Exponentially growing cell cultures were irradiated with 1 Gy X-rays. Following irradiation cells were allowed to repair for 1 to 5 h at 37°C to detect only the response of cells in G2 phase at the time of irradiation. Colcemid was added for 1 h aimed to block metaphases prior to harvesting the respective time point, except for the 1 h time point, where colcemid was added for 30 min before harvest to allow the migration of cells which were at the mitosis during irradiation. Cells were trypsinized, treated with hypotonic solution (75mM KCl) for 10 min at room temperature (RT) and fixed in Cornoy’s fixative (3X). The fixed cells were dropped on clean glass slide and stained with 3% Giemsa stain prepared in Sorenson’s buffer. For each experimental point about 300 cells were scored for chromatid damage from three independent experiments. Standard criteria were used for scoring. During scoring chromatid breaks and gaps were considered, the latter only when longer than the chromatid width. The chromosome exchanges were counted as two chromatid breaks. Bright field microscopy (Olympus VANOX-T, Japan) was employed to facilitate scoring. This protocol employed here is the modified protocol used by Bryant et al., 2008 (186).
Taken together these data support a model in which DEK has two distinct roles in the repair of DSBs by HR. First, DEK has an important function in γ H2AX activation by ATM, but this is unlikely to be sufficient for the degree of HR deficiency observed in DEK-deficient cells 44,66 . Therefore, DEK must have a second function
involving the DEK-RAD51 complex. With regard to a possible mechanism of action, DEK might play an early role in homologousrecombination by supporting the initial formation of the RAD51-DNA filament. However, since BRCA1 and BRCA2 play key roles in this step and, since BRCA1 was not detectable in the DEK-RAD51 complex (Fig. 5b), we do not favor this scenario. Based on previous structural and cell-free studies, DEK bind- ing to DNA stimulates self-multimerization and potential interactions with other proteins 22,67,68 . Furthermore,
The endeavor of profiling the molecular and genetic landscape of PCa and searching for novel predictive biomarkers that specify and optimize therapy has re- cently led to the discovery of multiple genomic alter- ations. Next generation sequencing studies of prostate tumors revealed a high number of recurrent gene mu- tations interfering with homologousrecombinationrepair (HRR). Among others, the Cancer Genome At- las (TCGA) Research and American Association for Cancer Research networks have identified HRR gene alterations in 19–23% of metastatic CRPC (mCRPC) samples [ 3 , 4 ]. Analyzing the TCGA prostate cancer database, Kim et al. found mutations and copy-num- ber variations of DNA damage repair (DDR) genes in 30% of localized (nonmetastatic) PCa indicating that DDR alterations might occur early during disease pro- gression [ 5 ]. Reported prevalence of mutations in- cluded HRR genes such as cyclin-dependent kinase 12 (CDK12), RAD51C, breast cancer 1 (BRCA1) or ataxia telangiectasia mutated (ATM). Interestingly, all studies found breast cancer 2 (BRCA2) to be the most com- monly mutated gene.
188.8.131.52 Methods – A Simple Strategy for Treating Gaps in Recco The large gap region in the multiple alignment makes the analysis of HERV-KX se- quences particularly challenging. As described in chapter 6, gaps can lead to several arti- facts and require a careful analysis. The analysis of gaps in Recco was actually motivated by the present study of HERV-KX sequences. Hence, we could not rely on the analysis of chapter 6 back then and only had a limited number of options. We could either (i) discard sites that contain a gap character, (ii) treat each gap character as a fifth nucleotide state or (iii) treat each consecutive run of columns containing gaps as a large polymorphism, as in Geneconv. All these options have certain disadvantages and may cause severe artifacts in the output. The first option results in an unacceptable loss of information, as the presence of the 96bp insertion already represents important information. More notably, different references that harbor the 96bp insertion also show a different sequence for the insertion. The second option may lead to an artificially high similarity or dissimilarity between se- quences in gap regions and can produce spurious recombination events. Finally, the third option prohibits recombinations in any run of columns containing a gap. A sequence with a long gap may therefore confound recombinations in other sequences. It is also difficult to choose an adequate scoring term for the large polymorphisms that are generated by this procedure. In conclusion, these approaches for treating gaps either discard a lot of infor- mation and thus miss recombination events or may infer spurious recombination events solely based on gap information.
Simple DNA lesions such as base damage (e.g. 8-Oxoguanine), AP sites and single strand breaks can be repaired via the BER pathway (Figure 1.2 A). BER is initiated by DNA glycosylases, which recognize damaged bases and excise them. So far there are 11 glycosylases known specific to different but also par- tially overlapping base damage. The formed AP site is cleaved by AP endonuclease APEX creating a single stranded break in the DNA backbone. In case of short-patch BER this gap is filled by polymerase and the nick is sealed by Ligase 3. Only one nucleotide gets replaced in this pathway. XRCC1 serves as a loading platform for this non-processive DNA synthesis reaction. In long-patch BER, polymerization is accomplished by polymerase or ✏. They synthesize up to 10 nucleotides, displacing the old strand and forming a DNA flap. Flap structure-specific endonuclease 1 (Fen1) removes this flap and Ligase 1 seals the nick. As loading platform for this reaction serves the processivity factor ’proliferating cell nuclear antigen’ (PCNA), which is also involved in other processive DNA repair pathways as well as in DNA replication (Klungland and Lindahl, 1997, Robertson et al., 2009).
Both processes, the three-body recombination (I) and the radiative two-body recombination (II) can be treat ed by the same theoretical model if a third body in case (II) is introduced as a “phonon field” 1. Mecha nism (I) can also result in “chemiluminescence” if a suitable radiative state of the stabilized molecule AB is populated during the recombination. The lifetime of [AB]* depends on the energy of the relative motion of the colliding particles, the angular momentum, and on the interaction of the internal energy states. Accord ing to recent calculations2, metastable “resonance” states of the “quasimolecule” [AB]* seem to be im portant, at least for the recombination of atoms. These states are assumed to be in quasi-equilibrium with the continuum states of the free atoms by quantum-mecha- nical tunnelling through a slight potential barrier. Pre vious calculations for [H2] * showed 3 that the lifetimes of such resonance states can be as long as several 10-10 sec. On the other hand, lifetimes of the collision pairs above the threshold of resonance states are in the order of 10~13 sec, the duration of one vibrational period.
Species-specific spread of retrotransposons was postulated as a main route enlarging plant genome (SanMiguel et al., 1996, 1998; Bennetzen and Kellog, 1997). Conversely, deletions might reduce genome size and counterbalance enlargements (Petrov, 1997). By using suitable restriction endonucleases for induction of breaks at the specific loci in eukaryotic genomes it has been possible to characterise DSB-induced DNA repair. In this study, due to the selection on 5-FC and kanamycin and the position of the PCR primer binding sites, deletion size in represented experiments could only be analysed in the range 0.2-2.5 kb. Although other kinds of repair events, like changes that are not linked to the loss of the codA gene or bigger deletions, could not be addressed with the current experimental set up, the presented data reveal a surprisingly strong difference in DSB repair between tobacco and Arabidopsis, two dicotyledonous plant species with a >20-fold difference in genome size (Bennet and Leitch, 1997).
In the original, very straightforward algorithm of the ARG described above, certain time consuming silent events are included, namely those recombination events that happen out- side of ancestral or trapped material. Trapped material is nonancestral material enclosed between two parts of ancestral material. Events happening in nonancestral material that is not trapped, neither affect the partitioning of ancestral material, nor the family of local trees. Moreover, if an event in such a regions occurs, the respective lineage will split into two lineages of which one does not share any genetic material with the sample. In order to keep the number of lines and events as small as possible, these silent events may thus be removed by increasing the memory capacity of the algorithm. Such modified algorithms, where all nonancestral lines are excluded and where for every sequence the information about the (continuous) region spanned by ancestral material is stored, belong to the class of reduced ARGs and are for example investigated in [95, 131]. A latest example in this class of modified algorithms is the sequential coalescent with recombination model (SCRM). The SCRM improved the algorithm studied in [95, 132] with regard to accuracy and efficiency .
Over the last five decades the fundamental properties of ZnO have been widely studied by many authors. 1 Its large band gap of 3.3 eV, large exciton binding energy of about 60 meV, and its structural compatibility with GaN are some the many advantages that make ZnO interesting concerning device applications. A renewed interest in this material sys- tem has emerged in the last years due to great improvements on its growth techniques. Its applications into piezotronics have been recently discussed since they represent the ulti- mate step for an efficient integration into novel devices. In addition, much effort has been put in fabricating low dimen- sional structures 2 such as nanodots, nanocrystals, tetrapods, nanorods, and nanowires 共NWs兲 due to the advantages of- fered by quantum confinement. Nevertheless, despite the great variety of nanostructures produced up to date, scaling ZnO or any material into the nanoscale present some com- plications as some of its physical properties depend on the dimensionality and, thus, need to be revisited. Recently, the mechanical properties of ZnO were found to be different to those of bulk material. 3 The importance of surface states as possible recombination channels in NWs was also discussed. 4 A complete revision of the optical, electrical, and mechanical properties is mandatory when the surface-to- volume ratio is not negligible. The recombination dynamics in ZnO NWs have been recently studied by many authors. 4 – 8 Different recombination times with single and biexponential decays have been reported for different nanostructured sys- tems. This situation is somehow dissapointing since it might lead to different interpretations of the possible recombination channels in these nanostructures. Although direct comparison between all these data is not possible since the lifetime de- pends on many different scattering processes 共which might differ for different growth techniques 兲, the knowledge of its dependence with the NWs size is desirable. In a recent letter, 8 the lifetime of the free exciton was investigated for
of RPA for the recombinase Rad51. This generates a highly coordinated right-handed nucleoprotein filament, commonly known as the presynaptic filament, which is the central entity of the HR pathway 45,46 . This functional unit then undergoes the search for a homologous template and eventually invades the donor, thereby forming the characteristic microscopic D-loop structure with one displaced donor strand 39,47 . The Rad54 protein stabilizes the D-loop and initiates removal of Rad51 from the heteroduplex to allow subsequent priming of DNA synthesis from the invading strand by DNA polymerase δ 48 . At this stage, the extended D-loop marks the branching point for a number of HR sub- pathways, of which one will be chosen depending on the cellular context 39 . In mitotic cells, seamless repair is guaranteed by synthesis-dependent strand annealing (SDSA) (Figure 3 B). Following D-loop extension, the heteroduplex DNA is reversed to allow annealing of the newly synthesized strand with the resected strand of the second DSB end. In contrast, the utilization of the Dmc1 recombinase in meiotic cells favors capturing of the second DSB end inside the D-loop. This results in the formation of a double Holliday junction (dHJ), representing the classical DSB repair model (DSBR) (Figure 3 C). Endonucleases such as Mus81-Mms4, Yen1 or Slx1-Slx4 are capable to trigger genetic exchange by resolving such structures in a crossover or non-crossover fashion 49 . Despite the fact that dHJs can form in mitotic cells at low frequency, crossover formation here is strongly suppressed by their dissolution via the STR complex 50 . Finally, a third variation of HR is able to deal with one-ended DSB, which among others form at replication forks, particularly when no second fork directly approaches from the opposite direction 51 . In this case, the free end in the D-loop will be replicated in a mutagenesis-prone, conservative mode known as break-induced replication (BIR) that involves the DNA helicase Pif1 52 (Figure 3 D).
The PDR-PL measurement routine as described has two limi- tations: ﬁrstly, it is necessary to calculate the photon recycling factor from known material properties (i.e. absorption and emission, see for example Refs. 1 and 5 ) and secondly, purely radiative recombina- tion has to be achieved for self-calibration. For the latter Auger and defect recombination have to be negligible compared to radiative recombination at high injection, which may or may not be the case depending on the material and its quality. Furthermore, band ﬁlling of the indirect bands as well as carrier escape out of the double- heterostructure (DH) via thermionic emission must both be negli- gible; fortunately for the AlGaAs/GaAs material system, the barrier height is sufﬁcient. 16 In other words, these constraints are not nec- essarily given for every sample investigated. Thus, two components are required to further validate the PDR-PL method: ﬁrst a direct method of extracting B eff rad and second, a calibration of the PDR-PL signal. For this purpose, TR-PL can be used to quantify the effective radiative efﬁciency under low injection conditions when combined with the knowledge of the effective radiative recombination coefﬁ- cient B eff rad . The result is a calibrated low-injection radiative efﬁciency used to calibrate the PDR-PL signal, which serves to quantify the high injection radiative efﬁciency of the system.
This precession is called “Rabi oscillation”. It is illustrated in fig. 3.3(b) and can be easily described in rotating reference frames as explained in appendix A.4. As already discussed in section 3.1.2, the observable of TSR experiments, the photocurrent, is fundamentally different than the observable of time–resolved ESR: The latter is always based on the measurement of microwave radiation produced by the polarisation of a precessing spin ensemble. Because the photocurrent in a TSR experiment reflects recombination and therefore singlet and triplet densities, only the symmetry state of the spin–pair ensemble becomes measurable and polarisation does not play an important role. Consequently, the relative spin motion of the two partners within a pair are of much greater importance than the absolute spin precession of any of the two spins itself. The relative periodic motion between two energy eigenstates, which means the coherent oscillation, is called a quantum–beat oscillation. Since the recombination rate is dependent on the relative motion between the two spin–pair partners, the recombination rate will reflect the beat oscillation of the two single precessions. Thus, when both partners carry out different Rabi oscillations, the frequency at which the permutation symmetry of the spin pair oscillates will be a beat frequency that is dependent on the two Rabi frequencies. In section 3.5 in which the theory of coherent spin propagation of the spin pairs is outlined in detail, this process is referred to as “Rabi–beat oscillation”. The Rabi–beat oscillation can become equal to the Rabi oscillation of one pair partner, when a strong difference between the Larmor frequencies of the two pair partners is present and the microwave is in resonance with one partner only. The different Rabi precessions of the two spin–pair partners and the resulting oscillation of the recombination rate is illustrated in time domain (b) of fig. 3.3. It takes place as long as the microwave is switched on and it is a purely coherent process when the microwave pulse length is much shorter than the recombination time as well as the spin–relaxation times.
In our approach to compute the recombination rate, we use density functional theory and a plane wave expansion within a pseudopotential method 12 implemented in the ABI- NIT software package 13 , 14 to calculate the band structure and wavefunctions ab initio. For all calculations, we use the LDA as exchange correlation functional and a plane wave cut-off of 40 Ha. In a first step, the energy dispersion is determined on a dense mesh of k points in the Brillouin zone, where we use 125 000 k points to converge the Auger rate within 10% with respect to the rate at a three times denser k point mesh. Momentum and energy conservation can then be exploited to search for possible Auger transitions on the mesh. The numerical effort of that search scales at least with the third power of the number of k points. Thus, it is sensible to use a cut-off energy E sum < E cut sum in the statisti-
This study sheds light onto the mechanism of RE-guided homology search (the search for a homologous sequence), using chromatin immunoprecipitation (ChIP) of the recombinase Rad51 as well as recombination efficiency measurements in S. cerevisiae. I demonstrate that the RE can be uncoupled from the mating type system and acts as a general recombinational enhancer of nearby donor sites during HR when placed at different genomic sites, independent of the DSB-inducing endonuclease. Furthermore, the RE guides ongoing homology search reflected by the Rad51 nucleoprotein filament, which assists in the recognition of the homologous donor site. Moreover, this study corroborates the essential function of Fkh1 and its phosphothreonine-binding forkhead associated (FHA) domain for the regulation of donor preference. Well-known factors involved in DSB repair like RPA and Mph1 are identified as Fkh1 interaction partners via a mass spectrometry based approach. I propose that these factors mediate the potential loop formation between the RE and the DSB via a phosphorylation-dependent interaction with Fkh1 bound to the RE. Finally, a role in donor preference regulation can be assigned to the checkpoint kinase Mec1.
2(d) mark the additional IQE losses due to the specific tem- perature dependence of the B and C coefficients. As the tem- perature dependence of B is minor, cf. Fig. 2(b) , Auger recombination is the major contributor to such kind of losses. We have found the efficiency losses to be much more pro- nounced for green LEDs, meaning that the contribution of Auger recombination to the green gap is most significant. The difference between the Auger losses in blue and green LEDs suggests that the more pronounced thermal activation of Auger processes directly correlates with the stronger localization occurring in green InGaN alloys. Naturally, any rise in charge carrier localization enhances the electron-pho- non-coupling 42 – 45 favoring phonon-assisted Auger processes and their impact on the IQE reduction. In conclusion, we have observed close to identical SRH RCs over the entire temperature range for blue- and green-emitting MQW, ruling out defect generation in InGaN with high In content as prin- cipal cause of the green gap. In contrast, the radiative and Auger RCs were found to decrease significantly in green- emitting LEDs, because of a reduced overlap between elec- tron and hole wave functions. The anomalous temperature dependence of the radiative recombination allows us to attri- bute a part of this reduction to hole localization induced by fluctuations of the InGaN composition, while the tempera- ture independent reduction stems from the QCSE. Here, even a simplified model for all recombination processes, involving delocalized electrons and localized holes, suffices to interpret the data. In addition, the strong rise with temper- ature of the Auger RC leads to a further reduction of
Our results show that, contrary to common belief, mixed HSV-1/HSV-2 infections have led to natural recombination events between HSV-1 and HSV-2 ( Thiry et al. 2005 ). These findings are not completely unexpected since such natural interspecies recombination events have already been docu- mented in other mammalian herpesviruses (equine herpesvi- ruses; Pagamjav et al. 2005 ; Greenwood et al. 2012 ). What may be more surprising is that recombination within HSV-1 and HSV-2 has often been investigated and detected during the last decade (for a complete review on recombination in alphaherpesviruses, see Loncoman et al. 2016 ). Yet, the re- combinant fragments in UL29 and UL30 went unnoticed, albeit being present in all partial and complete genomes pub- lished prior to our study. We note here that early studies focused on recombination used PCR-based approaches and typically covered 2–4% of the genome; to our knowledge, they did not target any of the coding sequences in which we identified recombinant HSV-1 fragments ( Bowden et al. 2004 ; Norberg et al. 2004 , 2007 ). More recently, a number of studies have produced several dozens of HSV-1 and HSV-2 complete (or nearly complete) genomes which were used to examine genome-wide patterns of recombination ( Kolb et al. 2011 , 2013, 2015 ; Szpara et al. 2014 ; Newman et al. 2015 ). However, it seems that these studies only used single species alignments, which of course prevents the identification of interspecies recombination. Our findings therefore highlight that taxonomically broader analysis of recombination is still warranted, even when studying relatively well-characterized viruses.