Mechanisms for Maintaining Eukaryotic Replisome Progression in the Presence of DNA Damage.

The eukaryotic replisome coordinates template unwinding and nascent-strand synthesis to drive DNA replication fork progression and complete efficient genome duplication. During its advancement along the parental template, each replisome may encounter an array of obstacles including damaged and structured DNA that impede its progression and threaten genome stability. A number of mechanisms exist to permit replisomes to overcome such obstacles, maintain their progression, and prevent fork collapse. A combination of recent advances in structural, biochemical, and single-molecule approaches have illuminated the architecture of the replisome during unperturbed replication, rationalised the impact of impediments to fork progression, and enhanced our understanding of DNA damage tolerance mechanisms and their regulation. This review focusses on these studies to provide an updated overview of the mechanisms that support replisomes to maintain their progression on an imperfect template

An investigation into the role, recruitment, and regulation of PrimPol in DNA replication restart

DNA replication is one of life's fundamental processes. This delicate task is performed by a complex of molecular machines, known collectively as the replisome. At the heart of the replisome lie the replicative DNA polymerases which catalyse synthesis of daughter DNA strands with astonishing accuracy and efficiency. Nevertheless, these enzymes are prone to stalling upon encountering DNA damage lesions and secondary structures. In order to restart replication, DNA damage tolerance mechanisms are required. This published article-format thesis focusses on a recently discovered primase-polymerase, and member of the archaeo-eukaryotic primase (AEP) superfamily, involved in DNA damage tolerance, known as PrimPol. The work presented here builds on the initial characterisation of the enzyme, which identified potential roles in the bypass of DNA damage through translesion synthesis (TLS) and repriming of replication. The first presented article consists of a review of the AEP superfamily, functionally repositioning the group under the category of primase-polymerases. In the second paper, evidence is presented to suggest that PrimPol's activity is regulated by single-strand binding proteins, required due to the enzyme's error-prone nature. In the third chapter, a novel PrimPol binding protein, polymerase delta-interacting protein 2 (PolDIP2), is identified and characterised as a stimulatory factor for PrimPol's primer extension activities. Chapter 4 focusses on the development and use of a gel-based fluorescent primase assay to assess PrimPol's ability to reprime downstream of DNA damage lesions and secondary structures. The fifth presented paper identifies the molecular basis for PrimPol's interaction with replication protein A (RPA). Using biophysical, biochemical, and cellular approaches, this paper identifies the mechanism by which PrimPol is recruited to reprime replication. Lastly, in Chapter 6, a review of the presented articles in the context of the wider literature is included. Together, this work supports a role for PrimPol in repriming and restarting DNA replication following stalling at impediments, as well as identifying mechanisms involved in the recruitment and regulation of the enzyme

PrimPol is required for replicative tolerance of G quadruplexes in vertebrate cells

G quadruplexes (G4s) can present potent blocks to DNA replication. Accurate and timely replication of G4s in vertebrates requires multiple specialized DNA helicases and polymerases to prevent genetic and epigenetic instability. Here we report that PrimPol, a recently described primase-polymerase (PrimPol), plays a crucial role in the bypass of leading strand G4 structures. While PrimPol is unable to directly replicate G4s, it can bind and reprime downstream of these structures. Disruption of either the catalytic activity or zinc-finger of PrimPol results in extreme G4-dependent epigenetic instability at the BU-1 locus in avian DT40 cells, indicative of extensive uncoupling of the replicative helicase and polymerase. Together, these observations implicate PrimPol in promoting restart of DNA synthesis downstream of, but closely coupled to, G4 replication impediments

PolDIP2 interacts with human PrimPol and enhances its DNA polymerase activities

Translesion synthesis (TLS) employs specialized DNA polymerases to bypass replication fork stalling lesions. PrimPol was recently identified as a TLS primase and polymerase involved in DNA damage tolerance. Here, we identify a novel PrimPol binding partner, PolDIP2, and describe how it regulates PrimPol's enzymatic activities. PolDIP2 stimulates the polymerase activity of PrimPol, enhancing both its capacity to bind DNA and the processivity of the catalytic domain. In addition, PolDIP2 stimulates both the efficiency and error-free bypass of 8-oxo-7,8-dihydrodeoxyguanosine (8-oxoG) lesions by PrimPol. We show that PolDIP2 binds to PrimPol's catalytic domain and identify potential binding sites. Finally, we demonstrate that depletion of PolDIP2 in human cells causes a decrease in replication fork rates, similar to that observed in PrimPol−/− cells. However, depletion of PolDIP2 in PrimPol−/− cells does not produce a further decrease in replication fork rates. Together, these findings establish that PolDIP2 can regulate the TLS polymerase and primer extension activities of PrimPol, further enhancing our understanding of the roles of PolDIP2 and PrimPol in eukaryotic DNA damage tolerance

Screening neonatal jaundice based on the sclera color of the eye using digital photography

A new screening technique for neonatal jaundice is proposed exploiting the yellow discoloration in the sclera. It involves taking digital photographs of newborn infants' eyes (n = 110) and processing the pixel colour values of the sclera to predict the total serum bilirubin (TSB) levels. This technique has linear and rank correlation coefficients of 0.75 and 0.72 (both p<0.01) with the measured TSB. The mean difference ( ± SD) is 0.00 ± 41.60 µmol/l. The receiver operating characteristic curve shows that this technique can identify subjects with TSB above 205 µmol/l with sensitivity of 1.00 and specificity of 0.50, showing its potential as a screening device

Primpol—prime time to reprime

The complex molecular machines responsible for genome replication encounter many obstacles during their progression along DNA. Tolerance of these obstructions is critical for efficient and timely genome duplication. In recent years, primase-polymerase (PrimPol) has emerged as a new player involved in maintaining eukaryotic replication fork progression. This versatile replicative enzyme, a member of the archaeo-eukaryotic primase (AEP) superfamily, has the capacity to perform a range of template-dependent and independent synthesis activities. Here, we discuss the emerging roles of PrimPol as a leading strand repriming enzyme and describe the mechanisms responsible for recruiting and regulating the enzyme during this process. This review provides an overview and update of the current PrimPol literature, as well as highlighting unanswered questions and potential future avenues of investigation

Primase-polymerases are a functionally diverse superfamily of replication and repair enzymes

Until relatively recently, DNA primases were viewed simply as a class of proteins that synthesize short RNA primers requisite for the initiation of DNA replication. However, recent studies have shown that this perception of the limited activities associated with these diverse enzymes can no longer be justified. Numerous examples can now be cited demonstrating how the term 'DNA primase' only describes a very narrow subset of these nucleotidyltransferases, with the vast majority fulfilling multifunctional roles from DNA replication to damage tolerance and repair. This article focuses on the archaeo-eukaryotic primase (AEP) superfamily, drawing on recently characterized examples from all domains of life to highlight the functionally diverse pathways in which these enzymes are employed. The broad origins, functionalities and enzymatic capabilities of AEPs emphasizes their previous functional misannotation and supports the necessity for a reclassification of these enzymes under a category called primase-polymerases within the wider functional grouping of polymerases. Importantly, the repositioning of AEPs in this way better recognizes their broader roles in DNA metabolism and encourages the discovery of additional functions for these enzymes, aside from those highlighted here

Development of an intelligent electronic load controller for stand-alone micro-hydropower systems

Abstract: People living in rural and remote areas of Sub- Saharan Africa generally lack access to electricity due to their geographical location and the costs associated with connecting these areas to the national electrical grid. A viable technology to supply electricity to some of these areas are stand-alone microhydropower systems which harnesses energy from flowing water. Self-excited induction generators (SEIGs) are commonly used for the generation of electricity in stand-alone micro-hydropower systems. The electricity supplied by a SEIG to the demand side i.e. the load needs to be maintained stable under various consumer load conditions. This is accomplished through the use of an electronic load controller (ELC). This paper presents the design and development of an intelligent ELC that is able to maintain stable voltage on the demand side of a 3-phase SEIG supplying varying single-phase consumer loads. The proposed intelligent ELC consists of an uncontrolled bridge rectifier, filtering capacitor, chopper switch, voltage sensor, optocoupler, Arduino microcontroller and a ballast load or storage, depending on site-specific requirements and economic viability. The fuzzy logic control method is implemented to maintain stable and reliable voltage. The ELC is designed and simulated under various consumer load conditions in Matlab/Simulink. Simulation results of the ELC model are verified experimentally in a laboratory setting. The proposed intelligent ELC will contribute towards providing reliable and cost-effective means of enhancing the proliferation of micro-hydropower particularly in rural and remote applications in Sub-Saharan Africa

R-loop formation during S phase is restricted by PrimPol-mediated repriming

During DNA replication conflicts with ongoing transcription are frequent and require careful management to avoid genetic instability. R-loops, three stranded nucleic acid structures comprising a DNA:RNA hybrid and displaced single stranded DNA, are important drivers of damage arising from such conflicts. How R-loops stall replication and the mechanisms that restrain their formation during S phase are incompletely understood. Here we show in vivo how R-loop formation drives a short purine-rich repeat, (GAA)10, to become a replication impediment that engages the repriming activity of the primase-polymerase PrimPol. Further, the absence of PrimPol leads to significantly increased R-loop formation around this repeat during S phase. We extend this observation by showing that PrimPol suppresses R-loop formation in genes harbouring secondary structure-forming sequences, exemplified by G quadruplex and H-DNA motifs, across the genome in both avian and human cells. Thus, R- loops promote the creation of replication blocks at susceptible structure-forming sequences, while PrimPol-dependent repriming limits the extent of unscheduled R-loop formation at these sequences, mitigating their impact on replication

A gate-tunable graphene Josephson parametric amplifier

With a large portfolio of elemental quantum components, superconducting quantum circuits have contributed to dramatic advances in microwave quantum optics. Of these elements, quantum-limited parametric amplifiers have proven to be essential for low noise readout of quantum systems whose energy range is intrinsically low (tens of $\mu$eV ). They are also used to generate non classical states of light that can be a resource for quantum enhanced detection. Superconducting parametric amplifiers, like quantum bits, typically utilize a Josephson junction as a source of magnetically tunable and dissipation-free nonlinearity. In recent years, efforts have been made to introduce semiconductor weak links as electrically tunable nonlinear elements, with demonstrations of microwave resonators and quantum bits using semiconductor nanowires, a two dimensional electron gas, carbon nanotubes and graphene. However, given the challenge of balancing nonlinearity, dissipation, participation, and energy scale, parametric amplifiers have not yet been implemented with a semiconductor weak link. Here we demonstrate a parametric amplifier leveraging a graphene Josephson junction and show that its working frequency is widely tunable with a gate voltage. We report gain exceeding 20 dB and noise performance close to the standard quantum limit. Our results complete the toolset for electrically tunable superconducting quantum circuits and offer new opportunities for the development of quantum technologies such as quantum computing, quantum sensing and fundamental science