Rad9 Mutants Research Articles

Congenital mirror movements are associated with defective polymerisation of RAD51

BackgroundMirror movements are involuntary movements of one hand that mirror intentional movements of the other hand. Congenital mirror movements (CMM) is a rare genetic disorder with autosomal dominant inheritance, in...

Single-molecule visualization of human RECQ5 interactions with single-stranded DNA recombination intermediates.

RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA). RECQ5 can also translocate on RAD51-coated ssDNA and readily dismantles RAD51–ssDNA filaments. RECQ5 interacts with RAD51 through protein–protein contacts, and disruption of this interface through a RECQ5–F666A mutation reduces translocation velocity by ∼50%. However, RECQ5 readily removes the ATP hydrolysis-deficient mutant RAD51–K133R from ssDNA, suggesting that filament disruption is not coupled to the RAD51 ATP hydrolysis cycle. RECQ5 also readily removes RAD51–I287T, a RAD51 mutant with enhanced ssDNA-binding activity, from ssDNA. Surprisingly, RECQ5 can bind to double-stranded DNA (dsDNA), but it is unable to translocate. Similarly, RECQ5 cannot dismantle RAD51-bound heteroduplex joint molecules. Our results suggest that the roles of RECQ5 in genome maintenance may be regulated in part at the level of substrate specificity.

155 - Loss of PRDX1 increases RAD51 Cys319 oxidation and decreases homologous recombination

Peroxiredoxin 1 (PRDX1) is an oxidative stress protein sensor, which can coordinate cell signaling of its binding partners. Loss of Prdx1 function in mice has been shown to enhance susceptibility to various cancers, including breast cancer. Homologous recombination (HR) is a critical process that enables template directed DNA repair to maintain genomic stability. RAD51 is a critical component of HR, which facilitates strand-exchange to repair DNA double-strand breaks (DSB). The role of PRDX1 in DNA damage and RAD51 directed repair was investigated. Irradiation (IR) of MEFs proficient or deficient of PRDX1 with doses up to 10 Gy found Prdx1-/- MEFs were more sensitive to IR and exhibited reduced homologous recombination in DR-GFP reporter assays. Prdx1-/- MEFs also displayed enhanced DNA damage as measured by nuclear ɣH2AX phosphorylation and reduced RAD51 foci formation following IR. As PRDX1 has been previously identified to bind proteins under heightened oxidative insult, immunoprecipitation assays were conducted, which found PRDX1 directly bound RAD51 with increasing IR. Oxidation of cysteine residues to the sulfenic acid form was specifically probed with DAZ2 and loss of PRDX1 enhanced sulfenylation of RAD51. Site-directed mutagenesis revealed C319 of RAD51 as a primary target of oxidation. Cells harboring overexpression of mutant RAD51 C319 revealed suppression of HR, IR-induced RAD51 foci formation, RAD51 enhanced malignancy in an anchorage-independent growth soft agar colony formation and proliferation. MDA-MB-231 breast cancer cells deficient of PRDX1 had decreased IR-induced RAD51 foci formation and were more sensitive to doxorubicin. In conclusion, PRDX1 binds RAD51 during heighted oxidative conditions and protects the functionally important RAD51 residue C319 from oxidation.

RFWD3-Mediated Ubiquitination Promotes Timely Removal of Both RPA and RAD51 from DNA Damage Sites to Facilitate Homologous Recombination

RFWD3 is a recently identified Fanconi anemia protein FANCW whose E3 ligase activity toward RPA is essential in homologous recombination (HR) repair. However, how RPA ubiquitination promotes HR remained unknown. Here, we identified RAD51, the central HR protein, as another target of RFWD3. We show that RFWD3 polyubiquitinates both RPA and RAD51 invitro and invivo. Phosphorylation by ATR and ATM kinases is required for this activity invivo. RFWD3 inhibits persistent mitomycin C (MMC)-induced RAD51 and RPA foci by promoting VCP/p97-mediated protein dynamics and subsequent degradation. Furthermore, MMC-induced chromatin loading of MCM8 and RAD54 is defective in cells with inactivated RFWD3 or expressing a ubiquitination-deficient mutant RAD51. Collectively, our data reveal a mechanism that facilitates timely removal of RPA and RAD51 from DNA damage sites, which is crucial for progression to the late-phase HR and suppression of the FA phenotype.

Mutant Rad50 enhances killing effects of radiation on nasopharyngeal carcinoma cell line CNE1

Objective: To investigate the killing effects of radiation and mutant Rad50 transfection on human nasopharyngeal carcinoma cell line CNE1. Methods: The experimental groups included: control group, Ad-Rad50-GFP group, Ad-EGFP group, irradiation group, Ad-Rad50-GFP combined with irradiation group, and Ad-EGFP combined with irradiation group. CNE1 cells were transfected with recombinant adenoviral vector Ad-Rad50-GFP carrying mutant Rad50 gene. The expressions of Mre11, Rad50, Nbs1, and relevant constituents composing MRN complex were detected by Western Blot. Neutral comet assay was used to detect the effect of mutant Rad50 on restoration process of DNA damage. Cell growth curve was used to evaluate the growth inhibition of CNE1 by mutant Rad50 and radiation. Results: Expressions of Mre11, Rad50, and Nbs1 in cells of Ad-Rad50-GFP group were less significantly than those in control group when irradiation was completed (0.48 vs 0.62, 0.42 vs 0.5, and 0.53 vs 0.69, respectively, P<0.05) and 24 hours after irradiation (0.41 vs 0.69, 0.46 vs 0.58, and 0.34 vs 0.78, respectively, P<0.05). The mean tail moment (MTM) in Ad-Rad50-GFP plus irradiation group was higher than that in irradiation group when irradiation was completed (16.06 vs 14.8, P<0.05), 24 hours after irradiation (58.23 vs 15.89, P<0.05) and 48 hours after irradiation: (45.12 vs 11.42, P<0.05). Seven days after irradiation, the cells in Ad-Rad50-GFP plus irradiation group was less than those in control group or irradiation group (both P<0.05). Conclusion: Mutant Rad50 enhances killing effects of radiation on nasopharyngeal carcinoma cell line CNE1.

Effects of Rad9 mutants with impaired DNA mismatch repair function on tumorigenesis of colorectal cancer

The aim of this study was to investigate the effects of Rad9 mutants with impaired DNA mismatch repair (MMR) function on the tumorigenesis of colorectal cancer. The colorectal cancer tumor samples were collected from 100 patients. The mutation profiles of human Rad9 (hRad9) gene in these samples were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and sequencing. The plasmid of pFLAG-hRad9 (L101M) was constructed following the QuickChange mutagenesis procedure and transfected into mRad9-deleted mouse cells (mRad9(-/-) cells). The expression of hRad9 protein was measured by western blot analysis. The MMR activity in live cells was detected by flow cytometry using the reporter plasmid for MMR function. Mutation from Leu to Met at the residue 101 (L101M) of hRad9 gene was detected in 7 of the 100 samples. The mismatch repair efficiency of mRad9(-/-)+ L101M cells (mRad9-deleted mouse cells with ectopic expression of L101M hRad9 gene) was (34.0±5.6)%, which was significantly lower than that in the mRad9(-/-)+ hRad9 cells [mRad9-deleted mouse cells with ectopic expression of hRad9 gene, (48.0±7.5)%, P<0.05]. After N-nitroso-N-methylurea (MNU) treatment, the survival rate of mRad9(-/-)+ L101M cells was (33.7±5.9)%, which was significantly higher than that in the mRad9(-/-)+ hRad9 cells [(21.3±4.7)%, P<0.05]. Thus, ectopic expression of L101M hRad9 gene resulted in significantly reduced MMR activity and increased resistance to MNU. Furthermore, ectopic expression of hRad9 gene with mutation at the target residues of post-translational modification in mRad9(-/-) cells also led to a reduced MMR activity. Rad9 mutants with impaired DNA mismatch repair function may promote tumorigenesis of colorectal cancer.

Targeting Rad50 sensitizes human nasopharyngeal carcinoma cells to radiotherapy.

BackgroundThe Mre11-Rad50-Nbs1 (MRN) complex is well known for its crucial role in initiating DNA double strand breaks (DSBs) repair pathways to resistant irradiation (IR) injury and thus facilitating radioresistance which severely reduces radiocurability of nasopharyngeal cancer (NPC). Targeting native cellular MRN function would sensitize NPC cells to IR.MethodsA recombinant adenovirus containing a mutant Rad50 gene (Ad-RAD50) expressing Rad50 zinc hook domain but lacking the ATPase domain and the Mre11 interaction domain was constructed to disrupt native cellular MRN functions. The effects of Ad-RAD50 on the MRN functions were assessed in NPC cells lines using western blot, co-immunoprecipitation and confocal microscopy analyses. The increased radiosensitivity of transient Ad-RAD50 to IR was examined in NPC cells, including MTT assay, colony formation. The molecular mechanisms of radiosensitization were confirmed by neutral comet assay and western bolts. Nude mice subcutaneous injection, tumor growth curve and TUNEL assay were used to evaluate tumor regression and apoptosis in vivo.ResultsRad50 is remarkably upregulated in NPC cells after IR, implying the critical role of Rad50 in MRN functions. The transient expression of this mutant Rad50 decreased the levels of native cellular Rad50, Mre11 and Nbs1, weakened the interactions among these proteins, abrogated the G2/M arrest induced by DSBs and reduced the DNA repair ability in NPC cells. A combination of IR and mutant RAD50 therapy produced significant tumor cytotoxicity in vitro, with a corresponding increase in DNA damage, prevented proliferation and cell viability. Furthermore, Ad-RAD50 sensitized NPC cells to IR by causing dramatic tumor regression and inducing apoptosis in vivo.ConclusionOur findings define a novel therapeutic approach to NPC radiosensitization via targeted native cellular Rad50 disruption.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2190-8) contains supplementary material, which is available to authorized users.

Suppression of Rad leads to arrhythmogenesis via PKA-mediated phosphorylation of ryanodine receptor activity in the heart

Ras-related small G-protein Rad plays a critical role in generating arrhythmias via regulation of the L-type Ca2+ channel (LTCC). The aim was to demonstrate the role of Rad in intracellular calcium homeostasis by cardiac-Specific dominant-negative suppression of Rad. Transgenic (TG) mice overexpressing dominant-negative mutant Rad (S105N Rad TG) were generated. To measure intracellular Ca2+ concentration ([Ca2+]i), we recorded [Ca2+]i transients and Ca2+ sparks from isolated cardiomyocytes using confocal microscopy. The mean [Ca2+]i transient amplitude was significantly increased in S105N Rad TG cardiomyocytes, compared with control littermate mouse cells. The frequency of Ca2+ sparks was also significantly higher in TG cells than in control cells, although there were no significant differences in amplitude. The sarcoplasmic reticulum Ca2+ content was not altered in the S105N Rad TG cells, as assessed by measuring caffeine-induced [Ca2+]i transient. In contrast, phosphorylation of Ser2809 on the cardiac ryanodine receptor (RyR2) was significantly enhanced in TG mouse hearts compared with controls. Additionally, the Rad-mediated RyR2 phosphorylation was regulated via a direct interaction of Rad with protein kinase A (PKA).

Interaction between Rad9-Hus1-Rad1 and TopBP1 activates ATR-ATRIP and promotes TopBP1 recruitment to sites of UV-damage.

The checkpoint clamp Rad9-Hus1-Rad1 (9-1-1) interacts with TopBP1 via two casein kinase 2 (CK2)-phosphorylation sites, Ser-341 and Ser-387 in Rad9. While this interaction is known to be important for the activation of ATR-Chk1 pathway, how the interaction contributes to their accumulation at sites of DNA damage remains controversial. Here, we have studied the contribution of the 9-1-1/TopBP1 interaction to the assembly and activation of checkpoint proteins at damaged DNA. UV-irradiation enhanced association of Rad9 with chromatin and its localization to sites of DNA damage without a direct interaction with TopBP1. TopBP1, as well as RPA and Rad17 facilitated Rad9 recruitment to DNA damage sites. Similar to Rad9, TopBP1 also localized to sites of UV-induced DNA damage. The DNA damage-induced TopBP1 redistribution was delayed in cells expressing a TopBP1 binding-deficient Rad9 mutant. Pharmacological inhibition of ATR recapitulated the delayed accumulation of TopBP1 in the cells, suggesting that ATR activation will induce more efficient accumulation of TopBP1. Taken together, TopBP1 and Rad9 can be independently recruited to damaged DNA. Once recruited, a direct interaction of 9-1-1/TopBP1 occurs and induces ATR activation leading to further TopBP1 accumulation and amplification of the checkpoint signal. Thus, we propose a new positive feedback mechanism that is necessary for successful formation of the damage-sensing complex and DNA damage checkpoint signaling in human cells.

Rad GTPase inhibits the NFκB pathway through interacting with RelA/p65 to impede its DNA binding and target gene transactivation

Rad is a Ras-related small GTPase shown to inhibit cancer cell migration, and its expression is frequently lost in lung cancer cells. Here we provide evidence that Rad can negatively regulate the NFκB pathway. Overexpressing Rad in cells lowered both the basal and TNFα-stimulated transcriptional activity of NFκB. Compared with control cells, Rad-overexpressing cells displayed more cytoplasmic distribution of the NFκB subunit RelA/p65, while Rad-knockdown cells had higher levels of nuclear RelA/p65. Depleting Rad did not affect the kinetics of TNFα-induced IκB degradation, suggesting that Rad-mediated regulation of NFκB was through an IκB-independent mechanism. Expression of a nucleus-localized mutant Rad was sufficient to inhibit the NFκB transcriptional activity, whereas expressing the scaffolding protein 14-3-3γ to retain Rad in the cytoplasm alleviated the suppressive effect of Rad on NFκB. GST pull-down assays showed that Rad could directly bind to RelA/p65, and co-immunoprecipitation demonstrated that the Rad–p65 interaction primarily occurred in the nucleus. Adding Rad-containing nuclear extracts or purified GST-Rad in the electrophoretic mobility shift assays dose-dependently decreased the binding of RelA/p65 to an oligonucleotide probe containing the NFκB response element, suggesting that Rad may directly impede the interaction between RelA/p65 and DNA. Rad depletion altered the expression of an array of NFκB target genes, including upregulating MMP9. Knockdown of Rad expression in cells increased both basal and TNFα-stimulated MMP9 activities and cell invasion. Collectively, our results disclose a novel role of nuclear Rad in inhibiting the NFκB pathway function.

Phosphorylation of Rad9 at Serine 328 by Cyclin A-Cdk2 Triggers Apoptosis via Interfering Bcl-xL

Cyclin A-Cdk2, a cell cycle regulated Ser/Thr kinase, plays important roles in a variety of apoptoticprocesses. However, the mechanism of cyclin A-Cdk2 regulated apoptosis remains unclear. Here, we demonstrated that Rad9, a member of the BH3-only subfamily of Bcl-2 proteins, could be phosphorylated by cyclin A-Cdk2 in vitro and in vivo. Cyclin A-Cdk2 catalyzed the phosphorylation of Rad9 at serine 328 in HeLa cells during apoptosis induced by etoposide, an inhibitor of topoisomeraseII. The phosphorylation of Rad9 resulted in its translocation from the nucleus to the mitochondria and its interaction with Bcl-xL. The forced activation of cyclin A-Cdk2 in these cells by the overexpression of cyclin A,triggered Rad9 phosphorylation at serine 328 and thereby promoted the interaction of Rad9 with Bcl-xL and the subsequent initiation of the apoptotic program. The pro-apoptotic effects regulated by the cyclin A-Cdk2 complex were significantly lower in cells transfected with Rad9S328A, an expression vector that encodes a Rad9 mutant that is resistant to cyclin A-Cdk2 phosphorylation. These findings suggest that cyclin A-Cdk2 regulates apoptosis through a mechanism that involves Rad9phosphorylation.

Expression and regulation of RAD51 mediate cellular responses to chemotherapeutics

There is evidence that RAD51 expression associates with resistance to commonly used chemotherapeutics. Our previous work demonstrated that inhibitors of thymidylate synthase (TS) induced RAD51-dependent homologous recombination (HR), and depleting the RAD51 recombinase sensitized cells to TS inhibitors. In this study, the consequences of RAD51 over-expression were studied. Over-expression of wild-type RAD51 (∼6-fold above endogenous RAD51) conferred resistance to TS inhibitors. In contrast, over-expression of a mutant RAD51 (T309A) that is incapable of being phosphorylated rendered cells more chemosensitive. Moreover, over-expression of the T309A mutant acted in a dominant negative manner over endogenous RAD51 by causing the reduced localization of RAD51 foci following treatment with TS inhibitors. To measure the effect of mutant RAD51 on the cellular response to other DNA damaging chemotherapeutics, the topoisomerase poison etoposide was utilized. Cells over-expressing wild-type RAD51 showed reduced DNA strand breaks, while cells over-expressing the mutant RAD51 showed more than twice as many strand breaks, suggesting that the mutant RAD51 was actively inhibiting strand break resolution. To directly demonstrate an effect on HR, wild-type RAD51 and T309A mutant RAD51 were transiently expressed in HeLa cells that contained an HR reporter construct. HR events provoked by DNA breaks induced by the I-SceI endonuclease increased in cells expressing wild-type RAD51 and decreased in cells expressing the T309A mutant. Collectively, the data suggest that interference with the activation of RAD51-mediated HR represents a potentially useful anticancer target for combination therapies.

An Interaction between the Walker A and D-loop Motifs Is Critical to ATP Hydrolysis and Cooperativity in Bacteriophage T4 Rad50

The ATP binding cassette (ABC) proteins make up a large superfamily with members coming from all kingdoms. The functional form of the ABC protein nucleotide binding domain (NBD) is dimeric with ATP binding sites shared between subunits. The NBD is defined by six motifs: the Walker A, Q-loop, Signature, Walker-B, D-loop, and H-loop. The D-loop contains a conserved aspartate whose function is not clear but has been proposed to be involved in cross-talk between ATP binding sites. Structures of various ABC proteins suggest an interaction between the D-loop aspartate and an asparagine residue located in Walker A loop of the opposing subunit. Here, we evaluate the functional role of the D-loop using a bacteriophage T4 ABC protein, Rad50 (gp46). Mutation of either the D-loop aspartate or the Walker A asparagine results in dramatic reductions in ATP affinity, hydrolysis rate, and cooperativity. The mutant proteins bind Mre11 (gp47) and DNA normally, but no longer support the ATP-dependent nuclease activities of Mre11. We propose that the D-loop aspartate functions to stabilize the Walker A asparagine in a position favorable for catalysis. We find that the asparagine is crucially important to the mechanism of ATP hydrolysis by increasing the affinity for ATP and positioning the γ-phosphate of ATP for catalysis. Additionally, we propose that the asparagine acts as a γ-phosphate sensor and, through its interaction with the conserved D-loop aspartate, transmits conformational changes across the dimer interface to the second ATP binding site.

Analyses of the yeast Rad51 recombinase A265V mutant reveal different in vivo roles of Swi2-like factors

The Saccharomyces cerevisiae Swi2-like factors Rad54 and Rdh54 play multifaceted roles in homologous recombination via their DNA translocase activity. Aside from promoting Rad51-mediated DNA strand invasion of a partner chromatid, Rad54 and Rdh54 can remove Rad51 from duplex DNA for intracellular recycling. Although the in vitro properties of the two proteins are similar, differences between the phenotypes of the null allele mutants suggest that they play different roles in vivo. Through the isolation of a novel RAD51 allele encoding a protein with reduced affinity for DNA, we provide evidence that Rad54 and Rdh54 have different in vivo interactions with Rad51. The mutant Rad51 forms a complex on duplex DNA that is more susceptible to dissociation by Rdh54. This Rad51 variant distinguishes the in vivo functions of Rad54 and Rdh54, leading to the conclusion that two translocases remove Rad51 from different substrates in vivo. Additionally, we show that a third Swi2-like factor, Uls1, contributes toward Rad51 clearance from chromatin in the absence of Rad54 and Rdh54, and define a hierarchy of action of the Swi2-like translocases for chromosome damage repair.

The Overexpression of S105N Mutant Rad Leads Intracellular Ca2+ Overload Via Up-Regulation of Cardiac Ryanodine Receptor Activity

Background: The ras-related small G-protein Rad was originally identified from skeletal muscle of patients with type 2 diabetes mellitus. We have recently reported that Rad plays a critical role in generating arrhythmias. The study was aimed to elucidate the role of Rad in intracellular calcium homeostasis. Methods and Results: We developed the transgenic mice that overexpress S105N mutant Rad driven by α-myosin heavy chain promoter. We measure intracellular Ca concentration ([Ca2+]i) from isolated cardiomyocytes by confocal microscopy. The amplitude of [Ca2+]i transient was significantly increased in S105N-Rad-TG cardiomyocytes, compared with littermate. Furthermore, the Ca2+ sparks and the spontaneous Ca2+ waves occurred with greater frequency in S105N-Rad-TG cells, implicating the enhanced activity of SERCA. We recorded L-type calcium currents (ICa-L) and action potentials (APs) from isolated cardiomyocytes using whole cell patch-clamp technique. The peak ICa-L was dramatically larger in the S105N-Rad-TG cells than in littermate. Early afterdepolarization (EAD) and delayed afterdepolarization (DAD) were frequently observed in S105N-Rad-TG. Then, the phosphorylation of RYR2 at Ser2809 and PKA was significantly enhanced in S105N-Rad-TG by Western blot. Conclusions: Our results provided the first evidence that Rad might regulate RYR2 activity possibly via downstream of PKA signaling pathway.

DDK Phosphorylates Checkpoint Clamp Component Rad9 and Promotes Its Release from Damaged Chromatin

When inappropriate DNA structures arise, they are sensed by DNA structure-dependent checkpoint pathways and subsequently repaired. Recruitment of checkpoint proteins to such structures precedes recruitment of proteins involved in DNA metabolism. Thus, checkpoints can regulate DNA metabolism. We show that fission yeast Rad9, a 9-1-1 heterotrimeric checkpoint-clamp component, is phosphorylated by Hsk1(Cdc7), the Schizosaccharomyces pombeDbf4-dependent kinase (DDK) homolog, in response to replication-induced DNA damage. Phosphorylation of Rad9 disrupts its interaction with replication protein A (RPA) and is dependent on 9-1-1 chromatin loading, the Rad9-associated protein Rad4/Cut5(TopBP1), and prior phosphorylation by Rad3(ATR). rad9 mutants defective in DDK phosphorylation show wild-type checkpoint responses but abnormal DNA repair protein foci and decreased viability after replication stress. We propose that Rad9 phosphorylation by DDK releases Rad9 from DNA damage sites to facilitate DNA repair.

Abstract 1411: A novel molecular approach for head and neck cancer radiosensitization

Abstract Purpose: Current combined external beam radiation therapy (XRT) and chemotherapy have demonstrated as an effective treatment against advanced head and neck cancers. However, combined modality treatments often result in unacceptable levels of toxicity to the patients. It has been well-known that enhanced DNA double strand break (DSB) repairing by Mre11, Rad50 and Nbs1 (MRN) complex is one of the most important mechanisms for developing radioresistance in the tumor cells. The present study investigates if the use of targeted Rad50 disruption could sensitize the tumor cells to XRT for the treatment of human head and neck squamous cell carcinoma (HNSCC). Our goal is to establish a most effective treatment strategy while minimizing XRT morbidity for the HNSCC. Method: Two human HNSCC tumor cell lines, JHU012 and JHU022, and an animal model with human HNSCC were used in this study. A dominant-negative recombinant adenoviral vector containing mutant Rad50 (Ad-Rad50) was constructed. The tumors were treated with Ad-Rad50, replication-defective virus (DL312), or untreated as mock control and a 2-Gy XRT. Cell viability and tumor volume were evaluated to analyze anti-tumor activity of different treatment groups. Neutral comet assay was performed for evaluating DNA DSB damage. Apoptosis in tumor cells was studied by using immunohistochemistry. Result: Our study demonstrates that the transgene expression of mutant Rad50 significantly interrupts the DNA DSB repairing and increases DNA damage in the tumor cells. Increased levels of tumor cell apoptosis were found associated with combined Ad-Rad50 and XRT treatment group. The combined Ad-Rad50 and XRT treatment shows the best anti-tumor result as compared to other groups both in vitro and in vivo. Conclusion: The present study suggests that Ad-Rad50 gene transfer effectively interrupts DNA DSB repairing and results in radiosensitizing in the HNSCC tumor cells treated with XRT. This strategy can be further investigated in the clinic for the treatment of advanced human head and neck cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1411.

Regulation of Rad51 Recombinase Presynaptic Filament Assembly via Interactions with the Rad52 Mediator and the Srs2 Anti-recombinase

Homologous recombination represents an important means for the error-free elimination of DNA double-strand breaks and other deleterious DNA lesions from chromosomes. The Rad51 recombinase, a member of the RAD52 group of recombination proteins, catalyzes the homologous recombination reaction in the context of a helical protein polymer assembled on single-stranded DNA (ssDNA) that is derived from the nucleolytic processing of a primary lesion. The assembly of the Rad51-ssDNA nucleoprotein filament, often referred to as the presynaptic filament, is prone to interference by the single-strand DNA-binding factor replication protein A (RPA). The Saccharomyces cerevisiae Rad52 protein facilitates presynaptic filament assembly by helping to mediate the displacement of RPA from ssDNA. On the other hand, disruption of the presynaptic filament by the Srs2 helicase leads to a net exchange of Rad51 for RPA. To understand the significance of protein-protein interactions in the control of Rad52- or Srs2-mediated presynaptic filament assembly or disassembly, we have examined two rad51 mutants, rad51 Y388H and rad51 G393D, that are simultaneously ablated for Rad52 and Srs2 interactions and one, rad51 A320V, that is differentially inactivated for Rad52 binding for their biochemical properties and also for functional interactions with Rad52 or Srs2. We show that these mutant rad51 proteins are impervious to the mediator activity of Rad52 or the disruptive function of Srs2 in concordance with their protein interaction defects. Our results thus provide insights into the functional significance of the Rad51-Rad52 and Rad51-Srs2 complexes in the control of presynaptic filament assembly and disassembly. Moreover, our biochemical studies have helped identify A320V as a separation-of-function mutation in Rad51 with regards to a differential ablation of Rad52 interaction.

Molecular disruption of RAD50 sensitizes human tumor cells to cisplatin-based chemotherapy

Platinum-based drugs that induce DNA damage are commonly used first-line chemotherapy agents for testicular, bladder, head and neck, lung, esophageal, stomach, and ovarian cancers. The inherent resistance of tumors to DNA damage often limits the therapeutic efficacy of these agents, such as cisplatin. An enhanced DNA repair and telomere maintenance response by the Mre11/Rad50/Nbs1 (MRN) complex is critical in driving this chemoresistance. We hypothesized therefore that the targeted impairment of native cellular MRN function could sensitize tumor cells to cisplatin. To test this, we designed what we believe to be a novel dominant-negative adenoviral vector containing a mutant RAD50 gene that significantly downregulated MRN expression and markedly disrupted MRN function in human squamous cell carcinoma cells. A combination of cisplatin and mutant RAD50 therapy produced significant tumor cytotoxicity in vitro, with a corresponding increase in DNA damage and telomere shortening. In cisplatin-resistant human squamous cell cancer xenografts in nude mice, this combination therapy caused dramatic tumor regression with increased apoptosis. Our findings suggest the use of targeted RAD50 disruption as what we believe to be a novel chemosensitizing approach for cancer therapy in the context of chemoresistance. This strategy is potentially applicable to several types of malignant tumors that demonstrate chemoresistance and may positively impact the treatment of these patients.

Saccharomyces cerevisiae G2 checkpoint and double‐strand break (DSB) repair genes synergistically suppress the formation of homology‐directed translocations

Checkpoint and DNA repair mutants exhibit more genetic instability compared to wild‐type yeast. We previously observed that G2 checkpoint and rad51 mutants exhibit elevated rates of spontaneous translocations. We measured rates of spontaneous translocations in diploid mutants defective in checkpoint and/or DSB repair genes. Directed translocation events were measured by selecting for His+ recombinants that result from mitotic recombination between two truncated his3 genes. The rate of spontaneous translocations in wild type is 4.4 x 10−8. Whereas rad51 and rad9 mutants exhibited fourfold and sevenfold higher rates of spontaneous translocations, rad51 rad9 double mutants exhibited 39‐fold higher rate. Compared to rad51 rad9, we observed similar rates in rad55 rad9 and rad57 rad9 mutants. While xrs2, rad50, and mre11 mutants exhibited 10‐20 fold higher rates, compared to wild type, xrs2 rad9, rad50 rad9, and mre11 rad9 exhibited similar rates compared to rad51 rad9. Interestingly, rate of spontaneous translocations in mec1‐21, a S phase checkpoint mutant that retains some G2 checkpoint function, was 23‐fold higher than wild type, while rates in mec1‐21 rad51 double mutants were 100‐fold higher. Pulse field gel electrophoresis confirmed that recombinants contain translocations. The results illustrate that both DSB repair pathways and G2 checkpoint pathways suppress translocation frequencies.Grant support: CA70105 from the National Cancer Institute