Cdt1 proteolysis is promoted by dual PIP degrons and is modulated by PCNA ubiquitylation – ScienceOpen
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      Cdt1 proteolysis is promoted by dual PIP degrons and is modulated by PCNA ubiquitylation

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          Abstract

          Cdt1 plays a critical role in DNA replication regulation by controlling licensing. In Metazoa, Cdt1 is regulated by CRL4 Cdt2-mediated ubiquitylation, which is triggered by DNA binding of proliferating cell nuclear antigen (PCNA). We show here that fission yeast Cdt1 interacts with PCNA in vivo and that DNA loading of PCNA is needed for Cdt1 proteolysis after DNA damage and in S phase. Activation of this pathway by ultraviolet (UV)-induced DNA damage requires upstream involvement of nucleotide excision repair or UVDE repair enzymes. Unexpectedly, two non-canonical PCNA-interacting peptide (PIP) motifs, which both have basic residues downstream, function redundantly in Cdt1 proteolysis. Finally, we show that poly-ubiquitylation of PCNA, which occurs after DNA damage, reduces Cdt1 proteolysis. This provides a mechanism for fine-tuning the activity of the CRL4 Cdt2 pathway towards Cdt1, allowing Cdt1 proteolysis to be more efficient in S phase than after DNA damage.

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          Most cited references61

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          RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO.

          The RAD6 pathway is central to post-replicative DNA repair in eukaryotic cells; however, the machinery and its regulation remain poorly understood. Two principal elements of this pathway are the ubiquitin-conjugating enzymes RAD6 and the MMS2-UBC13 heterodimer, which are recruited to chromatin by the RING-finger proteins RAD18 and RAD5, respectively. Here we show that UBC9, a small ubiquitin-related modifier (SUMO)-conjugating enzyme, is also affiliated with this pathway and that proliferating cell nuclear antigen (PCNA) -- a DNA-polymerase sliding clamp involved in DNA synthesis and repair -- is a substrate. PCNA is mono-ubiquitinated through RAD6 and RAD18, modified by lysine-63-linked multi-ubiquitination--which additionally requires MMS2, UBC13 and RAD5--and is conjugated to SUMO by UBC9. All three modifications affect the same lysine residue of PCNA, suggesting that they label PCNA for alternative functions. We demonstrate that these modifications differentially affect resistance to DNA damage, and that damage-induced PCNA ubiquitination is elementary for DNA repair and occurs at the same conserved residue in yeast and humans.
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            PCNA, the maestro of the replication fork.

            Inheritance requires genome duplication, reproduction of chromatin and its epigenetic information, mechanisms to ensure genome integrity, and faithful transmission of the information to progeny. Proliferating cell nuclear antigen (PCNA)-a cofactor of DNA polymerases that encircles DNA-orchestrates several of these functions by recruiting crucial players to the replication fork. Remarkably, many factors that are involved in replication-linked processes interact with a particular face of PCNA and through the same interaction domain, indicating that these interactions do not occur simultaneously during replication. Switching of PCNA partners may be triggered by affinity-driven competition, phosphorylation, proteolysis, and modification of PCNA by ubiquitin and SUMO.
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              DNA replication in eukaryotic cells.

              The maintenance of the eukaryotic genome requires precisely coordinated replication of the entire genome each time a cell divides. To achieve this coordination, eukaryotic cells use an ordered series of steps to form several key protein assemblies at origins of replication. Recent studies have identified many of the protein components of these complexes and the time during the cell cycle they assemble at the origin. Interestingly, despite distinct differences in origin structure, the identity and order of assembly of eukaryotic replication factors is highly conserved across all species. This review describes our current understanding of these events and how they are coordinated with cell cycle progression. We focus on bringing together the results from different organisms to provide a coherent model of the events of initiation. We emphasize recent progress in determining the function of the different replication factors once they have been assembled at the origin.
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                Author and article information

                Journal
                Nucleic Acids Res
                nar
                nar
                Nucleic Acids Research
                Oxford University Press
                0305-1048
                1362-4962
                August 2011
                August 2011
                14 April 2011
                14 April 2011
                : 39
                : 14
                : 5978-5990
                Affiliations
                Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS, UK
                Author notes
                *To whom correspondence should be addressed. Tel: +44 1865 271229; Fax +44 1865 310447; Email: stephen.kearsey@ 123456zoo.ox.ac.uk

                The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors

                Present address: Rachel Deegan, Department of Oncology, Cancer Research UK-Medical Research Council Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, OX3 7DQ, UK.

                Article
                gkr222
                10.1093/nar/gkr222
                3152358
                21493688
                4010a59c-ec26-465e-840d-ac4c10de3363
                © The Author(s) 2011. Published by Oxford University Press.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 15 December 2010
                : 11 March 2011
                : 28 March 2011
                Page count
                Pages: 13
                Categories
                Genome Integrity, Repair and Replication

                Genetics
                Genetics

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