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Identification of a candidate rad1 subunit for the kinetoplastid 9-1-1 (rad9-hus1-rad1) complex.

ABSTRACT: The trimeric 9-1-1 (Rad9-Hus1-Rad1) complex plays an important role in the eukaryotic DNA damage response by recruiting DNA repair factors and checkpoint mediators to damaged sites. Extensively characterised in mammals and yeast, evidence is now emerging that 9-1-1 function is conserved beyond the relatively narrow evolutionary range of the Opisthokonts. Kinetoplastid Rad9 and Hus1 proteins have been identified and shown to be involved in the DNA damage response but Rad1 has remained elusive. In this study, PSI-BLAST iterative database searching, phylogenetic and structural modeling techniques are used to identify and characterise candidate Rad1 proteins in kinetoplastid organisms.

SUBMITTER: MacNeill SA

PROVIDER: S-EPMC4280517 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Identification of a candidate rad1 subunit for the kinetoplastid 9-1-1 (rad9-hus1-rad1) complex.

MacNeill Stuart A SA

Biology 20141219 4

The trimeric 9-1-1 (Rad9-Hus1-Rad1) complex plays an important role in the eukaryotic DNA damage response by recruiting DNA repair factors and checkpoint mediators to damaged sites. Extensively characterised in mammals and yeast, evidence is now emerging that 9-1-1 function is conserved beyond the relatively narrow evolutionary range of the Opisthokonts. Kinetoplastid Rad9 and Hus1 proteins have been identified and shown to be involved in the DNA damage response but Rad1 has remained elusive. In ...[more]

PMID: 25534152

Similar Datasets

DNA binding by the Rad9A subunit of the Rad9-Rad1-Hus1 complex.

Project description:The Rad9-Rad1-Hus1 checkpoint clamp activates the DNA damage response and promotes DNA repair. DNA loading on the central channel of the Rad9-Rad1-Hus1 complex is required to execute its biological functions. Because Rad9A has the highest DNA affinity among the three subunits, we determined the domains and functional residues of human Rad9A that are critical for DNA interaction. The N-terminal globular domain (residues 1-133) had 3.7-fold better DNA binding affinity than the C-terminal globular domain (residues 134-266) of Rad9A1-266. Rad9A1-266 binds DNA 16-, 60-, and 30-fold better than Rad9A1-133, Rad9A134-266, and Rad9A94-266, respectively, indicating that different regions cooperatively contribute to DNA binding. We show that basic residues including K11, K15, R22, K78, K220, and R223 are important for DNA binding. The reductions on DNA binding of Ala substituted mutants of these basic residues show synergistic effect and are dependent on their residential Rad9A deletion constructs. Interestingly, deletion of a loop (residues 160-163) of Rad9A94-266 weakens DNA binding activity by 4.1-fold as compared to wild-type (WT) Rad9A94-266. Cellular sensitivity to genotoxin of rad9A knockout cells is restored by expressing WT-Rad9Afull. However, rad9A knockout cells expressing Rad9A mutants defective in DNA binding are more sensitive to H2O2 as compared to cells expressing WT-Rad9Afull. Only the rad9A knockout cells expressing loop-deleted Rad9A mutant are more sensitive to hydroxyurea than cells expressing WT-Rad9A. In addition, Rad9A-DNA interaction is required for DNA damage signaling activation. Our results indicate that DNA association by Rad9A is critical for maintaining cell viability and checkpoint activation under stress.

| S-EPMC9359528 | biostudies-literature

Structure and functional implications of the human rad9-hus1-rad1 cell cycle checkpoint complex.

Project description:Cellular DNA lesions are efficiently countered by DNA repair in conjunction with delays in cell cycle progression. Previous studies have demonstrated that Rad9, Hus1, and Rad1 can form a heterotrimeric complex (the 9-1-1 complex) that plays dual roles in cell cycle checkpoint activation and DNA repair in eukaryotic cells. Although the 9-1-1 complex has been proposed to form a toroidal structure similar to proliferating cell nuclear antigen (PCNA), which plays essential roles in DNA replication and repair, the structural basis by which it performs different functions has not been elucidated. Here we report the crystal structure of the human 9-1-1 complex at 3.2 A resolution. The crystal structure, together with biochemical assays, reveals that the interdomain connecting loops (IDC loop) of hRad9, hHus1, and hRad1 are largely divergent, and further cocrystallization study indicates that a PCNA-interacting box (PIP box)-containing peptide derived from hFen1 binds tightly to the interdomain connecting loop of hRad1, providing the molecular basis for the damage repair-specific activity of the 9-1-1 complex in contrast to PCNA. Furthermore, structural comparison with PCNA reveals other unique structural features of the 9-1-1 complex that are proposed to contribute to DNA damage recognition.

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The Rad9-Rad1-Hus1 DNA Repair Clamp is Found in Microsporidia.

Project description:DNA repair is an important component of genome integrity and organisms with reduced repair capabilities tend to accumulate mutations at elevated rates. Microsporidia are intracellular parasites exhibiting high levels of genetic divergence postulated to originate from the lack of several proteins, including the heterotrimeric Rad9-Rad1-Hus1 DNA repair clamp. Microsporidian species from the Encephalitozoonidae have undergone severe streamlining with small genomes coding for about 2,000 proteins. The highly divergent sequences found in Microsporidia render functional inferences difficult such that roughly half of these 2,000 proteins have no known function. Using a structural homology-based annotation approach combining protein structure prediction and tridimensional similarity searches, we found that the Rad9-Rad1-Hus1 DNA clamp is present in Microsporidia, together with many other components of the DNA repair machinery previously thought to be missing from these organisms. Altogether, our results indicate that the DNA repair machinery is present and likely functional in Microsporidia.

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A structural hinge in eukaryotic MutY homologues mediates catalytic activity and Rad9-Rad1-Hus1 checkpoint complex interactions.

Project description:The DNA glycosylase MutY homologue (MYH or MUTYH) removes adenines misincorporated opposite 8-oxoguanine as part of the base excision repair pathway. Importantly, defects in human MYH (hMYH) activity cause the inherited colorectal cancer syndrome MYH-associated polyposis. A key feature of MYH activity is its coordination with cell cycle checkpoint via interaction with the Rad9-Rad1-Hus1 (9-1-1) complex. The 9-1-1 complex facilitates cell cycle checkpoint activity and coordinates this activity with ongoing DNA repair. The interdomain connector (IDC, residues 295-350) between the catalytic domain and the 8-oxoguanine recognition domain of hMYH is a critical element that maintains interactions with the 9-1-1 complex. We report the first crystal structure of a eukaryotic MutY protein, a fragment of hMYH (residues 65-350) that consists of the catalytic domain and the IDC. Our structure reveals that the IDC adopts a stabilized conformation projecting away from the catalytic domain to form a docking scaffold for 9-1-1. We further examined the role of the IDC using Schizosaccharomyces pombe MYH as model system. In vitro studies of S. pombe MYH identified residues I261 and E262 of the IDC (equivalent to V315 and E316 of the hMYH IDC) as critical for maintaining the MYH/9-1-1 interaction. We determined that the eukaryotic IDC is also required for DNA damage selection and robust enzymatic activity. Our studies also provide the first evidence that disruption of the MYH/9-1-1 interaction diminishes the repair of oxidative DNA damage in vivo. Thus, preserving the MYH/9-1-1 interaction contributes significantly to minimizing the mutagenic potential of oxidative DNA damage.

| S-EPMC2953589 | biostudies-literature

The human checkpoint sensor Rad9-Rad1-Hus1 interacts with and stimulates NEIL1 glycosylase.

Project description:The checkpoint protein Rad9/Rad1/Hus1 heterotrimer (the 9-1-1 complex) is structurally similar to the proliferating cell nuclear antigen sliding clamp and has been proposed to sense DNA damage that leads to cell cycle arrest or apoptosis. Human (h) NEIL1 DNA glycosylase, an ortholog of bacterial Nei/Fpg, is involved in repairing oxidatively damaged DNA bases. In this study, we show that hNEIL1 interacts with hRad9, hRad1 and hHus1 as individual proteins and as a complex. Residues 290-350 of hNEIL1 are important for the 9-1-1 association. A significant fraction of the hNEIL1 nuclear foci co-localize with hRad9 foci in hydrogen peroxide treated cells. Human NEIL1 DNA glycosylase activity is significantly stimulated by hHus1, hRad1, hRad9 separately and the 9-1-1 complex. Thus, the 9-1-1 complex at the lesion sites serves as both a damage sensor to activate checkpoint control and a component of base excision repair.

| S-EPMC1885643 | biostudies-literature

Association of the Rad9-Rad1-Hus1 checkpoint clamp with MYH DNA glycosylase and DNA.

Project description:Cell cycle checkpoints provide surveillance mechanisms to activate the DNA damage response, thus preserving genomic integrity. The heterotrimeric Rad9-Rad1-Hus1 (9-1-1) clamp is a DNA damage response sensor and can be loaded onto DNA. 9-1-1 is involved in base excision repair (BER) by interacting with nearly every enzyme in BER. Here, we show that individual 9-1-1 components play distinct roles in BER directed by MYH DNA glycosylase. Analyses of Hus1 deletion mutants revealed that the interdomain connecting loop (residues 134-155) is a key determinant of MYH binding. Both the N-(residues 1-146) and C-terminal (residues 147-280) halves of Hus1, which share structural similarity, can interact with and stimulate MYH. The Hus1(K136A) mutant retains physical interaction with MYH but cannot stimulate MYH glycosylase activity. The N-terminal domain, but not the C-terminal half of Hus1 can also bind DNA with moderate affinity. Intact Rad9 expressed in bacteria binds to and stimulates MYH weakly. However, Rad9(1-266) (C-terminal truncated Rad9) can stimulate MYH activity and bind DNA with high affinity, close to that displayed by heterotrimeric 9(1-266)-1-1 complexes. Conversely, Rad1 has minimal roles in stimulating MYH activity or binding to DNA. Finally, we show that preferential recruitment of 9(1-266)-1-1 to 5'-recessed DNA substrates is an intrinsic property of this complex and is dependent on complex formation. Together, our findings provide a mechanistic rationale for unique contributions by individual 9-1-1 subunits to MYH-directed BER based on subunit asymmetry in protein-protein interactions and DNA binding events.

| S-EPMC4458174 | biostudies-literature

Opening pathways of the DNA clamps proliferating cell nuclear antigen and Rad9-Rad1-Hus1.

Project description:Proliferating cell nuclear antigen and the checkpoint clamp Rad9-Rad1-Hus1 topologically encircle DNA and act as mobile platforms in the recruitment of proteins involved in DNA damage response and cell cycle regulation. To fulfill these vital cellular functions, both clamps need to be opened and loaded onto DNA by a clamp loader complex-a process, which involves disruption of the DNA clamp's subunit interfaces. Herein, we compare the relative stabilities of the interfaces using the molecular mechanics Poisson-Boltzmann solvent accessible surface method. We identify the Rad9-Rad1 interface as the weakest and, therefore, most likely to open during clamp loading. We also delineate the dominant interface disruption pathways under external forces in multiple-trajectory steered molecular dynamics runs. We show that, similar to the case of protein folding, clamp opening may not proceed through a single interface breakdown mechanism. Instead, we identify an ensemble of opening pathways, some more prevalent than others, characterized by specific groups of contacts that differentially stabilize the regions of the interface and determine the spatial and temporal patterns of breakdown. In Rad9-Rad1-Hus1, the Rad9-Rad1 and Rad9-Hus1 interfaces share the same dominant unzipping pathway, whereas the Hus1-Rad1 interface is disrupted concertedly with no preferred directionality.

| S-EPMC3905852 | biostudies-literature

The RAD9-RAD1-HUS1 (9.1.1) complex interacts with WRN and is crucial to regulate its response to replication fork stalling.

Project description:The WRN protein belongs to the RecQ family of DNA helicases and is implicated in replication fork restart, but how its function is regulated remains unknown. We show that WRN interacts with the 9.1.1 complex, one of the central factors of the replication checkpoint. This interaction is mediated by the binding of the RAD1 subunit to the N-terminal region of WRN and is instrumental for WRN relocalization in nuclear foci and its phosphorylation in response to replication arrest. We also find that ATR-dependent WRN phosphorylation depends on TopBP1, which is recruited by the 9.1.1 complex in response to replication arrest. Finally, we provide evidence for a cooperation between WRN and 9.1.1 complex in preventing accumulation of DNA breakage and maintaining genome integrity at naturally occurring replication fork stalling sites. Taken together, our data unveil a novel functional interplay between WRN helicase and the replication checkpoint, contributing to shed light into the molecular mechanism underlying the response to replication fork arrest.

| S-EPMC3272477 | biostudies-literature

Physical and functional interactions between MutY glycosylase homologue (MYH) and checkpoint proteins Rad9-Rad1-Hus1.

Project description:The MYH (MutY glycosylase homologue) increases replication fidelity by removing adenines or 2-hydroxyadenine misincorporated opposite GO (7,8-dihydro-8-oxo-guanine). The 9-1-1 complex (Rad9, Rad1 and Hus1 heterotrimer complex) has been suggested as a DNA damage sensor. Here, we report that hMYH (human MYH) interacts with hHus1 (human Hus1) and hRad1 (human Rad1), but not with hRad9. In addition, interactions between MYH and the 9-1-1 complex, from both the fission yeast Schizosaccharomyces pombe and human cells, are partially interchangeable. The major Hus1-binding site is localized to residues 295-350 of hMYH and to residues 245-293 of SpMYH (S. pombe MYH). Val315 of hMYH and Ile261 of SpMYH play important roles for their interactions with Hus1. hHus1 protein and the 9-1-1 complex of S. pombe can enhance the glycosylase activity of SpMYH. Moreover, the interaction of hMYH-hHus1 is enhanced following ionizing radiation. A significant fraction of the hMYH nuclear foci co-localizes with hRad9 foci in H2O2-treated cells. These results reveal that the 9-1-1 complex plays a direct role in base excision repair.

| S-EPMC1635441 | biostudies-literature

Conditional inactivation of the DNA damage response gene Hus1 in mouse testis reveals separable roles for components of the RAD9-RAD1-HUS1 complex in meiotic chromosome maintenance.

Project description:The RAD9-RAD1-HUS1 (9-1-1) complex is a heterotrimeric PCNA-like clamp that responds to DNA damage in somatic cells by promoting DNA repair as well as ATR-dependent DNA damage checkpoint signaling. In yeast, worms, and flies, the 9-1-1 complex is also required for meiotic checkpoint function and efficient completion of meiotic recombination; however, since Rad9, Rad1, and Hus1 are essential genes in mammals, little is known about their functions in mammalian germ cells. In this study, we assessed the meiotic functions of 9-1-1 by analyzing mice with germ cell-specific deletion of Hus1 as well as by examining the localization of RAD9 and RAD1 on meiotic chromosomes during prophase I. Hus1 loss in testicular germ cells resulted in meiotic defects, germ cell depletion, and severely compromised fertility. Hus1-deficient primary spermatocytes exhibited persistent autosomal ?H2AX and RAD51 staining indicative of unrepaired meiotic DSBs, synapsis defects, an extended XY body domain often encompassing partial or whole autosomes, and an increase in structural chromosome abnormalities such as end-to-end X chromosome-autosome fusions and ruptures in the synaptonemal complex. Most of these aberrations persisted in diplotene-stage spermatocytes. Consistent with a role for the 9-1-1 complex in meiotic DSB repair, RAD9 localized to punctate, RAD51-containing foci on meiotic chromosomes in a Hus1-dependent manner. Interestingly, RAD1 had a broader distribution that only partially overlapped with RAD9, and localization of both RAD1 and the ATR activator TOPBP1 to the XY body and to unsynapsed autosomes was intact in Hus1 conditional knockouts. We conclude that mammalian HUS1 acts as a component of the canonical 9-1-1 complex during meiotic prophase I to promote DSB repair and further propose that RAD1 and TOPBP1 respond to unsynapsed chromatin through an alternative mechanism that does not require RAD9 or HUS1.

| S-EPMC3585019 | biostudies-literature

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