Vertebrate CtIP, and its fission yeast (Ctp1), budding yeast (Sae2) and plant (Com1) orthologs have emerged as key regulatory molecules in cellular responses to DNA double strand breaks (DSBs). By ...modulating the nucleolytic 5′-3′ resection activity of the Mre11/Rad50/Nbs1 (MRN) DSB repair processing and signaling complex, CtIP/Ctp1/Sae2/Com1 is integral to the channeling of DNA double strand breaks through DSB repair by homologous recombination (HR). Nearly two decades since its discovery, emerging new data are defining the molecular underpinnings for CtIP DSB repair regulatory activities. CtIP homologs are largely intrinsically unstructured proteins comprised of expanded regions of low complexity sequence, rather than defined folded domains typical of DNA damage metabolizing enzymes and nucleases. A compact structurally conserved N-terminus forms a functionally critical tetrameric helical dimer of dimers (THDD) region that bridges CtIP oligomers, and is flexibly appended to a conserved C-terminal Sae2-homology DNA binding and DSB repair pathway choice regulatory hub which influences nucleolytic activities of the MRN core nuclease complex. The emerging evidence from structural, biophysical, and biological studies converges on CtIP having functional roles in DSB repair that include: 1) dynamic DNA strand coordination through direct DNA binding and DNA bridging activities, 2) MRN nuclease complex cofactor functions that direct MRN endonucleolytic cleavage of protein-blocked DSB ends and 3) acting as a protein binding hub targeted by the cell cycle regulatory apparatus, which influences CtIP expression and activity via layers of post-translational modifications, protein–protein interactions and DNA binding.
DNA end resection plays a critical function in DNA double-strand break repair pathway choice. Resected DNA ends are refractory to end-joining mechanisms and are instead channeled to homology-directed ...repair. Using biochemical, genetic, and imaging methods, we show that phosphorylation of Saccharomyces cerevisiae Sae2 controls its capacity to promote the Mre11-Rad50-Xrs2 (MRX) nuclease to initiate resection of blocked DNA ends by at least two distinct mechanisms. First, DNA damage and cell cycle-dependent phosphorylation leads to Sae2 tetramerization. Second, and independently, phosphorylation of the conserved C-terminal domain of Sae2 is a prerequisite for its physical interaction with Rad50, which is also crucial to promote the MRX endonuclease. The lack of this interaction explains the phenotype of rad50S mutants defective in the processing of Spo11-bound DNA ends during meiotic recombination. Our results define how phosphorylation controls the initiation of DNA end resection and therefore the choice between the key DNA double-strand break repair mechanisms.
Abstract
The homodimeric PolG2 accessory subunit of the mitochondrial DNA polymerase gamma (Pol γ) enhances DNA binding and processive DNA synthesis by the PolG catalytic subunit. PolG2 also directly ...binds DNA, although the underlying molecular basis and functional significance are unknown. Here, data from Atomic Force Microscopy (AFM) and X-ray structures of PolG2–DNA complexes define dimeric and hexameric PolG2 DNA binding modes. Targeted disruption of PolG2 DNA-binding interfaces impairs processive DNA synthesis without diminishing Pol γ subunit affinities. In addition, a structure-specific DNA-binding role for PolG2 oligomers is supported by X-ray structures and AFM showing that oligomeric PolG2 localizes to DNA crossings and targets forked DNA structures resembling the mitochondrial D-loop. Overall, data indicate that PolG2 DNA binding has both PolG-dependent and -independent functions in mitochondrial DNA replication and maintenance, which provide new insight into molecular defects associated with PolG2 disruption in mitochondrial disease.
Graphical Abstract
Graphical Abstract
The Ctp1 protein in Schizosaccharomyces pombe is essential for DNA double-strand break (DSB) repair by homologous recombination. Fission yeast Ctp1 and its budding yeast (Sae2) and human (CtIP) ...homologs control Mre11–Rad50–Nbs1 nuclease complex activity and harbor DNA-binding and -bridging activities. However, the molecular basis for Ctp1–DNA transactions remains undefined. Here, we report atomic force microscopy (AFM) imaging of S. pombe Ctp1–DNA complexes revealing that Ctp1 polymerizes on dsDNA molecules and forms synaptic filaments that bridge two dsDNA strands. We observed that Ctp1 DNA filaments are typified by an average filament length of ∼180 bp of dsDNA and a Ctp1 tetramer footprint of ∼15 bp. Biochemical results characterizing Ctp1 variants with impaired DNA-binding or -bridging properties were consistent with Ctp1-mediated DNA bridging requiring the intact and correctly folded Ctp1 tetramer. Furthermore, mutations altering Ctp1 oligomerization and DNA bridging in vitro conferred cell sensitivity to DSB-producing agents. Together, these results support an important role for Ctp1-regulated DNA strand coordination required for DNA DSB repair in S. pombe.
A human XRCC4-XLF complex bridges DNA Andres, Sara N; Vergnes, Alexandra; Ristic, Dejan ...
Nucleic acids research,
02/2012, Letnik:
40, Številka:
4
Journal Article
Recenzirano
Odprti dostop
DNA double-strand breaks pose a significant threat to cell survival and must be repaired. In higher eukaryotes, such damage is repaired efficiently by non-homologous end joining (NHEJ). Within this ...pathway, XRCC4 and XLF fulfill key roles required for end joining. Using DNA-binding and -bridging assays, combined with direct visualization, we present evidence for how XRCC4-XLF complexes robustly bridge DNA molecules. This unanticipated, DNA Ligase IV-independent bridging activity by XRCC4-XLF suggests an early role for this complex during end joining, in addition to its more well-established later functions. Mutational analysis of the XRCC4-XLF C-terminal tail regions further identifies specialized functions in complex formation and interaction with DNA and DNA Ligase IV. Based on these data and the crystal structure of an extended protein filament of XRCC4-XLF at 3.94 Å, a model for XRCC4-XLF complex function in NHEJ is presented.
Lsr2 is a small nucleoid-associated protein found throughout the actinobacteria. Lsr2 functions similarly to the well-studied H-NS, in that it preferentially binds AT-rich sequences and represses ...gene expression. In Streptomyces venezuelae, Lsr2 represses the expression of many specialized metabolic clusters, including the chloramphenicol antibiotic biosynthetic gene cluster, and deleting
leads to significant upregulation of chloramphenicol cluster expression. We show here that Lsr2 likely exerts its repressive effects on the chloramphenicol cluster by polymerizing along the chromosome and by bridging sites within and adjacent to the chloramphenicol cluster. CmlR is a known activator of the chloramphenicol cluster, but expression of its associated gene is not upregulated in an
mutant strain. We demonstrate that CmlR is essential for chloramphenicol production, and further reveal that CmlR functions to "countersilence" Lsr2's repressive effects by recruiting RNA polymerase and enhancing transcription, with RNA polymerase effectively clearing bound Lsr2 from the chloramphenicol cluster DNA. Our results provide insight into the interplay between opposing regulatory proteins that govern antibiotic production in
, which could be exploited to maximize the production of bioactive natural products in other systems.
Specialized metabolic clusters in
are the source of many clinically prescribed antibiotics. However, many clusters are not expressed in the laboratory due to repression by the nucleoid-associated protein Lsr2. Understanding how Lsr2 represses cluster expression, and how repression can be alleviated, is key to accessing the metabolic potential of these bacteria. Using the chloramphenicol biosynthetic cluster from Streptomyces venezuelae as a model, we explored the mechanistic basis underlying Lsr2-mediated repression, and activation by the pathway-specific regulator CmlR. Lsr2 polymerized along the chromosome and bridged binding sites located within and outside the cluster, promoting repression. Conversely, CmlR was essential for chloramphenicol production and further functioned to countersilence Lsr2 repression by recruiting RNA polymerase and promoting transcription, ultimately removing Lsr2 polymers from the chromosome. Manipulating the activity of both regulators led to a >130× increase in chloramphenicol levels, suggesting that combinatorial regulatory strategies can be powerful tools for maximizing natural product yields.
The control of Wnt receptor abundance is critical for animal development and to prevent tumorigenesis, but the mechanisms that mediate receptor stabilization remain uncertain. We demonstrate that ...stabilization of the essential Wingless/Wnt receptor Arrow/LRP6 by the evolutionarily conserved Usp46-Uaf1-Wdr20 deubiquitylase complex controls signaling strength in Drosophila. By reducing Arrow ubiquitylation and turnover, the Usp46 complex increases cell surface levels of Arrow and enhances the sensitivity of target cells to stimulation by the Wingless morphogen, thereby increasing the amplitude and spatial range of signaling responses. Usp46 inactivation in Wingless-responding cells destabilizes Arrow, reduces cytoplasmic accumulation of the transcriptional coactivator Armadillo/β-catenin, and attenuates or abolishes Wingless target gene activation, which prevents the concentration-dependent regulation of signaling strength. Consequently, Wingless-dependent developmental patterning and tissue homeostasis are disrupted. These results reveal an evolutionarily conserved mechanism that mediates Wnt/Wingless receptor stabilization and underlies the precise activation of signaling throughout the spatial range of the morphogen gradient.
CSB (Cockayne syndrome group B) and SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent, regulator of chromatin, subfamily A-like 1) are DNA translocases that belong to the SNF2 helicase ...family. They both are enriched at stalled replication forks. While SMARCAL1 is recruited by RPA32 to stalled forks, little is known about whether RPA32 also regulates CSB's association with stalled forks. Here, we report that CSB directly interacts with RPA, at least in part via a RPA32C-interacting motif within the N-terminal region of CSB. Modeling of the CSB-RPA32C interaction suggests that CSB binds the RPA32C surface previously shown to be important for binding of UNG2 and SMARCAL1. We show that this interaction is necessary for promoting fork slowing and fork degradation in BRCA2-deficient cells but dispensable for mediating restart of stalled forks. CSB competes with SMARCAL1 for RPA32 at stalled forks and acts non-redundantly with SMARCAL1 to restrain fork progression in response to mild replication stress. In contrast to CSB stimulated restart of stalled forks, SMARCAL1 inhibits restart of stalled forks in BRCA2-deficient cells, likely by suppressing BIR-mediated repair of collapsed forks. Loss of CSB leads to re-sensitization of SMARCAL1-depleted BRCA2-deficient cells to chemodrugs, underscoring a role of CSB in targeted cancer therapy.
The Xenopus laevis APE2 (apurinic/apyrimidinic endonuclease 2) nuclease participates in 3′-5′ nucleolytic resection of oxidative DNA damage and activation of the ATR-Chk1 DNA damage response (DDR) ...pathway via ill-defined mechanisms. Here we report that APE2 resection activity is regulated by DNA interactions in its Zf-GRF domain, a region sharing high homology with DDR proteins Topoisomerase 3α (TOP3α) and NEIL3 (Nei-like DNA glycosylase 3), as well as transcription and RNA regulatory proteins, such as TTF2 (transcription termination factor 2), TFIIS, and RPB9. Biochemical and NMR results establish the nucleic acid-binding activity of the Zf-GRF domain. Moreover, an APE2 Zf-GRF X-ray structure and small-angle X-ray scattering analyses show that the Zf-GRF fold is typified by a crescent-shaped ssDNA binding claw that is flexibly appended to an APE2 endonuclease/exonuclease/phosphatase (EEP) catalytic core. Structure-guided Zf-GRF mutations impact APE2 DNA binding and 3′-5′ exonuclease processing, and also prevent efficient APE2-dependent RPA recruitment to damaged chromatin and activation of the ATR-Chk1 DDR pathway in response to oxidative stress in Xenopus egg extracts. Collectively, our data unveil the APE2 Zf-GRF domain as a nucleic acid interaction module in the regulation of a key single-strand break resection function of APE2, and also reveal topologic similarity of the Zf-GRF to the zinc ribbon domains of TFIIS and RPB9.
Translesion DNA synthesis pathways are necessary to ensure bacterial replication in the presence of DNA damage. Translesion DNA synthesis carried out by the PolV mutasome is well‐studied in ...Escherichia coli, but ~one third of bacteria use a functionally homologous protein complex, consisting of ImuA, ImuB, and ImuC (also called DnaE2). Numerous in vivo studies have shown that all three proteins are required for translesion DNA synthesis and that ImuC is the error‐prone polymerase, but the roles of ImuA and ImuB are unclear. Here we carry out biochemical characterization of ImuA and a truncation of ImuB from Myxococcus xanthus. We find that ImuA is an ATPase, with ATPase activity enhanced in the presence of DNA. The ATPase activity is likely regulated by the C‐terminus, as loss of the ImuA C‐terminus results in DNA‐independent ATP hydrolysis. We also find that ImuA binds a variety of DNA substrates, with DNA binding affinity affected by the addition of ADP or adenylyl‐imidodiphosphate. An ImuB truncation also binds DNA, with lower affinity than ImuA. In the absence of DNA, ImuA directly binds ImuB with moderate affinity. Finally, we show that ImuA and ImuB self‐interact, but that ImuA is predominantly a monomer, while truncated ImuB is a trimer in vitro. Together, with our findings and the current literature in the field, we suggest a model for translesion DNA synthesis, where a trimeric ImuB would provide sufficient binding sites for DNA, the β‐clamp, ImuC, and ImuA, and where ImuA ATPase activity may regulate assembly and disassembly of the translesion DNA synthesis complex.
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