication. S phase cells were examined by PFGE followed by Southern blotting with a probe specific to the rDNA locus on Chr XII. The relative levels of Chr XII signals in the gel and in the wells were quantified from two biological duplicates; means and SDs are plotted. Statistical significance is derived by Student’s t-test (* indicates p <0.05; ns = not statistically significant). (D) Cells unable to sumoylate Pol2 harbor shorter telomeres. Genomic DNA was digested using XhoI and PstI and separated on gels. Telomere and the associated 600-bp Y’ sub-telomere fragments were detected using telomere-specific probes. The mid-point of each fragment is indicated by a line and was used to deduce fragment size as marked under the blot (see method) (E) The CPT sensitivity of pol2-KR, dpb2-1 cells is suppressed by RNH1 overexpression. Cells with (+) and without (–) a Gal-inducible RNaseH1 enzyme were spotted at 10-fold serial dilution on plates containing galactose with or without CPT.
Fig 5
Fig 5. pol2-KR increases Rad52 foci and GCR levels in dpb2-1 cells.
(A) Examination of Rad52 foci levels. Cells of the indicated genotypes contained YFP-fused Rad52 at its endogenous locus. Left: Representative YFP and DIC images. White arrows indicate Rad52-YFP foci present in the background of diffuse nuclear Rad52-YFP signals as seen previously [57]. Right: Quantification of the percentage of cells containing Rad52-YFP foci. Statistical significance was determined by Chi-Square test (* indicates p<0.05 and ** indicates p<0.01). (B) Examination of GCR rates. Top: Schematic of the GCR assay as described [58]. Bottom: GCR rates for the strains indicated. Dot plot displays all data points collected for nine to twelve cultures from two biological replicates per genotype. The median and 95% confidence interval were indicated by a horizontal line and errors, respectively. Two-tailed Mann-Whitney test was performed to determine statistical significance. *, p<0.05; ****, p<0.0001.
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