Homologous recombination (HR) and nonhomologous end joining (NHEJ) are two distinct DNA double-stranded break (DSB) repair pathways. Here, we report that DNA-dependent protein kinase (DNA-PK), the core component of NHEJ, partnering with DNA-damage checkpoint kinases ataxia telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR), regulates HR repair of DSBs. The regulation was accomplished through modulation of the p53 and replication protein A (RPA) interaction. We show that upon DNA damage, p53 and RPA were freed from a p53-RPA complex by simultaneous phosphorylations of RPA at the N-terminus of RPA32 subunit by DNA-PK and of p53 at Ser37 and Ser46 in a Chk1/Chk2-independent manner by ATR and ATM, respectively. Neither the phosphorylation of RPA nor of p53 alone could dissociate p53 and RPA. Furthermore, disruption of the release significantly compromised HR repair of DSBs. Our results reveal a mechanism for the crosstalk between HR repair and NHEJ through the co-regulation of p53-RPA interaction by DNA-PK, ATM and ATR.

RPA (replication protein A), the eukaryotic ssDNA (single-stranded DNA)-binding protein, participates in most cellular processes in response to genotoxic insults, such as NER (nucleotide excision repair), DNA, DSB (double-strand break) repair and activation of cell cycle checkpoint signalling. RPA interacts with XPA (xeroderma pigmentosum A) and functions in early stage of NER. We have shown that in cells the RPA-XPA complex disassociated upon exposure of cells to high dose of UV irradiation. The dissociation required replication stress and was partially attributed to tRPA hyperphosphorylation. Treatment of cells with CPT (camptothecin) and HU (hydroxyurea), which cause DSB DNA damage and replication fork collapse respectively and also leads to the disruption of RPA-XPA complex. Purified RPA and XPA were unable to form complex in vitro in the presence of ssDNA. We propose that the competition-based RPA switch among different DNA metabolic pathways regulates the dissociation of RPA with XPA in cells after DNA damage. The biological significances of RPA-XPA complex disruption in relation with checkpoint activation, DSB repair and RPA hyperphosphorylation are discussed.

RPA (replication protein A) is an essential factor for DNA DSB (double-strand break) repair and cell cycle checkpoint activation. The 32 kDa subunit of RPA undergoes hyperphosphorylation in response to cellular genotoxic insults. However, the potential involvement of hyperphosphorylated RPA in DSB repair and check-point activation remains unclear. Using co-immunoprecipitation assays, we showed that cellular interaction of RPA with two DSB repair factors, Rad51 and Rad52, was predominantly mediated by the hyperphosphorylated species of RPA in cells after UV and camptothecin treatment. Moreover, Rad51 and Rad52 displayed higher affinity for the hyperphosphorylated RPA than native RPA in an in vitro binding assay. Checkpoint kinase ATR (ataxia telangiectasia mutated and Rad3-related) also interacted more efficiently with the hyperphosphorylated RPA than with native RPA following DNA damage. Consistently, immunofluorescence microscopy demonstrated that the hyperphosphorylated RPA was able to co-localize with Rad52 and ATR to form significant nuclear foci in cells. Our results suggest that hyperphosphorylated RPA is preferentially localized to DSB repair and the DNA damage checkpoint complexes in response to DNA damage.

Robotic Process Automation (RPA) är en allt mer framväxande teknologi och används i organisationer för att automatisera och effektivisera manuella affärsprocesser. Det finns en rad fördelar med att använda denna teknik. Implementeringen av RPA kan dock kopplas till blandade reaktioner hos anställda och det är inte ovanligt att en del anställda motsätter sig tekniken på grund av rädsla och okunskap för automatiseringens påverkan. Denna fallstudie syftar att undersöka hur implementeringen av RPA påverkar organisatoriska aspekter som arbetssätt, roller och ansvar samt att undersöka om det finns skillnader i hantering och upplevelse kring detta i konsult-, privat- och offentlig verksamhet. För att undersöka ovan nämnda områden skedde datainsamlingen genom semistrukturerade intervjuer hosorganisationer som implementerat RPA samt genom litteraturundersökning. Resultatet visar att det finns en hel del likheter i hur RPA påverkar arbetet i organisationer, men skillnaderna ligger i hur arbetet med RPA bedrivs. / Robotic Process Automation (RPA) is an increasingly emerging technology and is used in organizations to automate and streamline manual business processes. There are a number of advantages to using this technology. However, the implementation of RPA can be linked to mixed reactions among employees and it is not unusual for some employees to oppose the technology due to fear and ignorance of the influence of automation. This case study aims to investigate how the implementation of RPA affects organizational aspects such as working methods, roles and responsibilities, and examine whether there are differences in management and experience regarding this in consulting, private and public activities.In order to investigate the above-mentioned areas, the data collection was conducted through semistructured interviews with organizations that implemented RPA and through literature research. The result shows that there are a lot of similarities in how RPA affects the work in organizations, but the differences lie in how the work with RPA is conducted.

Detta examensarbete utforskar en implementation av digital automatisering i en entreprenörsverksamhet med hjälp av Robotic Process Autom ation utifrån ett holistiskt perspektiv. Utförandestrategin för arbetet har varit en aktionsforskning där författarna konfigurerat roboten under studien och intervjuat personer i verksamheten för att fånga intryck och känslor under implementationens gång. Resultatet av studien är ett kunskapsbidrag i form av nytt empiriskt material inom området, konceptvalidering och förslag på god praxis vid framtida implementationer. Studien har även resulterat i en för sitt syfte fullt fungerande prototyp. / This bachelor thesis examines an implementation of digital automation from a holistic perspective in an entrepreneurial business with Robotic Process Automation. The strategy for the thesis has been action research where the authors have configured the robot during the study and interviewed persons in the establishment to capture impressions and feelings during the implementation. The results of the study constitute a contribution of new empirical material within the area, proof of concept and suggestions for good practice in future implementations. The study has also resulted in a fully functional prototype fo r its purpose.

Denna studie ämnar identifiera utmaningar och aspekter som en offentlig verksamhet behöverta hänsyn till vid en RPA-implementation. Studien har utförts i en offentlig verksamhet som äri behov av att automatisera genom att implementera RPA. Förarbetet till denna RPAimplementation som skett iterativt har i denna studie observerats under workshops, observationoch intervju. Utförandet av studien har skett genom att genomföra en fallstudie i en offentligverksamhet med ett explorativt angreppssätt, kvalitativ datainsamling och en abduktiv ansats.Resultatet från datainsamlingstillfällena analyserades genom tematisk analys med två olikaperspektiv; teoridrivet och datadrivet. Utifrån det teoridrivna perspektivet analyserades empirinmot de teman som utvanns ifrån de teoretiska koncept som sammanfattats från tidigare studierom RPA och RPA-implementation. Efter att ställt teorin mot empirin blev resultatet att falletskaraktär var i linje med nio av de femton teoretiska koncepten. De koncept som inte påträffadesi detta fall samlades under temat ”Det är viktigt att välja rätt process för en RPAimplementation”. Det datadrivna perspektivet identifierade nya aspekter som offentligaverksamheter behöver beakta vid implementation av RPA. Den första slutsatsen är att detviktigt att välja rätt process inför en RPA-implementation. Den andra slutsatsen är aspektenom att offentliga verksamheter måste hantera mycket känsliga uppgifter och att det är enutmaning vid implementation av RPA.

Indiana University-Purdue University Indianapolis (IUPUI) / Accurate DNA replication is vital for maintaining genomic stability. Consequently, the machinery required to drive this process is designed to ensure the meticulous maintenance of information. However, random misincorporation of errors reduce the fidelity of the DNA and lead to pre-mature aging and age-related disorders such as cancer and neurodegenerative diseases. Some of the incorporated errors are the result of the error prone DNA polymerase alpha (Pol α), which initiates synthesis on both the leading and lagging strand. Lagging strand synthesis acquires an increased number of polymerase α tracks because of the number of Okazaki fragments synthesized per round of the cell cycle (~50 million in mammalian cells). The accumulation of these errors invariably reduces the fidelity of the genome. Previous work has shown that these pol α tracks can be removed by two redundant pathways referred to as the short and long flap pathway. The long flap pathway utilizes a complex network of proteins to remove more of the misincorporated nucleotides than the short flap pathway which mediates the removal of shorter flaps. Lysine acetylation has been reported to modulate the function of the nucleases implicated in flap processing. The cleavage activity of the long flap pathway nuclease, Dna2, is stimulated by lysine acetylation while conversely lysine acetylation of the short flap pathway nuclease, FEN1, inhibits its activity. The major protein players implicated during Okazaki fragment processing (OFP) are known, however, the choice of the processing pathway and its regulation by lysine acetylation of its main players is yet unknown. This dissertation identifies three main findings: 1) Saccharomyces cerevisiae helicase, petite integration frequency (Pif1) is lysine acetylated by Esa1 and deacetylated by Rpd3 regulating its viability and biochemical properties including helicase, binding and ATPase activity ii) the single stranded DNA binding protein, human replication protein A (RPA) is modified by p300 and this modification stimulates its primary binding function and iii) lysine acetylated human RPA directs OFP towards the long flap pathway even for a subset of short flaps.

This is an exploration of the potential for a citizen science project, with the goal to get the general public involved in microbial soil biodiversity around Uppsala, Sweden. Biodiversity serves an important role in how an ecosystem performs and functions. A large part of Earth's biodiversity exists below ground in soil, where microorganisms interact with plants. It would be beneficial to analyse the abundance and spread of some microorganisms in order to gain a better understanding of soil biodiversity. We suggest that one species family to study could be Phytophthora. Phytophthora is a genus of oomycetes that often are pathogenic, causing disease in various trees and other plants. It is unknown exactly how widespread the genus is today, making it extra interesting for the proposed study. For the general public to be able to do this a device needs to be developed that is easy to use and preferably could be used directly in the field. An isothermal amplification method is suitable for identifying the microorganism under these conditions. Many isothermal amplification methods are expensive, perhaps too expensive for a citizen science study, but have great potential for easy field testing. We propose a device utilizing RPA and lateral flow strips. RPA - Recombinase Polymerase Amplification is a method for amplification that might be suitable since it is simple, sensitive, and has a short run time. It is however expensive, which is an issue, but isothermal amplifications are expensive across the board. Lateral flow strips can be used to visualize the results. They utilize antibodies to detect the previously amplified amplicons, and give a positive or negative test answer that would be understandable to even untrained study participants. One of the biggest obstacles identified in this project concerns amplifying DNA from a soil sample, because an extraction step is necessary. The methods we have identified for extraction are not performable in the field, since they require centrifugation. In the proposition for a device a possible work-around for this is proposed, but since it has yet to be tested it is not yet known whether it will work or not.

Robotic Process Automation is a technology that is rapidly growing with continuous developments. Involving the use of software bots, it automates repetitive tasks and processes, resulting in saving both money and time. Simultaneously, the construction industry has been relatively slow to adapt to the quick digitalization of society. This study explores the possibility of implementing automation in the construction industry to automate repetitive tasks and processes in project planning. Based on semi-structured interviews with high-profile employees within the construction industry, RPA experts and an information security expert, a need for automation has been explored and identified. Based on the results, the industry is currently grasping the limit of carbon dioxide emissions. The project planning stage is described as time-consuming and repetitive with constant adjustments. Furthermore, it involves tasks such as listing materials, calculating budgets and conversion of units to present a building’s expected carbon dioxide emissions over its lifecycle. Therefore, there is a demand for a streamlined process where the calculations and transfers are done automatically by interconnecting software programs that are used in the currently individual tasks.  In summary, the study found that the process examined is suitable for automation based on feedback from RPA experts, developers, professionals in the construction industry, and an information security expert. Automating the process would also lead to greater efficiency and enable workers to focus on more critical tasks such as analysis and decision-making.

The genomes of all living cells are under constant attack from both endogenous and exogenous agents that damage DNA. In order to maintain genetic integrity a variety of response pathways have evolved to recognize and eliminate DNA damage. Replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA) binding protein, is a required factor for all major DNA metabolisms. Although much work has been done to elucidate the nature of the interaction between RPA and ssDNA currently there is no structural information on how the full-length protein binds to ssDNA. This study presents a novel examination of the full nucleoprotein complex formed between RPA and ssDNA. We identified three previously unknown contacts between ssDNA and lysine residues in DNA binding domain C located in the p70 subunit. This represents the first single amino-acid resolution determination of how full-length RPA contacts ssDNA. The Ataxia-Telangiectasia Mutated and RAD3-Related (ATR) mediated DNA damage checkpoint and nucleotide excision repair (NER) pathway are primarily responsible for repair of UV-C-induced photolesions in DNA. However, it is unclear how these two pathways are coordinated. We found the ATR-dependent checkpoint induces a rapid nuclear accumulation of the required NER factor Xeroderma pigmentosum group A (XPA) in both a dose- and time-dependent fashion. Also, using surface topology mapping we have defined an α-helix motif on XPA required for XPA-ATR complex formation necessary for XPA phosphorylation. In addition, we have determined that XPA phosphorylation promotes repair of persistent DNA lesions, such as cyclobutane pyrimidine dimers. The basis for initial damage recognition in NER is structural distortion of duplex DNA; however, the effects of adduct structure and sequence on strand opening and recognition are unclear. Using the E. coli NER system we determined that the identity of the adduct dictates the size of the strand opening generated by the UvrA2B complex. In addition we found that the sequence immediately surrounding the damaged nucleotide affects damage recognition by influencing the amount of helical distortion induced by the adduct. These effects are a result of the equilibrium conformation the adduct adopts in addition to the amount of hydrogen bonding available to maintain the structure.