PCNA Retention on DNA into G2/M Phase Causes Genome Instability in Cells Lacking Elg1

Acknowledgments We thank Richard Kolodner, Grant Brown, and Daniel Durocher for strains and plasmids. We thank Anne Donaldson, Alexander Lorenz, and Shin-ichiro Hiraga from University of Aberdeen for careful reading of the manuscript. Research in T.K.’s lab is supported by Medical Research Council Career Development Fellowship L019698/1. V.K.G. was supported by Biotechnology and Biological Sciences Research Council grant K006304/1. T.S.T. was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (nos. 23131507 and 25131712).Peer reviewedPublisher PD

Translesion synthesis DNA polymerase η exhibits a specific RNA extension activity and a transcription-associated function

We thank Andres Aguilera for providing the pCYC-LacZ plasmid for the GLRO experiments, and Szilvia Minorits for technical assistance. This work was also supported by grants from the National Research, Development and Innovation Office: GINOP-2.3.2-15-2016-00001 and GINOP-2.3.2-15-2016-00024.Peer reviewedPublisher PD

Def1 Promotes the Degradation of Pol3 for Polymerase Exchange to Occur During DNA-Damage–Induced Mutagenesis in Saccharomyces cerevisiae

The authors would like to thank Mark Hochstrasser for the MHY500 strain and Yasushi Saeki for the proteasome mutant and the corresponding wild-type strains. We also thank Sz. Minorits for technical assistance. This publication was also supported by the Dr. Rollin D. Hotchkiss Foundation. Funding: Wellcome Trust, 070247/Z/03/A. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

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

We thank members of the Donaldson, Kubota, and Lorenz labs for helpful discussion, Sophie Shaw at the University of Aberdeen for data upload to Array Express and Shin-ichiro Hiraga for help with Bioinformatic analysis. This work was supported by BBSRC Grant BB/K006304/1 and Cancer Research UK Programme Award A19059 to ADD, and Wellcome Trust Grant 095062 to TOH. KS was supported by Grant-in-Aid for Scientific Research on Priority Areas (15H05970 and 15K21761) from Ministry of Education, Culture, Sports, Science and Technology, Japan All raw-data files for MNase-Seq and ChIP-Seq data are uploaded to Array Express under accession number: E-MTAB-6985.Peer reviewedPublisher PD

The Zn-Finger of Saccharomyces cerevisiae Rad18 and Its Adjacent Region Mediate Interaction with Rad5

DNA damages that hinder the movement of the replication complex can ultimately lead to cell death. To avoid that, cells possess several DNA damage bypass mechanisms. The Rad18 ubiquitin ligase controls error-free and mutagenic pathways that help the replication complex to bypass DNA lesions by monoubiquitylating PCNA at stalled replication forks. In Saccharomyces cerevisiae, two of the Rad18 governed pathways are activated by monoubiquitylated PCNA and they involve translesion synthesis polymerases, whereas a third pathway needs subsequent polyubiquitylation of the same PCNA residue by another ubiquitin ligase the Rad5 protein, and it employs template switching. The goal of this study was to dissect the regulatory role of the multidomain Rad18 in DNA damage bypass using a structure-function based approach. Investigating deletion and point mutant RAD18 variants in yeast genetic and yeast two-hybrid assays we show that the Zn-finger of Rad18 mediates its interaction with Rad5, and the N-terminal adjacent region is also necessary for Rad5 binding. Moreover, results of the yeast two-hybrid and in vivo ubiquitylation experiments raise the possibility that direct interaction between Rad18 and Rad5 might not be necessary for the function of the Rad5 dependent pathway. The presented data also reveal that yeast Rad18 uses different domains to mediate its association with itself and with Rad5. Our results contribute to better understanding of the complex machinery of DNA damage bypass pathways

Rif1 acts through Protein Phosphatase 1 but independent of replication timing to suppress telomere extension in budding yeast

ACKNOWLEDGEMENTS We thank Katsunori Sugimoto for strains, plasmids and technical advice. We thank Amanda Williams for Illumina NextSeq 500 technical support and advice. Takashi Kubota provided helpful comments on the manuscript. FUNDING Cancer Research UK Programme Award [A19059 to A.D., S.H.]; Wellcome Trust Investigator Award [110064/Z/15/Z to C.A.N., R.H.C.W.]; SULSA PhD Studentship (to S.K.). Funding for open access charge: Cancer Research UK via University of Aberdeen Open Access Fund. Conflict of interest statement. None declared.Peer reviewedPublisher PD

Mutations at the Subunit Interface of Yeast Proliferating Cell Nuclear Antigen Reveal a Versatile Regulatory Domain

Acknowledgments We thank Szilvia Minorits for technical assistance. I.U. conceived and designed the project and wrote the manuscript. All authors participated in designing and performing the experiments, and analyzing the results. The authors declare no competing financial interests. This work was also supported by a grant from the National Research, Development and Innovation Office GINOP-2.3.2-15-2016-00001. Funding: This work was supported by Hungarian Science Foundation Grant OTKA 109521 and National Research Development and Innovation Office GINOP-2.3.2-15-2016-00001. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

DNA-damage–induced mutagenesis is abolished in <i>def1</i> deletion mutants.

<p>Forward mutation rates at the <i>CAN1</i> locus were determined after UV treatment. Where indicated, <i>def1</i> deletion was complemented by wild-type <i>DEF1</i> expressed under the control of the ADH1 promoter on a centromeric plasmid. The standard deviation is indicated above each bar. Experiments were repeated three times.</p

UV-dose–dependent degradation of Pol3.

<p>Cultures of <i>mms2</i> cells were synchronized by α-factor and irradiated with increasing doses of UV, as indicated. After released back to growth, 1 ml of cells was collected at the indicated time points, and cell extracts were analyzed by Western blotting. Anti-HA detected HA-tagged Pol3, and PGK served as a loading control. The level of Pol3 relative to PGK is shown at the bottom of each panel.</p

Model for polymerase exchange at a DNA damage site.

<p>DNA damage stalls the replication complex and triggers the ubiquitylation of PCNA by Rad6–Rad18 at the stalled fork. Monoubiquitylated PCNA promotes TLS, for which to occur first Pol3 is removed from the stalled complex through ubiquitylation-mediated proteasomal degradation, assisted by Def1. A TLS polymerase takes over the place of Pol3, and together with Pol31 and Pol32 carries out lesion bypass. After the deubiquitylation of PCNA, Pol3 regains its place at the replication complex, and normal replication resumes. For simplicity, only half of the replication fork is shown. The DNA damage site on the template strand is marked by a black diamond symbol.</p