Gene for the catalytic subunit of the human DNA-activated protein kinase maps to the site of the XRCC7 gene on chromosome 8.

The DNA-activated serine/threonine protein kinase (DNA-PK) is composed of a large (approximately 460 kDa) catalytic polypeptide (DNA-PKcs) and Ku, a heterodimeric DNA-binding component (p70/p80) that targets DNA-PKcs to DNA. A 41-kbp segment of the DNA-PKcs gene was isolated, and a 7902-bp segment was sequenced. The sequence contains a polymorphic Pvu II restriction enzyme site, and comparing the sequence with that of the cDNA revealed the positions of nine exons. The DNA-PKcs gene was mapped to band q11 of chromosome 8 by in situ hybridization. This location is coincident with that of XRCC7, the gene that complements the DNA double-strand break repair and V(D)J recombination defects (where V is variable, D is diversity, and J is joining) of hamster V3 and murine severe combined immunodeficient (scid) cells.

Aprataxin, a novel protein that protects against genotoxic stress

Ataxia-oculomotor apraxia (AOA1) is a neurological disorder with symptoms that overlap those of ataxia-telangiectasia, a syndrome characterized by abnormal responses to double-strand DNA breaks and genome instability. The gene mutated in AOA1, APTX, is predicted to code for a protein called aprataxin that contains domains of homology with proteins involved in DNA damage signalling and repair. We demonstrate that aprataxin is a nuclear protein, present in both the nucleoplasm and the nucleolus. Mutations in the APTX gene destabilize the aprataxin protein, and fusion constructs of enhanced green fluorescent protein and aprataxin, representing deletions of putative functional domains, generate highly unstable products. Cells from AOA1 patients are characterized by enhanced sensitivity to agents that cause single-strand breaks in DNA but there is no evidence for a gross defect in single-strand break repair. Sensitivity to hydrogen peroxide and the resulting genome instability are corrected by transfection with full-length aprataxin cDNA. We also demonstrate that aprataxin interacts with the repair proteins XRCC1, PARP-1 and p53 and that it co-localizes with XRCC1 along charged particle tracks on chromatin. These results demonstrate that aprataxin influences the cellular response to genotoxic stress very likely by its capacity to interact with a number of proteins involved in DNA repair.

Genetic Analysis of Mitotic Recombination in Saccharomyces cerevisiae

Mitotic genome instability can occur during the repair of double-strand breaks (DSBs) in DNA, which arise from endogenous and exogenous sources. Studying the mechanisms of DNA repair in the budding yeast, Saccharomyces cerevisiae has shown that Homologous Recombination (HR) is a vital repair mechanism for DSBs. HR can result in a crossover event, in which the broken molecule reciprocally exchanges information with a homologous repair template. The current model of double-strand break repair (DSBR) also allows for a tract of information to non-reciprocally transfer from the template molecule to the broken molecule. These “gene conversion” events can vary in size and can occur in conjunction with a crossover event or in isolation. The frequency and size of gene conversions in isolation and gene conversions associated with crossing over has been a source of debate due to the variation in systems used to detect gene conversions and the context in which the gene conversions are measured.

In Chapter 2, I use an unbiased system that measures the frequency and size of gene conversion events, as well as the association of gene conversion events with crossing over between homologs in diploid yeast. We show mitotic gene conversions occur at a rate of 1.3x10-6 per cell division, are either large (median 54.0kb) or small (median 6.4kb), and are associated with crossing over 43% of the time.

DSBs can arise from endogenous cellular processes such as replication and transcription. Two important RNA/DNA hybrids are involved in replication and transcription: R-loops, which form when an RNA transcript base pairs with the DNA template and displaces the non-template DNA strand, and ribonucleotides embedded into DNA (rNMPs), which arise when replicative polymerase errors insert ribonucleotide instead of deoxyribonucleotide triphosphates. RNaseH1 (encoded by RNH1) and RNaseH2 (whose catalytic subunit is encoded by RNH201) both recognize and degrade the RNA in within R-loops while RNaseH2 alone recognizes, nicks, and initiates removal of rNMPs embedded into DNA. Due to their redundant abilities to act on RNA:DNA hybrids, aberrant removal of rNMPs from DNA has been thought to lead to genome instability in an rnh201Δ background.

In Chapter 3, I characterize (1) non-selective genome-wide homologous recombination events and (2) crossing over on chromosome IV in mutants defective in RNaseH1, RNaseH2, or RNaseH1 and RNaseH2. Using a mutant DNA polymerase that incorporates 4-fold fewer rNMPs than wild type, I demonstrate that the primary recombinogenic lesion in the RNaseH2-defective genome is not rNMPs, but rather R-loops. This work suggests different in-vivo roles for RNaseH1 and RNaseH2 in resolving R-loops in yeast and is consistent with R-loops, not rNMPs, being the the likely source of pathology in Aicardi-Goutières Syndrome patients defective in RNaseH2.

Biochemical Characterization of Human Exonuclease 1 Protein-protein Interactions

Human Exonuclease 1 (Exo1) plays important roles in numerous DNA metabolic/repair pathways including DNA mismatch repair, DNA double strand break repair, Okazaki fragment maturation etc. The nuclease activity of Exo1 is tightly regulated in vivo. The regulation of Exo1 in different pathways is achieved by interactions with different protein partners. The focus of this dissertation will be on characterization of Exo1 interactions with traditional protein partners and providing experimental evidences for new Exo1 interactions.

Molecular cloning, biochemical assays, collaborative nuclear magnetic resonance and X-ray crystallography have been employed to study Exo1 interactions with protein partners. This work contains: (i) the experimental evidence for new Exo1 interactions, and (ii) the detailed characterization of Exo1 interactions with PCNA, MLH1 and MutSα/β.

Taken together, the research progress presented in this dissertation further advances our understanding of traditional Exo1 interaction network and probably provides new insights to new functions and new regulations of Exo1.

Phosphorylation of Exo1 modulates homologous recombination repair of DNA double-strand breaks

DNA double-strand break (DSB) repair via the homologous recombination pathway is a multi-stage process, which results in repair of the DSB without loss of genetic information or fidelity. One essential step in this process is the generation of extended single-stranded DNA (ssDNA) regions at the break site. This ssDNA serves to induce cell cycle checkpoints and is required for Rad51 mediated strand invasion of the sister chromatid. Here, we show that human Exonuclease 1 (Exo1) is required for the normal repair of DSBs by HR. Cells depleted of Exo1 show chromosomal instability and hypersensitivity to ionising radiation (IR) exposure. We find that Exo1 accumulates rapidly at DSBs and is required for the recruitment of RPA and Rad51 to sites of DSBs, suggesting a role for Exo1 in ssDNA generation. Interestingly, the phosphorylation of Exo1 by ATM appears to regulate the activity of Exo1 following resection, allowing optimal Rad51 loading and the completion of HR repair. These data establish a role for Exo1 in resection of DSBs in human cells, highlighting the critical requirement of Exo1 for DSB repair via HR and thus the maintenance of genomic stability.

Distinct Roles of FANCO/RAD51C Protein in DNA Damage Signaling and Repair Implications for Fanconi Anemia and Breast Cancer Susceptibility

RAD51C, a RAD51 paralog, has been implicated in homologous recombination (HR), and germ line mutations in RAD51C are known to cause Fanconi anemia (FA)-like disorder and breast and ovarian cancers. The role of RAD51C in the FA pathway of DNA interstrand cross-link (ICL) repair and as a tumor suppressor is obscure. Here, we report that RAD51C deficiency leads to ICL sensitivity, chromatid-type errors, and G(2)/M accumulation, which are hallmarks of the FA phenotype. We find that RAD51C is dispensable for ICL unhooking and FANCD2 monoubiquitination but is essential for HR, confirming the downstream role of RAD51C in ICL repair. Furthermore, we demonstrate that RAD51C plays a vital role in the HR-mediated repair of DNA lesions associated with replication. Finally, we show that RAD51C participates in ICL and double strand break-induced DNA damage signaling and controls intra-S-phase checkpoint through CHK2 activation. Our analyses with pathological mutants of RAD51C that were identified in FA and breast and ovarian cancers reveal that RAD51C regulates HR and DNA damage signaling distinctly. Together, these results unravel the critical role of RAD51C in the FA pathway of ICL repair and as a tumor suppressor.

Comet sensitivity in assessing DNA damage and repair in different cell cycle stages

The comet assay is a sensitive tool for estimation of DNA damage and repair at the cellular level, requiring only a very small number of cells. In comparing the levels of damage or repair in different cell samples, it is possible that small experimental effects could be confounded by different cell cycle states in the samples examined, if sensitivity to DNA damage, and repair capacity, varies with the cell cycle. We assessed this by arresting HeLa cells in various cell cycle stages and then exposing them to ionizing radiation. Unirradiated cells demonstrated significant differences in strand break levels measured by the comet assay (predominantly single-strand breaks) at different cell cycle stages, increasing from G1 into S and falling again in G2. Over and above this variation in endogenous strand break levels, a significant difference in susceptibility to breaks induced by 3.5 Gy ionizing radiation was also evident in different cell cycle phases. Levels of induced DNA damage fluctuate throughout the cycle, with cells in G1 showing slightly lower levels of damage than an asynchronous population. Damage increases as cells progress through S phase before falling again towards the end of S phase and reaching lowest levels in M phase. The results from repair experiments (where cells were allowed to repair for 10 min after exposure to ionizing radiation) also showed differences throughout the cell cycle with G1-phase cells apparently being the most efficient at repair and M-phase cells the least efficient. We suggest, therefore, that in experiments where small differences in DNA damage and repair are to be investigated with the comet assay, it may be desirable to arrest cells in a specific stage of the cell cycle or to allow for differential cycle distribution.

Variations in the Processing of DNA Double-Strand Breaks Along 60-MeV Therapeutic Proton Beams

PURPOSE: To investigate the variations in induction and repair of DNA damage along the proton path, after a previous report on the increasing biological effectiveness along clinically modulated 60-MeV proton beams.

METHODS AND MATERIALS: Human skin fibroblast (AG01522) cells were irradiated along a monoenergetic and a modulated spread-out Bragg peak (SOBP) proton beam used for treating ocular melanoma at the Douglas Cyclotron, Clatterbridge Centre for Oncology, Wirral, Liverpool, United Kingdom. The DNA damage response was studied using the 53BP1 foci formation assay. The linear energy transfer (LET) dependence was studied by irradiating the cells at depths corresponding to entrance, proximal, middle, and distal positions of SOBP and the entrance and peak position for the pristine beam.

RESULTS: A significant amount of persistent foci was observed at the distal end of the SOBP, suggesting complex residual DNA double-strand break damage induction corresponding to the highest LET values achievable by modulated proton beams. Unlike the directly irradiated, medium-sharing bystander cells did not show any significant increase in residual foci.

CONCLUSIONS: The DNA damage response along the proton beam path was similar to the response of X rays, confirming the low-LET quality of the proton exposure. However, at the distal end of SOBP our data indicate an increased complexity of DNA lesions and slower repair kinetics. A lack of significant induction of 53BP1 foci in the bystander cells suggests a minor role of cell signaling for DNA damage under these conditions.

Chk1 phosphorylation of Metnase enhances DNA repair but inhibits replication fork restart

Chk1 both arrests replication forks and enhances repair of DNA damage by phosphorylating downstream effectors. Although there has been a concerted effort to identify effectors of Chk1 activity, underlying mechanisms of effector action are still being identified. Metnase (also called SETMAR) is a SET and transposase domain protein that promotes both DNA double-strand break (DSB) repair and restart of stalled replication forks. In this study, we show that Metnase is phosphorylated only on Ser495 (S495) in vivo in response to DNA damage by ionizing radiation. Chk1 is the major mediator of this phosphorylation event. We had previously shown that wild-type (wt) Metnase associates with chromatin near DSBs and methylates histone H3 Lys36. Here we show that a Ser495Ala (S495A) Metnase mutant, which is not phosphorylated by Chk1, is defective in DSB-induced chromatin association. The S495A mutant also fails to enhance repair of an induced DSB when compared with wt Metnase. Interestingly, the S495A mutant demonstrated increased restart of stalled replication forks compared with wt Metnase. Thus, phosphorylation of Metnase S495 differentiates between these two functions, enhancing DSB repair and repressing replication fork restart. In summary, these data lend insight into the mechanism by which Chk1 enhances repair of DNA damage while at the same time repressing stalled replication fork restart. Oncogene (2012) 31, 4245-4254; doi:10.1038/onc.2011.586; published online 9 January 2012

THE ROLE OF E2F1 IN THE RESPONSE TO DNA DOUBLE STRAND BREAKS

The importance of E2F transcription factors in the processes of proliferation and apoptosis are well established. E2F1, but not other E2F family members, is also phosphorylated and stabilized in response to various forms of DNA damage to regulate the expression of cell cycle and pro-apoptotic genes. E2F1 also relocalizes and forms foci at sites of DNA double-strand breaks but the function of E2F1 at sites of damage is still unknown. Here I reveal that E2F1 deficiency leads to increased spontaneous DNA break and impaired recovery following exposure to ionizing radiation. In response to DNA double-strand breaks, NBS1 phosphorylation and foci formation are defective in cells lacking E2F1, but NBS1 expression levels are unaffected. Moreover, it was observed that an association between NBS1 and E2F1 is increased in response to DNA damage, suggesting that E2F1 may promote NBS1 foci formation through a direct or indirect interaction at sites of DNA breaks. E2F1 deficient cells also display impaired foci formation of RPA and Rad51, which suggests a defect in DNA end resection and formation of single-stranded DNA at DNA double-strand breaks. I also found E2F1 status affects foci formation of the histone acetyltransferase GCN5 in response to DNA double-strand breaks. E2F1 is phosphorylated at serine 31 (serine 29 in mouse) by the ATM kinase as part of the DNA damage response. To investigate the importance of this event, our lab developed an E2F1 serine 29 mutant mouse model. I find that E2F1 serine 29 mutant cells show loss of E2F1 foci formation in response to DNA double-strand breaks. Furthermore, DNA repair and NBS1 foci formation are impaired in E2f1S29A/S29A cells. Taken together, my results indicate novel roles for E2F1 in the DNA damage response, which may directly promote DNA repair and genome maintenance.

hSSB1 rapidly binds at the sites of DNA double-strand breaks and is required for the efficient recruitment of the MRN complex

hSSB1 is a newly discovered single-stranded DNA (ssDNA)-binding protein that is essential for efficient DNA double-strand break signalling through ATM. However, the mechanism by which hSSB1 functions to allow efficient signalling is unknown. Here, we show that hSSB1 is recruited rapidly to sites of double-strand DNA breaks (DSBs) in all interphase cells (G1, S and G2) independently of, CtIP, MDC1 and the MRN complex (Rad50, Mre11, NBS1). However expansion of hSSB1 from the DSB site requires the function of MRN. Strikingly, silencing of hSSB1 prevents foci formation as well as recruitment of MRN to sites of DSBs and leads to a subsequent defect in resection of DSBs as evident by defective RPA and ssDNA generation. Our data suggests that hSSB1 functions upstream of MRN to promote its recruitment at DSBs and is required for efficient resection of DSBs. These findings, together with previous work establish essential roles of hSSB1 in controlling ATM activation and activity, and subsequent DSB resection and homologous recombination (HR).

Promotion of Homologous Recombination and Genomic Stability by RAD51AP1 via RAD51 Recombinase Enhancement

Homologous recombination (HR) repairs chromosome damage and is indispensable for tumor suppression in humans. RAD51 mediates the DNA strand-pairing step in HR. RAD51 associated protein 1 (RAD51AP1) is a RAD51-interacting protein whose function has remained elusive. Knockdown of RAD51AP1 in human cells by RNA interference engenders sensitivity to different types of genotoxic stress, and RAD51AP1 is epistatic to the HR protein XRCC3. Moreover, RAD51AP1-depleted cells are impaired for the recombinational repair of a DNA double-strand break and exhibit chromatid breaks both spontaneously and upon DNA-damaging treatment. Purified RAD51AP1 binds both dsDNA and a D loop structure and, only when able to interact with RAD51, greatly stimulates the RAD51-mediated D loop reaction. Biochemical and cytological results show that RAD51AP1 functions at a step subsequent to the assembly of the RAD51-ssDNA nucleoprotein filament. Our findings provide evidence that RAD51AP1 helps maintain genomic integrity via RAD51 recombinase enhancement.

Rad51 supports triple negative breast cancer metastasis

In contrast to extensive studies on familial breast cancer, it is currently unclear whether defects in DNA double strand break (DSB) repair genes play a role in sporadic breast cancer development and progression. We performed analysis of immunohistochemistry in an independent cohort of 235 were sporadic breast tumours. This analysis suggested that RAD51 expression is increased during breast cancer progression and metastasis and an oncogenic role for RAD51 when deregulated. Subsequent knockdown of RAD51 repressed cancer cell migration in vitro and reduced primary tumor growth in a syngeneic mouse model in vivo. Loss of RAD51 also inhibited associated metastasis not only in syngeneic mice but human xenografts and changed the metastatic gene expression profile of cancer cells, consistent with inhibition of distant metastasis. This demonstrates for the first time a new function of RAD51 that may underlie the proclivity of patients with RAD51 overexpression to develop distant metastasis. RAD51 is a potential biomarker and attractive drug target for metastatic triple negative breast cancer, with the capability to extend the survival of patients, which is less than 6 months.

γ-radiation-induced γH2AX formation occurs preferentially in actively transcribing euchromatic loci

The central dogma in radiation biology is that nuclear DNA is the critical target with respect to radiosensitivity. In accordance with the theoretical expectations, and in the absence of a conclusive model, the general consensus in the field has been to view chromatin as a homogeneous template for DNA damage and repair. This paradigm has been called into question by recent findings indicating a disparity in γ-irradiation-induced γH2AX foci formation in euchromatin and heterochromatin. Here, we have extended those studies and provide evidence that γH2AX foci form preferentially in actively transcribing euchromatin following γ-irradiation.