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      Replication-Coupled DNA-Protein Crosslink Repair by SPRTN and the Proteasome in Xenopus Egg Extracts

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          Summary

          DNA-protein crosslinks (DPCs) are bulky lesions that interfere with DNA metabolism and therefore threaten genomic integrity. Recent studies implicate the metalloprotease SPRTN in S phase removal of DPCs, but how SPRTN is targeted to DPCs during DNA replication is unknown. Using Xenopus egg extracts that recapitulate replication-coupled DPC proteolysis, we show that DPCs can be degraded by SPRTN or the proteasome, which act as independent DPC proteases. Proteasome recruitment requires DPC polyubiquitylation, which is partially dependent on the ubiquitin ligase activity of TRAIP. In contrast, SPRTN-mediated DPC degradation does not require DPC polyubiquitylation but instead depends on nascent strand extension to within a few nucleotides of the lesion, implying that polymerase stalling at the DPC activates SPRTN on both leading and lagging strand templates. Our results demonstrate that SPRTN and proteasome activities are coupled to DNA replication by distinct mechanisms that promote replication across immovable protein barriers.

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          Highlights

          • The proteasome and SPRTN can each degrade DPCs during DNA replication

          • Efficient DPC ubiquitylation and proteasome targeting requires TRAIP

          • SPRTN can degrade DPCs that are not ubiquitylated

          • SPRTN-mediated DPC degradation is a post-replicative process

          Abstract

          DNA-protein crosslinks (DPCs) pose major threats to genomic integrity. Using a cell-free system, Larsen et al. show that SPRTN and the proteasome operate as independent replication-coupled DPC proteases. The existence of two DPC proteases with distinct modes of action facilitates the degradation of chemically diverse DPCs.

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          Most cited references53

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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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            Geminin, an inhibitor of DNA replication, is degraded during mitosis.

            We describe a novel 25 kDa protein, geminin, which inhibits DNA replication and is degraded during the mitotic phase of the cell cycle. Geminin has a destruction box sequence and is ubiquitinated anaphase-promoting complex (APC) in vitro. In synchronized HeLa cells, geminin is absent during G1 phase, accumulates during S, G2, and M phases, and disappears at the time of the metaphase-anaphase transition. Geminin inhibits DNA replication by preventing the incorporation of MCM complex into prereplication complex (pre-RC). We propose that geminin inhibits DNA replication during S, G2, and M phases and that geminin destruction at the metaphase-anaphase transition permits replication in the succeeding cell cycle.
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              Mechanism of replication-coupled DNA interstrand crosslink repair.

              DNA interstrand crosslinks (ICLs) are toxic DNA lesions whose repair occurs in the S phase of metazoans via an unknown mechanism. Here, we describe a cell-free system based on Xenopus egg extracts that supports ICL repair. During DNA replication of a plasmid containing a site-specific ICL, two replication forks converge on the crosslink. Subsequent lesion bypass involves advance of a nascent leading strand to within one nucleotide of the ICL, followed by incisions, translesion DNA synthesis, and extension of the nascent strand beyond the lesion. Immunodepletion experiments suggest that extension requires DNA polymerase zeta. Ultimately, a significant portion of the input DNA is fully repaired, but not if DNA replication is blocked. Our experiments establish a mechanism for ICL repair that reveals how this process is coupled to DNA replication.
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                Author and article information

                Contributors
                Journal
                Mol Cell
                Mol. Cell
                Molecular Cell
                Cell Press
                1097-2765
                1097-4164
                07 February 2019
                07 February 2019
                : 73
                : 3
                : 574-588.e7
                Affiliations
                [1 ]Faculty of Health and Medical Sciences, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, DK-2200 Copenhagen, Denmark
                [2 ]Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
                [3 ]Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany
                [4 ]Department of Molecular Biotechnology and Systems Biology, Technical University of Kaiserslautern, 67653 Kaiserslautern, Germany
                [5 ]Howard Hughes Medical Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
                Author notes
                []Corresponding author johannes_walter@ 123456hms.harvard.edu
                [∗∗ ]Corresponding author julien.duxin@ 123456cpr.ku.dk
                [6]

                These authors contributed equally

                [7]

                Lead Contact

                Article
                S1097-2765(18)30994-8
                10.1016/j.molcel.2018.11.024
                6375733
                30595436
                906acc89-2e27-444d-b277-2ba734048c75
                © 2018 The Authors

                This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

                History
                : 9 March 2018
                : 20 September 2018
                : 15 November 2018
                Categories
                Article

                Molecular biology
                dna-portein crosslink (dpc),dna replication,dna repair,ubiquitin,proteasome,sprtn,traip,translesion synthesis (tls)

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