The role of BRCA2 in the maintenance of genome stability in response to replication stress

A fundamental aspect of living organisms is the accurate replication and maintenance of the genome to ensure the high-fidelity inheritance of genetic information throughout many cell generations. The molecular machinery that replicates the DNA – acting at the so-called replication fork – can be frequently hindered by obstacles of both extracellular and intracellular origin, such as chemicals or UV radiations on one hand, and collision with other processes occurring on the DNA, like gene transcription, on the other. These challenges to DNA replication may lead to transient slowing or stalling of replication forks: we refer to this as replication stress. Cells evolved a variety of mechanisms, which fall under the definition of DNA damage response, that allow them to respond to replication stress. Failure of these mechanisms can lead to DNA damage, and ultimately result in genomic instability, a major driving force of cancer. On the other hand, many chemotherapeutic compounds are designed to induce replication stress, exploiting the stringent requirement of highly replicating cancer cells to continuously replicate their genome. Therefore, investigating the mechanisms underlying replication stress has emerged as a key tool to both understand cancer onset, and to develop new therapeutic approaches. Our laboratory has recently reported that, upon cellular exposure to genotoxic treatments, replication forks slow down and are frequently remodeled to form a detectable four-way junction at the replication fork, called reversed fork. This transient molecular transaction is considered to be a protective response, required to limit fork breakage in conditions of replication stress. In addition, our laboratory reported that, besides exogenous genotoxic treatments, fork reversal frequently occurs also in response to endogenous molecular processes, which are known to undermine genomic integrity, such as the activation (overexpression or amplification) of cellular proto-oncogenes. In the current thesis, I will present our efforts to understand what triggers reversed fork formation in different contexts, and what are the factors that contribute to its formation and stability. In the first part, I describe strategies we designed for the generation of new inducible oncogene overexpression systems in different cellular models, with the aim of describing the common or different molecular consequences downstream of aberrant activation of different oncogenes. This part has led to date to inconclusive results, mainly due to technical difficulties in establishing an efficient and robust oncogene induction system. The importance of this question (oncogene- induced replication stress in cancer) demands additional future work, and fine-tuning of the techniques for a time-controlled oncogene overexpression. In the second part, I successfully investigated the function in replication fork remodeling of the proteins involved in homologous recombination, with particular attention on the tumor suppressors RAD51 and BRCA2. Besides their established role in double strand break repair via homologous recombination, RAD51 and BRCA2 were in fact known to protect stalled replication forks from extensive nucleolytic degradation. Underlying the importance of this alternative function for these genes, defects in fork protection lead to chromosomal instability, and contribute to the sensitivity of BRCA2-defective tumors to chemotherapeutics by yet-unknown mechanisms. Our results showed that RAD51 contributes to reverse fork formation. Moreover, we found that these structures are progressively degraded in the absence of BRCA2 (loss-of-function mutations in BRCA2 are a common feature of many cancer types). Inhibiting MRE11 nuclease activity or its recruitment to the reversed fork can restore fork integrity and prevent chromosomal breakage. On the contrary, preventing fork degradation by impairing the formation of reversed forks, leads to increased chromosomal breakage in BRCA2-defective cells, being thus detrimental for genome stability. Collectively, our study reveals that fork reversal has a crucial physiological relevance in protecting genome stability upon replication stress, and that a complex interplay of HR factors co- operate to remodel and stabilize stalled DNA replication forks

Platelet Activation In Situ in Breasts at High Risk of Cancer : Relationship with Mammographic Density and Estradiol

Context: High mammographic density in postmenopausal women is an independent risk factor for breast cancer by undetermined mechanisms. No preventive therapy for this risk group is available. Activated platelets release growth factors that modulate the microenvironment into a protumorigenic state. Estrogens may affect the risk of breast cancer and platelet function. Whether platelets are activated in situ in breast cancer or in normal breast tissue at high risk of breast cancer and the association to estradiol remains elusive. Objective: To investigate whether platelets are activated in situ in breast cancers and in dense breast tissue of postmenopausal women and explore correlations between estradiol, released platelet factors, and inflammatory proteins. Setting and design: Sampling of in vivo proteins was performed using microdialysis in a total of 71 women: 10 with breast cancer, 42 healthy postmenopausal women with different breast densities, and 19 premenopausal women. Results: Our data demonstrate increased levels of coagulation factors in dense breast tissue similar to that found in breast cancers, indicating excessive platelet activation. Premenopausal breasts exhibited similar levels of coagulation factors as postmenopausal dense breasts. Out of 13 coagulations factors that were upregulated in dense breasts, 5 exhibited significant correlations with estradiol, both locally in the breast and systemically. In breast tissue, positive correlations between coagulation factors and key inflammatory proteins and matrix metalloproteinases were detected. Conclusions: Breast density, not estradiol, is the major determinant of local platelet activation. Inactivation of platelets may be a therapeutic strategy for cancer prevention in postmenopausal women with dense breasts.Funding Agencies|Swedish Cancer SocietySwedish Cancer Society [2018/464]; Swedish Research CouncilSwedish Research CouncilEuropean Commission [2018-02584]; LiU-Cancer; ALF of Linkoping University Hospital</p

CDC7 kinase promotes MRE11 fork processing, modulating fork speed and chromosomal breakage

The CDC7 kinase is essential for the activation of DNA replication origins and has been implicated in the replication stress response. Using a highly specific chemical inhibitor and a chemical genetic approach, we now show that CDC7 activity is required to coordinate multiple MRE11-dependent processes occurring at replication forks, independently from its role in origin firing. CDC7 localizes at replication forks and, similarly to MRE11, mediates active slowing of fork progression upon mild topoisomerase inhibition. Both proteins are also retained on stalled forks, where they promote fork processing and restart. Moreover, MRE11 phosphorylation and localization at replication factories are progressively lost upon CDC7 inhibition. Finally, CDC7 activity at reversed forks is required for their pathological MRE11-dependent degradation in BRCA2-deficient cells. Thus, upon replication interference CDC7 is a key regulator of fork progression, processing and integrity. These results highlight a dual role for CDC7 in replication, modulating both initiation and elongation steps of DNA synthesis, and identify a key intervention point for anticancer therapies exploiting replication interference

Sequential role of RAD51 paralog complexes in replication fork remodeling and restart

Homologous recombination (HR) factors were recently implicated in DNA replication fork remodeling and protection. While maintaining genome stability, HR-mediated fork remodeling promotes cancer chemoresistance, by as-yet elusive mechanisms. Five HR cofactors - the RAD51 paralogs RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3 - recently emerged as crucial tumor suppressors. Albeit extensively characterized in DNA repair, their role in replication has not been addressed systematically. Here, we identify all RAD51 paralogs while screening for modulators of RAD51 recombinase upon replication stress. Single-molecule analysis of fork progression and architecture in isogenic cellular systems shows that the BCDX2 subcomplex restrains fork progression upon stress, promoting fork reversal. Accordingly, BCDX2 primes unscheduled degradation of reversed forks in BRCA2-defective cells, boosting genomic instability. Conversely, the CX3 subcomplex is dispensable for fork reversal, but mediates efficient restart of reversed forks. We propose that RAD51 paralogs sequentially orchestrate clinically relevant transactions at replication forks, cooperatively promoting fork remodeling and restart

Deregulated origin licensing leads to chromosomal breaks by rereplication of a gapped DNA template

Deregulated origin licensing and rereplication promote genome instability and tumorigenesis by largely elusive mechanisms. Investigating the consequences of Early mitotic inhibitor 1 (Emi1) depletion in human cells, previously associated with rereplication, we show by DNA fiber labeling that origin reactivation occurs rapidly, well before accumulation of cells with >4N DNA, and is associated with checkpoint-blind ssDNA gaps and replication fork reversal. Massive RPA chromatin loading, formation of small chromosomal fragments, and checkpoint activation occur only later, once cells complete bulk DNA replication. We propose that deregulated origin firing leads to undetected discontinuities on newly replicated DNA, which ultimately cause breakage of rereplicating forks

Deregulated origin licensing leads to chromosomal breaks by rereplication of a gapped DNA template

Deregulated origin licensing and rereplication promote genome instability and tumorigenesis by largely elusive mechanisms. Investigating the consequences of Early mitotic inhibitor 1 (Emi1) depletion in human cells, previously associated with rereplication, we show by DNA fiber labeling that origin reactivation occurs rapidly, well before accumulation of cells with >4N DNA, and is associated with checkpoint-blind ssDNA gaps and replication fork reversal. Massive RPA chromatin loading, formation of small chromosomal fragments, and checkpoint activation occur only later, once cells complete bulk DNA replication. We propose that deregulated origin firing leads to undetected discontinuities on newly replicated DNA, which ultimately cause breakage of rereplicating forks

Sequential role of RAD51 paralog complexes in replication fork remodeling and restart

International audienceHomologous recombination (HR) factors were recently implicated in DNA replication fork remodeling and protection. While maintaining genome stability, HR-mediated fork remodeling promotes cancer chemoresistance, by as-yet elusive mechanisms. Five HR cofactors - the RAD51 paralogs RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3 - recently emerged as crucial tumor suppressors. Albeit extensively characterized in DNA repair, their role in replication has not been addressed systematically. Here, we identify all RAD51 paralogs while screening for modulators of RAD51 recombinase upon replication stress. Single-molecule analysis of fork progression and architecture in isogenic cellular systems shows that the BCDX2 subcomplex restrains fork progression upon stress, promoting fork reversal. Accordingly, BCDX2 primes unscheduled degradation of reversed forks in BRCA2-defective cells, boosting genomic instability. Conversely, the CX3 subcomplex is dispensable for fork reversal, but mediates efficient restart of reversed forks. We propose that RAD51 paralogs sequentially orchestrate clinically relevant transactions at replication forks, cooperatively promoting fork remodeling and restart

"I got mad about that book" : A study of Vietnamese children's views on reading

The aim of this Bachelor thesis and Minor Field Study is, from the perspective of the Vietnamese children at The General Science Library in Ho Chi Minh City, to obtain a deeper understanding of the ways in which the reading environment of The Children's Room supports children's interest in reading and their reading experience. It is assumed that reading is a dynamic and social activity made possible by internal and external conditions. In this context limited freedom of expression is one of the external conditions that is taken into consideration. In order to obtain understanding of Vietnamese children's experiences of reading, I used Aidan Chambers' model of The Reading Circle as a theoretical framework. The methods used in this study were semi-structured interviews with Vietnamese young people at the library, four girls and five boys, aged between 10 and 15 years. The children in this study describe two types of reading experiences: 1) reading that gives feelings of excitement and joy, and makes them want to reread a book, talk about it with others, think of it, remember and analyse it. And 2) reading they describe as developing, either spiritually or intellectually, a form of reading for improvement. Four reading environments are identified that both enable and obstruct reading experiences, their homes, school, library and the bookstore. Primarily, The Children's Room enables reading