Publications by authors named "Johannes C Walter"

88 Publications

Mechanisms of Vertebrate DNA Interstrand Cross-Link Repair.

Annu Rev Biochem 2021 Apr 21. Epub 2021 Apr 21.

Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts 02115, USA; email:

DNA interstrand cross-links (ICLs) covalently connect the two strands of the double helix and are extremely cytotoxic. Defective ICL repair causes the bone marrow failure and cancer predisposition syndrome, Fanconi anemia, and upregulation of repair causes chemotherapy resistance in cancer. The central event in ICL repair involves resolving the cross-link (unhooking). In this review, we discuss the chemical diversity of ICLs generated by exogenous and endogenous agents. We then describe how proliferating and nonproliferating vertebrate cells unhook ICLs. We emphasize fundamentally new unhooking strategies, dramatic progress in the structural analysis of the Fanconi anemia pathway, and insights into how cells govern the choice between different ICL repair pathways. Throughout, we highlight the many gaps that remain in our knowledge of these fascinating DNA repair pathways. Expected final online publication date for the , Volume 90 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
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http://dx.doi.org/10.1146/annurev-biochem-080320-112510DOI Listing
April 2021

Single-strand DNA breaks cause replisome disassembly.

Mol Cell 2021 03 22;81(6):1309-1318.e6. Epub 2021 Jan 22.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Blavatnik Institute, Boston, MA 02115, USA; Howard Hughes Medical Institute, Boston, MA 02115, USA. Electronic address:

DNA damage impedes replication fork progression and threatens genome stability. Upon encounter with most DNA adducts, the replicative CMG helicase (CDC45-MCM2-7-GINS) stalls or uncouples from the point of synthesis, yet eventually resumes replication. However, little is known about the effect on replication of single-strand breaks or "nicks," which are abundant in mammalian cells. Using Xenopus egg extracts, we reveal that CMG collision with a nick in the leading strand template generates a blunt-ended double-strand break (DSB). Moreover, CMG, which encircles the leading strand template, "runs off" the end of the DSB. In contrast, CMG collision with a lagging strand nick generates a broken end with a single-stranded overhang. In this setting, CMG translocates along double-stranded DNA beyond the break and is then ubiquitylated and removed from chromatin by the same pathway used during replication termination. Our results show that nicks are uniquely dangerous DNA lesions that invariably cause replisome disassembly, and they suggest that CMG cannot be stored on dsDNA while cells resolve replication stress.
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http://dx.doi.org/10.1016/j.molcel.2020.12.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7979477PMC
March 2021

The Ubiquitin Ligase TRAIP: Double-Edged Sword at the Replisome.

Trends Cell Biol 2021 Feb 11;31(2):75-85. Epub 2020 Dec 11.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Blavatnik Institute, Boston, MA 02115, USA; Howard Hughes Medical Institute, Cambridge, MA, 02139, USA. Electronic address:

In preparation for cell division, the genome must be copied with high fidelity. However, replisomes often encounter obstacles, including bulky DNA lesions caused by reactive metabolites and chemotherapeutics, as well as stable nucleoprotein complexes. Here, we discuss recent advances in our understanding of TRAIP, a replisome-associated E3 ubiquitin ligase that is mutated in microcephalic primordial dwarfism. In interphase, TRAIP helps replisomes overcome DNA interstrand crosslinks and DNA-protein crosslinks, whereas in mitosis it triggers disassembly of all replisomes that remain on chromatin. We describe a model to explain how TRAIP performs these disparate functions and how they help maintain genome integrity.
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http://dx.doi.org/10.1016/j.tcb.2020.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7856240PMC
February 2021

Publisher Correction: The cooperative action of CSB, CSA, and UVSSA target TFIIH to DNA damage-stalled RNA polymerase II.

Nat Commun 2020 Nov 6;11(1):5734. Epub 2020 Nov 6.

Department of Human Genetics, Leiden University Medical Center, Einthovenweg 20, 2333 ZC, Leiden, the Netherlands.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-020-19643-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7648835PMC
November 2020

The DNA replication fork suppresses CMG unloading from chromatin before termination.

Genes Dev 2020 11 17;34(21-22):1534-1545. Epub 2020 Sep 17.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Blavatnik Institute, Boston, Massachusetts 02115, USA.

When converging replication forks meet during replication termination, the CMG (Cdc45-MCM2-7-GINS) helicase is polyubiquitylated by CRL2 and unloaded from chromatin by the p97 ATPase. Here, we investigate the signal that triggers CMG unloading in egg extracts using single-molecule and ensemble approaches. We show that converging CMGs pass each other and keep translocating at the same speed as before convergence, whereafter they are rapidly and independently unloaded. When CMG unloading is blocked, diverging CMGs do not support DNA synthesis, indicating that after bypass CMGs encounter the nascent lagging strands of the converging fork and then translocate along double-stranded DNA (dsDNA). However, translocation on dsDNA is not required for CMG's removal from chromatin because in the absence of nascent strand synthesis, converging CMGs are still unloaded. Moreover, recombinant CMG added to nuclear extract undergoes ubiquitylation and disassembly in the absence of any DNA, and DNA digestion triggers CMG ubiquitylation at stalled replication forks. Our findings suggest that DNA suppresses CMG ubiquitylation during elongation and that this suppression is relieved when CMGs converge, leading to CMG unloading.
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http://dx.doi.org/10.1101/gad.339739.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7608748PMC
November 2020

The Histone Chaperone FACT Induces Cas9 Multi-turnover Behavior and Modifies Genome Manipulation in Human Cells.

Mol Cell 2020 07 29;79(2):221-233.e5. Epub 2020 Jun 29.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Biology, ETH Zürich, 8093 Zürich, Switzerland. Electronic address:

Cas9 is a prokaryotic RNA-guided DNA endonuclease that binds substrates tightly in vitro but turns over rapidly when used to manipulate genomes in eukaryotic cells. Little is known about the factors responsible for dislodging Cas9 or how they influence genome engineering. Unbiased detection through proximity labeling of transient protein interactions in cell-free Xenopus laevis egg extract identified the dimeric histone chaperone facilitates chromatin transcription (FACT) as an interactor of substrate-bound Cas9. FACT is both necessary and sufficient to displace dCas9, and FACT immunodepletion converts Cas9's activity from multi-turnover to single turnover. In human cells, FACT depletion extends dCas9 residence times, delays genome editing, and alters the balance between indel formation and homology-directed repair. FACT knockdown also increases epigenetic marking by dCas9-based transcriptional effectors with a concomitant enhancement of transcriptional modulation. FACT thus shapes the intrinsic cellular response to Cas9-based genome manipulation most likely by determining Cas9 residence times.
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http://dx.doi.org/10.1016/j.molcel.2020.06.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7398558PMC
July 2020

The cooperative action of CSB, CSA, and UVSSA target TFIIH to DNA damage-stalled RNA polymerase II.

Nat Commun 2020 04 30;11(1):2104. Epub 2020 Apr 30.

Department of Human Genetics, Leiden University Medical Center, Einthovenweg 20, 2333 ZC, Leiden, the Netherlands.

The response to DNA damage-stalled RNA polymerase II (RNAPIIo) involves the assembly of the transcription-coupled repair (TCR) complex on actively transcribed strands. The function of the TCR proteins CSB, CSA and UVSSA and the manner in which the core DNA repair complex, including transcription factor IIH (TFIIH), is recruited are largely unknown. Here, we define the assembly mechanism of the TCR complex in human isogenic knockout cells. We show that TCR is initiated by RNAPIIo-bound CSB, which recruits CSA through a newly identified CSA-interaction motif (CIM). Once recruited, CSA facilitates the association of UVSSA with stalled RNAPIIo. Importantly, we find that UVSSA is the key factor that recruits the TFIIH complex in a manner that is stimulated by CSB and CSA. Together these findings identify a sequential and highly cooperative assembly mechanism of TCR proteins and reveal the mechanism for TFIIH recruitment to DNA damage-stalled RNAPIIo to initiate repair.
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http://dx.doi.org/10.1038/s41467-020-15903-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7192910PMC
April 2020

A new varietal of DNA interstrand crosslink repair.

Cell Res 2020 06;30(6):459-460

Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA, 02115, USA.

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http://dx.doi.org/10.1038/s41422-020-0321-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7264340PMC
June 2020

A Mechanism to Minimize Errors during Non-homologous End Joining.

Mol Cell 2020 03 17;77(5):1080-1091.e8. Epub 2019 Dec 17.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Enzymatic processing of DNA underlies all DNA repair, yet inappropriate DNA processing must be avoided. In vertebrates, double-strand breaks are repaired predominantly by non-homologous end joining (NHEJ), which directly ligates DNA ends. NHEJ has the potential to be highly mutagenic because it uses DNA polymerases, nucleases, and other enzymes that modify incompatible DNA ends to allow their ligation. Using frog egg extracts that recapitulate NHEJ, we show that end processing requires the formation of a "short-range synaptic complex" in which DNA ends are closely aligned in a ligation-competent state. Furthermore, single-molecule imaging directly demonstrates that processing occurs within the short-range complex. This confinement of end processing to a ligation-competent complex ensures that DNA ends undergo ligation as soon as they become compatible, thereby minimizing mutagenesis. Our results illustrate how the coordination of enzymatic catalysis with higher-order structural organization of substrate maximizes the fidelity of DNA repair.
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http://dx.doi.org/10.1016/j.molcel.2019.11.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060804PMC
March 2020

Mitotic CDK Promotes Replisome Disassembly, Fork Breakage, and Complex DNA Rearrangements.

Mol Cell 2019 03;73(5):915-929.e6

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Blavatnik Institute, Boston, MA 02115, USA; Howard Hughes Medical Institute, Boston, MA 02115, USA. Electronic address:

DNA replication errors generate complex chromosomal rearrangements and thereby contribute to tumorigenesis and other human diseases. One mechanism that triggers these errors is mitotic entry before the completion of DNA replication. To address how mitosis might affect DNA replication, we used Xenopus egg extracts. When mitotic CDK (Cyclin B1-CDK1) is used to drive interphase egg extracts into a mitotic state, the replicative CMG (CDC45/MCM2-7/GINS) helicase undergoes ubiquitylation on its MCM7 subunit, dependent on the E3 ubiquitin ligase TRAIP. Whether replisomes have stalled or undergone termination, CMG ubiquitylation is followed by its extraction from chromatin by the CDC48/p97 ATPase. TRAIP-dependent CMG unloading during mitosis is also seen in C. elegans early embryos. At stalled forks, CMG removal results in fork breakage and end joining events involving deletions and templated insertions. Our results identify a mitotic pathway of global replisome disassembly that can trigger replication fork collapse and DNA rearrangements.
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http://dx.doi.org/10.1016/j.molcel.2018.12.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6410736PMC
March 2019

TRAIP is a master regulator of DNA interstrand crosslink repair.

Nature 2019 03 6;567(7747):267-272. Epub 2019 Mar 6.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.

Cells often use multiple pathways to repair the same DNA lesion, and the choice of pathway has substantial implications for the fidelity of genome maintenance. DNA interstrand crosslinks covalently link the two strands of DNA, and thereby block replication and transcription; the cytotoxicity of these crosslinks is exploited for chemotherapy. In Xenopus egg extracts, the collision of replication forks with interstrand crosslinks initiates two distinct repair pathways. NEIL3 glycosylase can cleave the crosslink; however, if this fails, Fanconi anaemia proteins incise the phosphodiester backbone that surrounds the interstrand crosslink, generating a double-strand-break intermediate that is repaired by homologous recombination. It is not known how the simpler NEIL3 pathway is prioritized over the Fanconi anaemia pathway, which can cause genomic rearrangements. Here we show that the E3 ubiquitin ligase TRAIP is required for both pathways. When two replisomes converge at an interstrand crosslink, TRAIP ubiquitylates the replicative DNA helicase CMG (the complex of CDC45, MCM2-7 and GINS). Short ubiquitin chains recruit NEIL3 through direct binding, whereas longer chains are required for the unloading of CMG by the p97 ATPase, which enables the Fanconi anaemia pathway. Thus, TRAIP controls the choice between the two known pathways of replication-coupled interstrand-crosslink repair. These results, together with our other recent findings establish TRAIP as a master regulator of CMG unloading and the response of the replisome to obstacles.
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http://dx.doi.org/10.1038/s41586-019-1002-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6417926PMC
March 2019

The CMG Helicase Bypasses DNA-Protein Cross-Links to Facilitate Their Repair.

Cell 2019 01 27;176(1-2):167-181.e21. Epub 2018 Dec 27.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Howard Hughes Medical Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Covalent DNA-protein cross-links (DPCs) impede replication fork progression and threaten genome integrity. Using Xenopus egg extracts, we previously showed that replication fork collision with DPCs causes their proteolysis, followed by translesion DNA synthesis. We show here that when DPC proteolysis is blocked, the replicative DNA helicase CMG (CDC45, MCM2-7, GINS), which travels on the leading strand template, bypasses an intact leading strand DPC. Single-molecule imaging reveals that GINS does not dissociate from CMG during bypass and that CMG slows dramatically after bypass, likely due to uncoupling from the stalled leading strand. The DNA helicase RTEL1 facilitates bypass, apparently by generating single-stranded DNA beyond the DPC. The absence of RTEL1 impairs DPC proteolysis, suggesting that CMG must bypass the DPC to enable proteolysis. Our results suggest a mechanism that prevents inadvertent CMG destruction by DPC proteases, and they reveal CMG's remarkable capacity to overcome obstacles on its translocation strand.
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http://dx.doi.org/10.1016/j.cell.2018.10.053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6475077PMC
January 2019

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

Mol Cell 2019 02 27;73(3):574-588.e7. Epub 2018 Dec 27.

Faculty of Health and Medical Sciences, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, DK-2200 Copenhagen, Denmark. Electronic address:

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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http://dx.doi.org/10.1016/j.molcel.2018.11.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6375733PMC
February 2019

A single XLF dimer bridges DNA ends during nonhomologous end joining.

Nat Struct Mol Biol 2018 09 3;25(9):877-884. Epub 2018 Sep 3.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.

Nonhomologous end joining (NHEJ) is the primary pathway of DNA double-strand-break repair in vertebrate cells, yet how NHEJ factors assemble a synaptic complex that bridges DNA ends remains unclear. To address the role of XRCC4-like factor (XLF) in synaptic-complex assembly, we used single-molecule fluorescence imaging in Xenopus laevis egg extract, a system that efficiently joins DNA ends. We found that a single XLF dimer binds DNA substrates just before the formation of a ligation-competent synaptic complex between DNA ends. The interaction of both globular head domains of the XLF dimer with XRCC4 is required for efficient formation of this synaptic complex. Our results indicate that, in contrast to a model in which filaments of XLF and XRCC4 bridge DNA ends, binding of a single XLF dimer facilitates the assembly of a stoichiometrically well-defined synaptic complex.
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http://dx.doi.org/10.1038/s41594-018-0120-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6128732PMC
September 2018

Replication Fork Reversal during DNA Interstrand Crosslink Repair Requires CMG Unloading.

Cell Rep 2018 06;23(12):3419-3428

Howard Hughes Medical Institute and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

DNA interstrand crosslinks (ICLs) are extremely cytotoxic, but the mechanism of their repair remains incompletely understood. Using Xenopus egg extracts, we previously showed that repair of a cisplatin ICL is triggered when two replication forks converge on the lesion. After CDC45/MCM2-7/GINS (CMG) ubiquitylation and unloading by the p97 segregase, FANCI-FANCD2 promotes DNA incisions by XPF-ERCC1, leading to ICL unhooking. Here, we report that, during this cell-free ICL repair reaction, one of the two converged forks undergoes reversal. Fork reversal fails when CMG unloading is inhibited, but it does not require FANCI-FANCD2. After one fork has undergone reversal, the opposing fork that still abuts the ICL undergoes incisions. Our data show that replication fork reversal at an ICL requires replisome disassembly. We present a revised model of ICL repair that involves a reversed fork intermediate.
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http://dx.doi.org/10.1016/j.celrep.2018.05.061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6086610PMC
June 2018

Extracts for Analysis of DNA Replication in a Nucleus-Free System.

Cold Spring Harb Protoc 2019 03 1;2019(3). Epub 2019 Mar 1.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115;

Frog egg extracts represent a powerful approach with which to dissect molecular mechanisms of vertebrate DNA replication and repair. In the classical approach, sperm chromatin is added to a crude egg lysate to form replication-competent nuclei. We subsequently described a procedure that bypasses the requirement for nuclear assembly in DNA replication. In this method, DNA is first added to a high-speed supernatant (HSS) of egg lysate, which mimics the G phase of the cell cycle in that it supports replication licensing. Subsequent addition of a concentrated nucleoplasmic extract (NPE) leads to replication initiation followed by a single complete round of DNA replication. The advantage of the nucleus-free system is that it supports efficient replication of model DNA templates such as plasmids and lambda DNA that can be modified with specific features such as LacI arrays, DNA protein cross-links, or DNA interstrand cross-links. Here, we describe our current protocol for preparation of HSS and NPE. Methods for their use in DNA replication and repair are described elsewhere.
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http://dx.doi.org/10.1101/pdb.prot097154DOI Listing
March 2019

Ensemble and Single-Molecule Analysis of Non-Homologous End Joining in Frog Egg Extracts.

Methods Enzymol 2017 15;591:233-270. Epub 2017 May 15.

Harvard Medical School, Boston, MA, United States. Electronic address:

Non-homologous end joining (NHEJ) repairs the majority of DNA double-strand breaks in human cells, yet the detailed order of events in this process has remained obscure. Here, we describe how to employ Xenopus laevis egg extract for the study of NHEJ. The egg extract is easy to prepare in large quantities, and it performs efficient end joining that requires the core end joining proteins Ku, DNA-PKcs, XLF, XRCC4, and DNA ligase IV. These factors, along with the rest of the soluble proteome, are present at endogenous concentrations, allowing mechanistic analysis in a system that begins to approximate the complexity of cellular end joining. We describe an ensemble assay that monitors covalent joining of DNA ends and fluorescence assays that detect joining of single pairs of DNA ends. The latter assay discerns at least two discrete intermediates in the bridging of DNA ends.
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http://dx.doi.org/10.1016/bs.mie.2017.03.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6404771PMC
April 2018

Mechanisms of DNA replication termination.

Nat Rev Mol Cell Biol 2017 08 24;18(8):507-516. Epub 2017 May 24.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.

Genome duplication is carried out by pairs of replication forks that assemble at origins of replication and then move in opposite directions. DNA replication ends when converging replication forks meet. During this process, which is known as replication termination, DNA synthesis is completed, the replication machinery is disassembled and daughter molecules are resolved. In this Review, we outline the steps that are likely to be common to replication termination in most organisms, namely, fork convergence, synthesis completion, replisome disassembly and decatenation. We briefly review the mechanism of termination in the bacterium Escherichia coli and in simian virus 40 (SV40) and also focus on recent advances in eukaryotic replication termination. In particular, we discuss the recently discovered E3 ubiquitin ligases that control replisome disassembly in yeast and higher eukaryotes, and how their activity is regulated to avoid genome instability.
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http://dx.doi.org/10.1038/nrm.2017.42DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6386472PMC
August 2017

CRL2 promotes unloading of the vertebrate replisome from chromatin during replication termination.

Genes Dev 2017 02 24;31(3):275-290. Epub 2017 Feb 24.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.

A key event during eukaryotic replication termination is the removal of the CMG helicase from chromatin. CMG unloading involves ubiquitylation of its Mcm7 subunit and the action of the p97 ATPase. Using a proteomic screen in egg extracts, we identified factors that are enriched on chromatin when CMG unloading is blocked. This approach identified the E3 ubiquitin ligase CRL2, a specific p97 complex, other potential regulators of termination, and many replisome components. We show that Mcm7 ubiquitylation and CRL2 binding to chromatin are temporally linked and occur only during replication termination. In the absence of CRL2, Mcm7 is not ubiquitylated, CMG unloading is inhibited, and a large subcomplex of the vertebrate replisome that includes DNA Pol ε is retained on DNA. Our data identify CRL2 as a master regulator of replisome disassembly during vertebrate DNA replication termination.
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http://dx.doi.org/10.1101/gad.291799.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5358724PMC
February 2017

Replication-Dependent Unhooking of DNA Interstrand Cross-Links by the NEIL3 Glycosylase.

Cell 2016 Oct 29;167(2):498-511.e14. Epub 2016 Sep 29.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Howard Hughes Medical Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

During eukaryotic DNA interstrand cross-link (ICL) repair, cross-links are resolved ("unhooked") by nucleolytic incisions surrounding the lesion. In vertebrates, ICL repair is triggered when replication forks collide with the lesion, leading to FANCI-FANCD2-dependent unhooking and formation of a double-strand break (DSB) intermediate. Using Xenopus egg extracts, we describe here a replication-coupled ICL repair pathway that does not require incisions or FANCI-FANCD2. Instead, the ICL is unhooked when one of the two N-glycosyl bonds forming the cross-link is cleaved by the DNA glycosylase NEIL3. Cleavage by NEIL3 is the primary unhooking mechanism for psoralen and abasic site ICLs. When N-glycosyl bond cleavage is prevented, unhooking occurs via FANCI-FANCD2-dependent incisions. In summary, we identify an incision-independent unhooking mechanism that avoids DSB formation and represents the preferred pathway of ICL repair in a vertebrate cell-free system.
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http://dx.doi.org/10.1016/j.cell.2016.09.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5237264PMC
October 2016

Assays to Study Mitotic Centrosome and Spindle Pole Assembly and Regulation.

Methods Mol Biol 2016 ;1413:207-35

Department of Medicine, Harvard Medical School, Boston, MA, USA.

Faithful chromosome segregation during cell division requires proper bipolar spindle assembly and critically depends on spindle pole integrity. In most animal cells, spindle poles form as the result of the concerted action of various factors operating in two independent pathways of microtubule assembly mediated by chromatin/RanGTP and by centrosomes. Mutation or deregulation of a number of spindle pole-organizing proteins has been linked to human diseases, including cancer and microcephaly. Our knowledge on how the spindle pole-organizing factors function at the molecular level and cooperate with one another is still quite limited. As the list of these factors expands, so does the need for the development of experimental approaches to study their function. Cell-free extracts from Xenopus laevis eggs have played an instrumental role in the dissection of the mechanisms of bipolar spindle assembly and have recently allowed the reconstitution of the key steps of the centrosome-driven microtubule nucleation pathway (Joukov et al., Mol Cell 55:578-591, 2014). Here we describe assays to study both centrosome-dependent and centrosome-independent spindle pole formation in Xenopus egg extracts. We also provide experimental procedures for the use of artificial centrosomes, such as microbeads coated with an anti-Aurora A antibody or a recombinant fragment of the Cep192 protein, to model and study centrosome maturation in egg extract. In addition, we detail the protocol for a microtubule regrowth assay that allows assessment of the centrosome-driven spindle microtubule assembly in mammalian cells.
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http://dx.doi.org/10.1007/978-1-4939-3542-0_14DOI Listing
December 2017

Two-Stage Synapsis of DNA Ends during Non-homologous End Joining.

Mol Cell 2016 Mar;61(6):850-8

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Repair of DNA double-strand breaks (DSBs) is essential for genomic stability. The most common DSB repair mechanism in human cells, non-homologous end joining (NHEJ), rejoins broken DNA ends by direct ligation. It remains unclear how components of the NHEJ machinery assemble a synaptic complex that bridges DNA ends. Here, we use single-molecule imaging in a vertebrate cell-free extract to show that synapsis of DNA ends occurs in at least two stages that are controlled by different NHEJ factors. DNA ends are initially tethered in a long-range complex whose formation requires the Ku70/80 heterodimer and the DNA-dependent protein kinase catalytic subunit. The ends are then closely aligned, which requires XLF, a non-catalytic function of XRCC4-LIG4, and DNA-PK activity. These results reveal a structural transition in the synaptic complex that governs alignment of DNA ends. Our approach provides a means of studying physiological DNA DSB repair at single-molecule resolution.
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http://dx.doi.org/10.1016/j.molcel.2016.02.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4799494PMC
March 2016

What is the DNA repair defect underlying Fanconi anemia?

Curr Opin Cell Biol 2015 Dec 11;37:49-60. Epub 2015 Nov 11.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Howard Hughes Medical Institute. Electronic address:

Fanconi anemia (FA) is a rare human genetic disease characterized by bone marrow failure, cancer predisposition, and genomic instability. It has been known for many years that FA patient-derived cells are exquisitely sensitive to DNA interstrand cross-linking agents such as cisplatin and mitomycin C. On this basis, it was widely assumed that failure to repair endogenous interstrand cross-links (ICLs) causes FA, although the endogenous mutagen that generates these lesions remained elusive. Recent genetic evidence now suggests that endogenous aldehydes are the driving force behind FA. Importantly, aldehydes cause a variety of DNA lesions, including ICLs and DNA protein cross-links (DPCs), re-kindling the debate about which DNA lesions cause FA. In this review, we discuss new developments in our understanding of DPC and ICL repair, and how these findings bear on the question of which DNA lesion underlies FA.
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http://dx.doi.org/10.1016/j.ceb.2015.09.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4688103PMC
December 2015

The mechanism of DNA replication termination in vertebrates.

Nature 2015 Sep 31;525(7569):345-50. Epub 2015 Aug 31.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.

Eukaryotic DNA replication terminates when replisomes from adjacent replication origins converge. Termination involves local completion of DNA synthesis, decatenation of daughter molecules and replisome disassembly. Termination has been difficult to study because termination events are generally asynchronous and sequence nonspecific. To overcome these challenges, we paused converging replisomes with a site-specific barrier in Xenopus egg extracts. Upon removal of the barrier, forks underwent synchronous and site-specific termination, allowing mechanistic dissection of this process. We show that DNA synthesis does not slow detectably as forks approach each other, and that leading strands pass each other unhindered before undergoing ligation to downstream lagging strands. Dissociation of the replicative CMG helicase (comprising CDC45, MCM2-7 and GINS) occurs only after the final ligation step, and is not required for completion of DNA synthesis, strongly suggesting that converging CMGs pass one another and dissociate from double-stranded DNA. This termination mechanism allows rapid completion of DNA synthesis while avoiding premature replisome disassembly.
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http://dx.doi.org/10.1038/nature14887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4575634PMC
September 2015

Regulation of the Rev1-pol ζ complex during bypass of a DNA interstrand cross-link.

EMBO J 2015 Jul 12;34(14):1971-85. Epub 2015 Jun 12.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Howard Hughes Medical Institute, Boston, MA, USA

DNA interstrand cross-links (ICLs) are repaired in S phase by a complex, multistep mechanism involving translesion DNA polymerases. After replication forks collide with an ICL, the leading strand approaches to within one nucleotide of the ICL ("approach"), a nucleotide is inserted across from the unhooked lesion ("insertion"), and the leading strand is extended beyond the lesion ("extension"). How DNA polymerases bypass the ICL is incompletely understood. Here, we use repair of a site-specific ICL in Xenopus egg extracts to study the mechanism of lesion bypass. Deep sequencing of ICL repair products showed that the approach and extension steps are largely error-free. However, a short mutagenic tract is introduced in the vicinity of the lesion, with a maximum mutation frequency of ~1%. Our data further suggest that approach is performed by a replicative polymerase, while extension involves a complex of Rev1 and DNA polymerase ζ. Rev1-pol ζ recruitment requires the Fanconi anemia core complex but not FancI-FancD2. Our results begin to illuminate how lesion bypass is integrated with chromosomal DNA replication to limit ICL repair-associated mutagenesis.
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http://dx.doi.org/10.15252/embj.201490878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4547899PMC
July 2015

DNA repair. Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links.

Science 2015 May 30;348(6234):1253671. Epub 2015 Apr 30.

Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany. Novo Nordisk Foundation Center for Protein Research, Proteomics Program, University of Copenhagen, DK-2200 Copenhagen, Denmark.

DNA interstrand cross-links (ICLs) block replication fork progression by inhibiting DNA strand separation. Repair of ICLs requires sequential incisions, translesion DNA synthesis, and homologous recombination, but the full set of factors involved in these transactions remains unknown. We devised a technique called chromatin mass spectrometry (CHROMASS) to study protein recruitment dynamics during perturbed DNA replication in Xenopus egg extracts. Using CHROMASS, we systematically monitored protein assembly and disassembly on ICL-containing chromatin. Among numerous prospective DNA repair factors, we identified SLF1 and SLF2, which form a complex with RAD18 and together define a pathway that suppresses genome instability by recruiting the SMC5/6 cohesion complex to DNA lesions. Our study provides a global analysis of an entire DNA repair pathway and reveals the mechanism of SMC5/6 relocalization to damaged DNA in vertebrate cells.
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http://dx.doi.org/10.1126/science.1253671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5331883PMC
May 2015

Single-Molecule Visualization of MCM2-7 DNA Loading: Seeing Is Believing.

Cell 2015 Apr;161(3):429-430

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Howard Hughes Medical Institute. Electronic address:

The first event in the initiation of eukaryotic DNA replication is the recruitment of the MCM2-7 ATPase, the core of the replicative DNA helicase, to origins. Ticau et al. use single-molecule imaging to reveal how ORC, Cdc6, and Cdt1 cooperate to load MCM2-7 onto DNA, enabling bidirectional replication.
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http://dx.doi.org/10.1016/j.cell.2015.04.006DOI Listing
April 2015

DNA interstrand cross-link repair requires replication-fork convergence.

Nat Struct Mol Biol 2015 Mar 2;22(3):242-7. Epub 2015 Feb 2.

Howard Hughes Medical Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.

DNA interstrand cross-links (ICLs) prevent strand separation during DNA replication and transcription and therefore are extremely cytotoxic. In metazoans, a major pathway of ICL repair is coupled to DNA replication, and it requires the Fanconi anemia pathway. In most current models, collision of a single DNA replication fork with an ICL is sufficient to initiate repair. In contrast, we show here that in Xenopus egg extracts two DNA replication forks must converge on an ICL to trigger repair. When only one fork reaches the ICL, the replicative CMG helicase fails to unload from the stalled fork, and repair is blocked. Arrival of a second fork, even when substantially delayed, rescues repair. We conclude that ICL repair requires a replication-induced X-shaped DNA structure surrounding the lesion, and we speculate on how this requirement helps maintain genomic stability in S phase.
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http://dx.doi.org/10.1038/nsmb.2956DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4351167PMC
March 2015

Repair of a DNA-protein crosslink by replication-coupled proteolysis.

Cell 2014 Oct;159(2):346-57

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Howard Hughes Medical Institute. Electronic address:

DNA-protein crosslinks (DPCs) are caused by environmental, endogenous, and chemotherapeutic agents and pose a severe threat to genome stability. We use Xenopus egg extracts to recapitulate DPC repair in vitro and show that this process is coupled to DNA replication. A DPC on the leading strand template arrests the replisome by stalling the CMG helicase. The DPC is then degraded on DNA, yielding a peptide-DNA adduct that is bypassed by CMG. The leading strand subsequently resumes synthesis, stalls again at the adduct, and then progresses past the adduct using DNA polymerase ζ. A DPC on the lagging strand template only transiently stalls the replisome, but it too is degraded, allowing Okazaki fragment bypass. Our experiments describe a versatile, proteolysis-based mechanism of S phase DPC repair that avoids replication fork collapse.
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http://dx.doi.org/10.1016/j.cell.2014.09.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4229047PMC
October 2014

BRCA1 promotes unloading of the CMG helicase from a stalled DNA replication fork.

Mol Cell 2014 Oct 11;56(1):174-85. Epub 2014 Sep 11.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

The tumor suppressor protein BRCA1 promotes homologous recombination (HR), a high-fidelity mechanism to repair DNA double-strand breaks (DSBs) that arise during normal replication and in response to DNA-damaging agents. Recent genetic experiments indicate that BRCA1 also performs an HR-independent function during the repair of DNA interstrand crosslinks (ICLs). Here we show that BRCA1 is required to unload the CMG helicase complex from chromatin after replication forks collide with an ICL. Eviction of the stalled helicase allows leading strands to be extended toward the ICL, followed by endonucleolytic processing of the crosslink, lesion bypass, and DSB repair. Our results identify BRCA1-dependent helicase unloading as a critical, early event in ICL repair.
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http://dx.doi.org/10.1016/j.molcel.2014.08.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4185004PMC
October 2014