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1.
Cell Rep ; 40(9): 111297, 2022 08 30.
Article in English | MEDLINE | ID: mdl-36044844

ABSTRACT

A critical determinant of DNA repair pathway choice is REV7, an adaptor that binds to various DNA repair proteins through its C-terminal seatbelt domain. The REV7 seatbelt binds to either REV3, activating translesion synthesis, or to SHLD3, activating non-homologous end joining (NHEJ) repair. Recent studies have identified another REV7 seatbelt-binding protein, CHAMP1 (chromosome alignment-maintaining phosphoprotein 1), though its possible role in DNA repair is unknown. Here, we show that binding of CHAMP1 to REV7 activates homologous recombination (HR) repair. Mechanistically, CHAMP1 binds directly to REV7 and reduces the level of the Shieldin complex, causing an increase in double-strand break end resection. CHAMP1 also interacts with POGZ in a heterochromatin complex further promoting HR repair. Importantly, in human tumors, CHAMP1 overexpression promotes HR, confers poly (ADP-ribose) polymerase inhibitor resistance, and correlates with poor prognosis. Thus, by binding to either SHLD3 or CHAMP1 through its seatbelt, the REV7 protein can promote either NHEJ or HR repair, respectively.


Subject(s)
Cell Cycle Proteins , Chromosomal Proteins, Non-Histone , Mad2 Proteins , Recombinational DNA Repair , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA End-Joining Repair , DNA Repair/genetics , Homologous Recombination , Humans , Mad2 Proteins/metabolism , Phosphoproteins/genetics , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Recombinational DNA Repair/genetics , Transposases/metabolism
2.
Proc Natl Acad Sci U S A ; 117(43): 26795-26803, 2020 10 27.
Article in English | MEDLINE | ID: mdl-33051298

ABSTRACT

The repair of DNA double strand breaks (DSBs) that arise from external mutagenic agents and routine cellular processes is essential for life. DSBs are repaired by two major pathways, homologous recombination (HR) and classical nonhomologous end joining (C-NHEJ). DSB repair pathway choice is largely dictated at the step of 5'-3' DNA end resection, which is promoted during S phase, in part by BRCA1. Opposing end resection is the 53BP1 protein, which recruits the ssDNA-binding REV7-Shieldin complex to favor C-NHEJ repair. We recently identified TRIP13 as a proresection factor that remodels REV7, causing its dissociation from the Shieldin subunit SHLD3. Here, we identify p31comet, a negative regulator of MAD2 and the spindle assembly checkpoint, as an important mediator of the TRIP13-REV7 interaction. p31comet binds to the REV7-Shieldin complex in cells, promotes REV7 inactivation, and causes PARP inhibitor resistance. p31comet also participates in the extraction of REV7 from the chromatin. Furthermore, p31comet can counteract REV7 function in translesion synthesis (TLS) by releasing it from REV3 in the Pol ζ complex. Finally, p31comet, like TRIP13, is overexpressed in many cancers and this correlates with poor prognosis. Thus, we reveal a key player in the regulation of HR and TLS with significant clinical implications.


Subject(s)
ATPases Associated with Diverse Cellular Activities/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Cell Cycle Proteins/metabolism , Mad2 Proteins/metabolism , Nuclear Proteins/metabolism , Recombinational DNA Repair , Cell Line, Tumor , HEK293 Cells , Humans , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/mortality
3.
Cell Cycle ; 19(13): 1565-1575, 2020 07.
Article in English | MEDLINE | ID: mdl-32420796

ABSTRACT

In the past decade, the study of the major DNA double strand break (DSB) repair pathways, homologous recombination (HR) and classical non-homologous end joining (C-NHEJ), has revealed a vast and intricate network of regulation.  The choice between HR and C-NHEJ is largely controlled at the step of DNA end-resection. A pro-C-NHEJ cascade commencing with 53BP1 and culminating in the newly discovered REV7-Shieldin complex impedes end resection and therefore HR. Importantly, loss of any component of this pathway confers PARP inhibitor resistance in BRCA1-deficient cells; hence, their study is of great clinical importance. The newest entrant on the scene of end resection regulation is the ATPase TRIP13 that disables the pro-C-NHEJ cascade by promoting a novel conformational change of the HORMA protein REV7. Here, we tie these new findings and factors with previous research on the regulation of DSB repair and HORMA proteins, and suggest testable hypotheses for how TRIP13 could specifically inactivate REV7-Shieldin to promote HR. We also discuss these biological questions in the context of clinical therapeutics.


Subject(s)
Cell Cycle Proteins/metabolism , Multiprotein Complexes/metabolism , Amino Acid Sequence , Animals , Genomic Instability , Humans , Models, Molecular , Multiprotein Complexes/chemistry , Protein Binding
4.
Nat Cell Biol ; 22(1): 87-96, 2020 01.
Article in English | MEDLINE | ID: mdl-31915374

ABSTRACT

DNA double-strand breaks (DSBs) are repaired through homology-directed repair (HDR) or non-homologous end joining (NHEJ). BRCA1/2-deficient cancer cells cannot perform HDR, conferring sensitivity to poly(ADP-ribose) polymerase inhibitors (PARPi). However, concomitant loss of the pro-NHEJ factors 53BP1, RIF1, REV7-Shieldin (SHLD1-3) or CST-DNA polymerase alpha (Pol-α) in BRCA1-deficient cells restores HDR and PARPi resistance. Here, we identify the TRIP13 ATPase as a negative regulator of REV7. We show that REV7 exists in active 'closed' and inactive 'open' conformations, and TRIP13 catalyses the inactivating conformational change, thereby dissociating REV7-Shieldin to promote HDR. TRIP13 similarly disassembles the REV7-REV3 translesion synthesis (TLS) complex, a component of the Fanconi anaemia pathway, inhibiting error-prone replicative lesion bypass and interstrand crosslink repair. Importantly, TRIP13 overexpression is common in BRCA1-deficient cancers, confers PARPi resistance and correlates with poor prognosis. Thus, TRIP13 emerges as an important regulator of DNA repair pathway choice-promoting HDR, while suppressing NHEJ and TLS.


Subject(s)
ATPases Associated with Diverse Cellular Activities/genetics , BRCA1 Protein/deficiency , Cell Cycle Proteins/genetics , DNA Repair/genetics , Recombinational DNA Repair/genetics , ATPases Associated with Diverse Cellular Activities/drug effects , Cell Cycle Proteins/drug effects , Cell Cycle Proteins/metabolism , DNA Damage/drug effects , DNA End-Joining Repair/genetics , DNA Repair/drug effects , DNA Replication/drug effects , DNA Replication/genetics , Humans , Mad2 Proteins/genetics , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Telomere-Binding Proteins/drug effects , Telomere-Binding Proteins/genetics
5.
Nat Rev Mol Cell Biol ; 17(6): 337-49, 2016 06.
Article in English | MEDLINE | ID: mdl-27145721

ABSTRACT

The Fanconi anaemia pathway repairs DNA interstrand crosslinks (ICLs) in the genome. Our understanding of this complex pathway is still evolving, as new components continue to be identified and new biochemical systems are used to elucidate the molecular steps of repair. The Fanconi anaemia pathway uses components of other known DNA repair processes to achieve proper repair of ICLs. Moreover, Fanconi anaemia proteins have functions in genome maintenance beyond their canonical roles of repairing ICLs. Such functions include the stabilization of replication forks and the regulation of cytokinesis. Thus, Fanconi anaemia proteins are emerging as master regulators of genomic integrity that coordinate several repair processes. Here, we summarize our current understanding of the functions of the Fanconi anaemia pathway in ICL repair, together with an overview of its connections with other repair pathways and its emerging roles in genome maintenance.


Subject(s)
DNA Repair , Fanconi Anemia Complementation Group Proteins/physiology , Fanconi Anemia/genetics , Animals , DNA Damage , DNA Replication , Humans
6.
Trends Biochem Sci ; 40(4): 233-42, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25778614

ABSTRACT

Sumoylation has important roles during DNA damage repair and responses. Recent broad-scope and substrate-based studies have shed light on the regulation and significance of sumoylation during these processes. An emerging paradigm is that sumoylation of many DNA metabolism proteins is controlled by DNA engagement. Such 'on-site modification' can explain low substrate modification levels and has important implications in sumoylation mechanisms and effects. New studies also suggest that sumoylation can regulate a process through an ensemble effect or via major substrates. Additionally, we describe new trends in the functional effects of sumoylation, such as bi-directional changes in biomolecule binding and multilevel coordination with other modifications. These emerging themes and models will stimulate our thinking and research in sumoylation and genome maintenance.


Subject(s)
DNA Repair/physiology , SUMO-1 Protein/metabolism , Animals , DNA Repair/genetics , Humans , Protein Processing, Post-Translational , SUMO-1 Protein/genetics , Sumoylation/genetics , Sumoylation/physiology
7.
Nucleic Acids Res ; 43(5): 2666-77, 2015 Mar 11.
Article in English | MEDLINE | ID: mdl-25690888

ABSTRACT

Many genome maintenance factors have multiple enzymatic activities. In most cases, how their distinct activities functionally relate with each other is unclear. Here we examined the conserved budding yeast Rad5 protein that has both ubiquitin ligase and DNA helicase activities. The Rad5 ubiquitin ligase activity mediates PCNA poly-ubiquitination and subsequently recombination-based DNA lesion tolerance. Interestingly, the ligase domain is embedded in a larger helicase domain comprising seven consensus motifs. How features of the helicase domain influence ligase function is controversial. To clarify this issue, we use genetic, 2D gel and biochemical analyses and show that a Rad5 helicase motif important for ATP binding is also required for PCNA poly-ubiquitination and recombination-based lesion tolerance. We determine that this requirement is due to a previously unrecognized contribution of the motif to the PCNA and ubiquitination enzyme interaction, and not due to its canonical role in supporting helicase activity. We further show that Rad5's helicase-mediated contribution to replication stress survival is separable from recombination. These findings delineate how two Rad5 enzymatic domains concertedly influence PCNA modification, and unveil their discrete contributions to stress tolerance.


Subject(s)
DNA Damage , DNA Helicases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Binding Sites/genetics , DNA Helicases/genetics , DNA Replication/genetics , Electrophoresis, Gel, Two-Dimensional , Immunoblotting , Mutation , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism , Protein Binding , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Sumoylation , Ubiquitin-Protein Ligases/genetics , Ubiquitination
8.
PLoS Genet ; 11(1): e1004899, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25569253

ABSTRACT

Protein modifications regulate both DNA repair levels and pathway choice. How each modification achieves regulatory effects and how different modifications collaborate with each other are important questions to be answered. Here, we show that sumoylation regulates double-strand break repair partly by modifying the end resection factor Sae2. This modification is conserved from yeast to humans, and is induced by DNA damage. We mapped the sumoylation site of Sae2 to a single lysine in its self-association domain. Abolishing Sae2 sumoylation by mutating this lysine to arginine impaired Sae2 function in the processing and repair of multiple types of DNA breaks. We found that Sae2 sumoylation occurs independently of its phosphorylation, and the two modifications act in synergy to increase soluble forms of Sae2. We also provide evidence that sumoylation of the Sae2-binding nuclease, the Mre11-Rad50-Xrs2 complex, further increases end resection. These findings reveal a novel role for sumoylation in DNA repair by regulating the solubility of an end resection factor. They also show that collaboration between different modifications and among multiple substrates leads to a stronger biological effect.


Subject(s)
DNA End-Joining Repair/genetics , DNA Repair/genetics , Endonucleases/genetics , Saccharomyces cerevisiae Proteins/genetics , Sumoylation/genetics , DNA Breaks, Double-Stranded , DNA Damage/genetics , DNA-Binding Proteins/genetics , Endodeoxyribonucleases/genetics , Exodeoxyribonucleases/genetics , Humans , Phosphorylation , Saccharomyces cerevisiae , Solubility
9.
Cell Rep ; 9(1): 143-152, 2014 Oct 09.
Article in English | MEDLINE | ID: mdl-25263559

ABSTRACT

DNA repair scaffolds mediate specific DNA and protein interactions in order to assist repair enzymes in recognizing and removing damaged sequences. Many scaffold proteins are dedicated to repairing a particular type of lesion. Here, we show that the budding yeast Saw1 scaffold is more versatile. It helps cells cope with base lesions and protein-DNA adducts through its known function of recruiting the Rad1-Rad10 nuclease to DNA. In addition, it promotes UV survival via a mechanism mediated by its sumoylation. Saw1 sumoylation favors its interaction with another nuclease Slx1-Slx4, and this SUMO-mediated role is genetically separable from two main UV lesion repair processes. These effects of Saw1 and its sumoylation suggest that Saw1 is a multifunctional scaffold that can facilitate diverse types of DNA repair through its modification and nuclease interactions.


Subject(s)
DNA Damage , DNA Repair , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Endonucleases/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Endonucleases/genetics , Saccharomyces cerevisiae/cytology , Sumoylation , Survival Analysis
10.
Nucleic Acids Res ; 42(10): 6393-404, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24753409

ABSTRACT

The Saccharomyces cerevisiae Rad1-Rad10 complex is a conserved, structure-specific endonuclease important for repairing multiple types of DNA lesions. Upon recruitment to lesion sites, Rad1-Rad10 removes damaged sequences, enabling subsequent gap filling and ligation. Acting at mid-steps of repair, the association and dissociation of Rad1-Rad10 with DNA can influence repair efficiency. We show that genotoxin-enhanced Rad1 sumoylation occurs after the nuclease is recruited to lesion sites. A single lysine outside Rad1's nuclease and Rad10-binding domains is sumoylated in vivo and in vitro. Mutation of this site to arginine abolishes Rad1 sumoylation and impairs Rad1-mediated repair at high doses of DNA damage, but sustains the repair of a single double-stranded break. The timing of Rad1 sumoylation and the phenotype bias toward high lesion loads point to a post-incision role for sumoylation, possibly affecting Rad1 dissociation from DNA. Indeed, biochemical examination shows that sumoylation of Rad1 decreases the complex's affinity for DNA without affecting other protein properties. These findings suggest a model whereby sumoylation of Rad1 promotes its disengagement from DNA after nuclease cleavage, allowing it to efficiently attend to large numbers of DNA lesions.


Subject(s)
DNA Repair Enzymes/metabolism , DNA Repair , DNA/metabolism , Endonucleases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Sumoylation , DNA Damage , DNA Repair Enzymes/chemistry , DNA Repair Enzymes/genetics , Endonucleases/chemistry , Endonucleases/genetics , Intracellular Signaling Peptides and Proteins/physiology , Lysine/metabolism , Mutation , Protein Serine-Threonine Kinases/physiology , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/physiology , Ubiquitin-Protein Ligases/physiology
11.
Nucleic Acids Res ; 41(10): 5341-53, 2013 May 01.
Article in English | MEDLINE | ID: mdl-23571759

ABSTRACT

Non-homologous end-joining (NHEJ) repairs DNA double-strand breaks by tethering and ligating the two DNA ends. The mechanisms regulating NHEJ efficiency and interplay between its components are not fully understood. Here, we identify and characterize the SUMOylation of budding yeast Lif1 protein, which is required for the ligation step in NHEJ. We show that Lif1 SUMOylation occurs throughout the cell cycle and requires the Siz SUMO ligases. Single-strand DNA, but not double-strand DNA or the Lif1 binding partner Nej1, is inhibitory to Lif1 SUMOylation. We identify lysine 301 as the major conjugation site and demonstrate that its replacement with arginine completely abolishes Lif1 SUMOylation in vivo and in vitro. The lif1-K301R mutant cells exhibit increased levels of NHEJ repair compared with wild-type cells throughout the cell cycle. This is likely due to the inhibitory effect of Lif1 SUMOylation on both its self-association and newly observed single-strand DNA binding activity. Taken together, these findings suggest that SUMOylation of Lif1 represents a new regulatory mechanism that downregulates NHEJ in a cell cycle phase-independent manner.


Subject(s)
DNA End-Joining Repair , DNA-Binding Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Sumoylation , DNA/metabolism , DNA Ligase ATP , DNA Ligases/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Lysine/metabolism , Mutation , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Ubiquitin-Protein Ligases/genetics
12.
Nucleic Acids Res ; 40(16): 7831-43, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22705796

ABSTRACT

The Srs2 DNA helicase of Saccharomyces cerevisiae affects recombination in multiple ways. Srs2 not only inhibits recombination at stalled replication forks but also promotes the synthesis-dependent strand annealing (SDSA) pathway of recombination. Both functions of Srs2 are regulated by sumoylation--sumoylated PCNA recruits Srs2 to the replication fork to disfavor recombination, and sumoylation of Srs2 can be inhibitory to SDSA in certain backgrounds. To understand Srs2 function, we characterize the mechanism of its sumoylation in vitro and in vivo. Our data show that Srs2 is sumoylated at three lysines, and its sumoylation is facilitated by the Siz SUMO ligases. We also show that Srs2 binds to SUMO via a C-terminal SUMO-interacting motif (SIM). The SIM region is required for Srs2 sumoylation, likely by binding to SUMO-charged Ubc9. Srs2's SIM also cooperates with an adjacent PCNA-specific interaction site in binding to sumoylated PCNA to ensure the specificity of the interaction. These two functions of Srs2's SIM exhibit a competitive relationship: sumoylation of Srs2 decreases the interaction between the SIM and SUMO-PCNA, and the SUMO-PCNA-SIM interaction disfavors Srs2 sumoylation. Our findings suggest a potential mechanism for the equilibrium of sumoylated and PCNA-bound pools of Srs2 in cells.


Subject(s)
DNA Helicases/chemistry , DNA Helicases/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Sumoylation , Amino Acid Sequence , Lysine/metabolism , Molecular Sequence Data , Proliferating Cell Nuclear Antigen/metabolism , Protein Interaction Domains and Motifs , SUMO-1 Protein/metabolism , Saccharomyces cerevisiae/enzymology , Ubiquitin-Protein Ligases/metabolism
13.
Mol Cell ; 45(3): 422-32, 2012 Feb 10.
Article in English | MEDLINE | ID: mdl-22285753

ABSTRACT

The cellular response to DNA damage employs multiple dynamic protein modifications to exert rapid and adaptable effects. Substantial work has detailed the roles of canonical checkpoint-mediated phosphorylation in this program. Recent studies have also implicated sumoylation in the DNA damage response; however, a systematic view of the contribution of sumoylation to replication and repair and its interplay with checkpoints is lacking. Here, using a biochemical screen in yeast, we establish that DNA damage-induced sumoylation occurs on a large scale. We identify MRX (Mre11-Rad50-Xrs2) as a positive regulator of this induction for a subset of repair targets. In addition, we find that defective sumoylation results in failure to complete replication of a damaged genome and impaired DNA end processing, highlighting the importance of the SUMO-mediated response in genome integrity. We also show that DNA damage-induced sumoylation does not require Mec1 checkpoint signaling, and the presence of both enables optimal DNA damage resistance.


Subject(s)
DNA Repair Enzymes/metabolism , DNA Repair , DNA Replication , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/physiology , Sumoylation , Cell Cycle Checkpoints , DNA Damage , DNA-Binding Proteins/metabolism , Gene Knockout Techniques , Genome, Fungal , Genomic Instability , Intracellular Signaling Peptides and Proteins/genetics , Microbial Viability , Multiprotein Complexes/metabolism , Phosphorylation , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics
14.
Biomolecules ; 2(3): 376-388, 2012 Sep.
Article in English | MEDLINE | ID: mdl-24926426

ABSTRACT

The cellular response to DNA damage involves multiple pathways that work together to promote survival in the face of increased genotoxic lesions. Proteins in these pathways are often posttranslationally modified, either by small groups such as phosphate, or by protein modifiers such as ubiquitin or SUMO. The recent discovery of many more SUMO substrates that are modified at higher levels in damage conditions adds weight to the accumulated evidence suggesting that sumoylation plays an important functional role in the DNA damage response. Here we discuss the significance of DNA damage-induced sumoylation, the effects of sumoylation on repair proteins, sumoylation dynamics, and crosstalk with other posttranslational modifications in the DNA damage response.

15.
Mol Cell ; 35(5): 657-68, 2009 Sep 11.
Article in English | MEDLINE | ID: mdl-19748359

ABSTRACT

The Smc5/6 complex is an evolutionarily conserved chromosomal ATPase required for cell growth and DNA repair. Its Mms21 subunit supports both functions by docking to the arm region of Smc5 and providing SUMO ligase activity. Here, we report the crystal structure of Mms21 in complex with the Smc5 arm. Our structure revealed two distinct structural and functional domains of the Smc5-bound Mms21: its N-terminal half is dedicated to Smc5 binding by forming a helix bundle with a coiled-coil structure of Smc5; its C-terminal half includes the SUMO ligase domain, which adopts a new type of RING E3 structure. Mutagenesis and structural analyses showed that the Mms21-Smc5 interface is required for cell growth and resistance to DNA damage, while the unique Mms21 RING domain confers specificity to the SUMO E2-E3 interaction. Through structure-based dissection of Mms21 functions, our studies establish a framework for understanding its roles in the Smc5/6 complex.


Subject(s)
Cell Cycle Proteins/metabolism , SUMO-1 Protein/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Amino Acid Sequence , Animals , Binding Sites , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Crystallography, X-Ray , DNA Damage , Humans , Models, Molecular , Molecular Sequence Data , Multiprotein Complexes , Mutation , Protein Conformation , Protein Structure, Tertiary , Rats , SUMO-1 Protein/chemistry , SUMO-1 Protein/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Structure-Activity Relationship , Ubiquitin-Activating Enzymes/metabolism , Ubiquitin-Conjugating Enzymes/metabolism
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