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High-risk childhood leukemia has a poor prognosis because of treatment failure and toxic side effects of therapy. Drug encapsulation into liposomal nanocarriers has shown clinical success at improving biodistribution and tolerability of chemotherapy. However, enhancements in drug efficacy have been limited because of a lack of selectivity of the liposomal formulations for the cancer cells. Here, we report on the generation of bispecific antibodies (BsAbs) with dual binding to a leukemic cell receptor, such as CD19, CD20, CD22, or CD38, and methoxy polyethylene glycol (PEG) for the targeted delivery of PEGylated liposomal drugs to leukemia cells. This liposome targeting system follows a "mix-and-match" principle where BsAbs were selected on the specific receptors expressed on leukemia cells. BsAbs improved the targeting and cytotoxic activity of a clinically approved and low-toxic PEGylated liposomal formulation of doxorubicin (Caelyx) toward leukemia cell lines and patient-derived samples that are immunophenotypically heterogeneous and representative of high-risk subtypes of childhood leukemia. BsAb-assisted improvements in leukemia cell targeting and cytotoxic potency of Caelyx correlated with receptor expression and were minimally detrimental in vitro and in vivo toward expansion and functionality of normal peripheral blood mononuclear cells and hematopoietic progenitors. Targeted delivery of Caelyx using BsAbs further enhanced leukemia suppression while reducing drug accumulation in the heart and kidneys and extended overall survival in patient-derived xenograft models of high-risk childhood leukemia. Our methodology using BsAbs therefore represents an attractive targeting platform to potentiate the therapeutic efficacy and safety of liposomal drugs for improved treatment of high-risk leukemia.

Context: Increases in fire frequency, intensity and extent are occurring globally. Relative to historical, Indigenous managed conditions, contemporary landscapes are often characterised by younger age classes of vegetation and a much smaller representation of long-unburnt habitat. Objectives: We argue that, to conserve many threatened vertebrate species in Australia, landscape management should emphasise the protection of existing long-unburnt patches from fire, as well as facilitate the recruitment of additional long-unburnt habitat, while maintaining historically relevant age distributions of more recently burned patches. Methods: We use a range of case studies and ecosystem types to illustrate three lines of evidence: (1) that many threatened vertebrate species depend on mid- to late-successional ecosystem attributes; (2) disturbance to long-unburnt habitat tends to increase risk of future disturbance and ecosystem collapse; and (3) contemporary landscapes exhibit a range of characteristics that differ to historical conditions and require context-specific management. Conclusions: It is crucial that we adequately consider the implications of altered contemporary landscapes for management activities that aim to conserve threatened vertebrates. Contemporary landscapes often lack a range of critical structural and compositional components typical of late-successional habitat that are required for the persistence of threatened vertebrates. We need to shift towards strategic, objective-driven approaches that identify and protect long-unburnt habitats and promote their recruitment to enable recovery of many declining and threatened species.

In response to Australia’s current extinction crisis, substantial research efforts have been targeted towards some of the most imperilled species. One such species is the northern quoll (Dasyurus hallucatus), a marsupial predator that has recently suffered substantial declines in range and is now listed as Endangered. We conducted a systematic review of all literature relevant to the conservation and ecology of northern quolls. We reviewed 143 studies, including research articles, government and industry reports, theses, and books, and quantified research effort in terms of topic, location, and publication period. We then summarised research relevant to northern quoll taxonomy, genetics, distribution, habitat associations, diet, reproduction, movement, threats, management, and Indigenous knowledge. Research effort was higher between 2011 and 2020 than the previous four decades combined. Northern quolls in the Northern Territory were the most studied, followed by the Pilbara, the Kimberley, and Queensland populations. Most studies focused on northern quoll distribution and habitat, management, and threats – primarily cane toads, predation, and fire. We conclude with a non-exhaustive list of ten future research directions. If pursued, these future research directions should provide information critical to managing and conserving northern quolls.

Earth's rapidly warming climate is propelling us towards an increasingly fire-prone future. Currently, knowledge of the extent and characteristics of animal mortality rates during fire remains rudimentary, hindering our ability to predict how animal populations may be impacted in the future. To address this knowledge gap, we conducted a global systematic review of the direct effects of fire on animal mortality rates, based on studies that unequivocally determined the fate of animals during fire. From 31 studies spanning 1984-2020, we extracted data on the direct impacts of fire on the mortality of 31 species from 23 families. From these studies, there were 43 instances where direct effects were measured by reporting animal survival from pre- to post-fire. Most studies were conducted in North America (52%) and Oceania (42%), focused largely on mammals (53%) and reptiles (30%), and reported mostly on animal survival in planned (82%) and/or low severity (70%) fires. We found no studies from Asia, Europe or South America. Although there were insufficient data to conduct a formal meta-analysis, we tested the effect of fire type, fire severity, fire regime, animal body mass, ecological attributes and class on survival. Only fire severity affected animal mortality, with a higher proportion of animals being killed by high than low severity fires. Recent catastrophic fires across the globe have drawn attention to the plight of animals exposed to wildfire. Yet, our systematic review suggests that a relatively low proportion of animals (mean predicted mortality [95% CI] = 3% [1%-9%]) are killed during fire. However, our review also underscores how little we currently know about the direct effects of fire on animal mortality, and highlights the critical need to understand the effects of high severity fire on animal populations.

Mammal declines across northern Australia are one of the major biodiversity loss events occurring globally. There has been no regional assessment of the implications of these species declines for genomic diversity. To address this, we conducted a species-wide assessment of genomic diversity in the northern quoll (Dasyurus hallucatus), an Endangered marsupial carnivore. We used next generation sequencing methods to genotype 10,191 single nucleotide polymorphisms (SNPs) in 352 individuals from across a 3220-km length of the continent, investigating patterns of population genomic structure and diversity, and identifying loci showing signals of putative selection. We found strong heterogeneity in the distribution of genomic diversity across the continent, characterized by (i) biogeographical barriers driving hierarchical population structure through long-term isolation, and (ii) severe reductions in diversity resulting from population declines, exacerbated by the spread of introduced toxic cane toads (Rhinella marina). These results warn of a large ongoing loss of genomic diversity and associated adaptive capacity as mammals decline across northern Australia. Encouragingly, populations of the northern quoll established on toad-free islands by translocations appear to have maintained most of the initial genomic diversity after 16 years. By mapping patterns of genomic diversity within and among populations, and investigating these patterns in the context of population declines, we can provide conservation managers with data critical to informed decision-making. This includes the identification of populations that are candidates for genetic management, the importance of remnant island and insurance/translocated populations for the conservation of genetic diversity, and the characterization of putative evolutionarily significant units.

(1) Background: MALDI imaging is a technique that still largely depends on time of flight (TOF)-based instrument such as the Bruker UltrafleXtreme. While capable of performing targeted MS/MS, these instruments are unable to perform fragmentation while imaging a tissue section necessitating the reliance of MS1 values for peptide level identifications. With this premise in mind, we have developed a hybrid bioinformatic/image-based method for the identification and validation of viral biomarkers. (2) Methods: Formalin-Fixed Paraffin-Embedded (FFPE) mouse samples were sectioned, mounted and prepared for mass spectrometry imaging using our well-established methods. Peptide identification was achieved by first extracting confident images corresponding to theoretical viral peptides. Next, those masses were used to perform a Peptide Mmass Fingerprint (PMF) searched against known viral FASTA sequences against a background mouse FASTA database. Finally, a correlational analysis was performed with imaging data to confirm pixel-by-pixel colocalization and intensity of viral peptides. (3) Results: 14 viral peptides were successfully identified with significant PMF Scores and a correlational result of >0.79 confirming the presence of the virus and distinguishing it from the background mouse proteins. (4) Conclusions: this novel approach leverages the power of mass spectrometry imaging and provides confident identifications for viral proteins without requiring MS/MS using simple MALDI Time Of Flight/Time Of Flight (TOF/TOF) instrumentation.

The 2019–2020 Australian megafires were unprecedented in their intensity and extent. These wildfires may have caused high mortality of adult broad-headed snakes Hoplocephalus bungaroides which shelter inside tree hollows during summer. We evaluated the impacts of two high-intensity wildfires (2002 Touga Fire and 2020 Morton Fire) on a broad-headed snake population in Morton National Park, south-eastern Australia. We analysed a 29-year mark–recapture data set to estimate survival rates of adults in years with and without wildfires, and with and without human disturbance to rock outcrops. To examine the short-term effects of fire on occupancy, we analysed presence–absence data collected from 25 sites during 2019 and 2020. Estimates of occupancy were higher for 2020 (0.93 ± 0.09) than for 2019 (0.66 ± 0.14), while detection rates were constant (0.40 ± 0.06). Over the period 1992–2020, the best supported Cormack–Jolly–Seber model was one in which adult survival rates were high and stable (0.81 ± 0.04), but were 23% lower in years when humans disturbed rocks (0.63 ± 0.08). A model in which adult mortality was 20% higher in years with human disturbance and 14% higher in years with wildfires was also supported. Estimates of abundance revealed that the population declined by 34% after the Touga Fire, and 26% after the Morton Fire. Over the 29-year study, the population has declined by 60%. Our results highlight how mortality events from wildfires need to be evaluated in the context of other threatening processes. For this population, the removal of snakes and associated habitat disturbance poses a more serious threat to population viability than infrequent wildfires.

Cultural adaptation is one means by which conservationists may help populations adapt to threats. A learned behavior may protect an individual from a threat, and the behavior can be transmitted horizontally (within generations) and vertically (between generations), rapidly conferring population-level protection. Although possible in theory, it remains unclear whether such manipulations work in a conservation setting; what conditions are required for them to work; and how they might affect the evolutionary process. We examined models in which a population can adapt through both genetic and cultural mechanisms. Our work was motivated by the invasion of highly toxic cane toads (Rhinella marina) across northern Australia and the resultant declines of endangered northern quolls (Dasyurus hallucatus), which attack and are fatally poisoned by the toxic toads. We examined whether a novel management strategy in which wild quolls are trained to avoid toads can reduce extinction probability. We used a simulation model tailored to quoll life history. Within simulations, individuals were trained and a continuous evolving trait determined innate tendency to attack toads. We applied this model in a population viability setting. The strategy reduced extinction probability only when heritability of innate aversion was low (<20%) and when trained mothers trained >70% of their young to avoid toads. When these conditions were met, genetic adaptation was slower, but rapid cultural adaptation kept the population extant while genetic adaptation was completed. To gain insight into the evolutionary dynamics (in which we saw a transitory peak in cultural adaptation over time), we also developed a simple analytical model of evolutionary dynamics. This model showed that the strength of natural selection declined as the cultural transmission rate increased and that adaptation proceeded only when the rate of cultural transmission was below a critical value determined by the relative levels of pr

The arrival of novel predators can trigger trophic cascades driven by shifts in prey numbers. Predators also elicit behavioral change in prey populations, via phenotypic plasticity and/or rapid evolution, and such changes may also contribute to trophic cascades. Here, we document rapid demographic and behavioral changes in populations of a prey species (grassland melomys Melomys burtoni, a granivorous rodent) following the introduction of a novel marsupial predator (northern quoll Dasyurus hallucatus). Within months of quolls appearing, populations of melomys exhibited reduced survival and population declines relative to control populations. Quoll-invaded populations were also significantly shyer than nearby, quoll-free populations of conspecifics. This rapid but generalized response to a novel threat was replaced over the following 2 yr with more threat-specific antipredator behaviors (i.e., predator-scent aversion). Predator-exposed populations, however, remained more neophobic than predator-free populations throughout the study. These behavioral responses manifested rapidly in changed rates of seed predation by melomys across treatments. Quoll-invaded melomys populations exhibited lower per-capita seed take rates, and rapidly developed an avoidance of seeds associated with quoll scent, with discrimination playing out over a spatial scale of tens of meters. Presumably the significant and novel predation pressure induced by quolls drove melomys populations to fine-tune behavioral responses to be more predator specific through time. These behavioral shifts could reflect individual plasticity (phenotypic flexibility) in behavior or may be adaptive shifts from natural selection imposed by quoll predation. Our study provides a rare insight into the rapid ecological and behavioral shifts enacted by prey to mitigate the impacts of a novel predator and shows that trophic cascades can be strongly influenced by behavioral as well as numerical responses.

Global wildlife trade is a multibillion-dollar industry and a significant driver of vertebrate extinction risk. Yet, few studies have quantified the impact of wild harvesting for the illicit pet trade on populations. Long-lived species, by virtue of their slow life history characteristics, may be unable to sustain even low levels of collecting. Here, we assessed the impact of illegal collecting on populations of endangered broad-headed snakes (Hoplocephalus bungaroides) at gated (protected) and ungated (unprotected) sites. Because broad-headed snakes are long-lived, grow slowly and reproduce infrequently, populations are likely vulnerable to increases in adult mortality. Long-term data revealed that annual survival rates of snakes were significantly lower in the ungated population than the gated population, consistent with the hypothesis of human removal of snakes for the pet trade. Population viability analysis showed that the ungated population has a strongly negative population growth rate and is only prevented from ultimate extinction by dispersal of small numbers of individuals from the gated population. Sensitivity analyses showed that the removal of a small number of adult females was sufficient to impose negative population growth and suggests that threatened species with slow life histories are likely to be especially vulnerable to illegal collecting.

Mouse kidney parvovirus (MKPV) is a member of the provisional genus Chapparvovirus that causes renal disease in immune-compromised mice, with a disease course reminiscent of polyomavirus-associated nephropathy in immune-suppressed kidney transplant patients. Here we map four major MKPV transcripts, created by alternative splicing, to a common initiator region, and use mass spectrometry to identify "p10" and "p15" as novel chapparvovirus accessory proteins produced in MKPV-infected kidneys. p15 and the splicing-dependent putative accessory protein NS2 are conserved in all near-complete amniote chapparvovirus genomes currently available (from mammals, birds and a reptile). In contrast, p10 may be encoded only by viruses with >60% amino acid identity to MKPV. We show that MKPV is kidney-tropic and that the bat chapparvovirus DrPV-1 and a non-human primate chapparvovirus, CKPV, are also found in the kidneys of their hosts. We propose, therefore, that many mammal chapparvoviruses are likely to be nephrotropic.

Mouse kidney parvovirus (MKPV) is a member of the provisional genus Chapparvovirus that causes renal disease in immune-compromised mice, with a disease course reminiscent of polyomavirus-associated nephropathy in immune-suppressed kidney transplant patients. Here we map four major MKPV transcripts, created by alternative splicing, to a common initiator region, and use mass spectrometry to identify "p10" and "p15" as novel chapparvovirus accessory proteins produced in MKPV-infected kidneys. p15 and the splicing-dependent putative accessory protein NS2 are conserved in all near-complete amniote chapparvovirus genomes currently available (from mammals, birds and a reptile). In contrast, p10 may be encoded only by viruses with >60% amino acid identity to MKPV. We show that MKPV is kidney-tropic and that the bat chapparvovirus DrPV-1 and a non-human primate chapparvovirus, CKPV, are also found in the kidneys of their hosts. We propose, therefore, that many mammal chapparvoviruses are likely to be nephrotropic.

© The Author(s) 2020. Attempts to reintroduce threatened species from ex situ populations (zoos or predator-free sanctuaries) regularly fail because of predation. When removed from their natural predators, animals may lose their ability to recognize predators and thus fail to adopt appropriate antipredator behaviors. Recently, northern quolls (Dasyurus hallucatus; Dasyuromorpha: Dasyuridae) conserved on a predator-free "island ark"for 13 generations were found to have no recognition of dingoes, a natural predator with which they had coevolved on mainland Australia for about 8,000 years. A subsequent reintroduction attempt using quolls acquired from this island ark failed due to predation by dingoes. In this study, we tested whether instrumental conditioning could be used to improve predator recognition in captive quolls sourced from a predator-free "island ark."We used a previously successful scent-recognition assay (a giving-up density experiment) to compare predator-scent recognition of captive-born island animals before and after antipredator training. Our training was delivered by pairing live predators (dingo and domestic dog) with an electrified cage floor in repeat trials such that, when the predators were present, foraging animals would receive a shock. Our training methodology did not result in any discernible change in the ability of quolls to recognize and avoid dingo scent after training. We conclude either that our particular training method was ineffective (though ethically permissible); or that because these quolls appear unable to recognize natural predators, predator recognition may be extremely difficult to impart in a captive setting given ethical constraints. Our results point to the difficulty of reinstating lost behaviors, and to the value of maintaining antipredator behaviors in conservation populations before they are lost.

© 2020, Springer Nature B.V. Targeted gene flow is a novel conservation strategy that involves translocating individuals with favourable genes to areas where they will have a conservation benefit. One oft-cited risk of the strategy is the potential for outbreeding depression. Here, we used the northern quoll (Dasyurus hallucatus) as a model to test this possibility for the first time in a field setting. Northern quolls are endangered by the spread of the invasive cane toad (Rhinella marina), which they are fatally poisoned by, if they mistakenly attempt to consume them. There are, however, a small number of quolls that are “toad-smart”—they possess a heritable trait that means they innately do not attack toads. It is this trait we hoped to promote through targeted gene flow. We established a hybrid population (54 toad-smart and toad-naïve northern quolls) and introduced this population onto a small offshore, toad-infested island in 2017. Genetic data suggests an increase in the toad-smart proportion of the genome increasing from 29.4% in the release population to 40.2% in the first island generation (F2). Our data demonstrate successful in situ hybridisation between populations, with viable F2 hybrids and backcrosses observed, and some evidence of heterosis (hybrid vigour) in F1 hybrids. The population experienced significant reductions in size over the two years, however, through a combination of toad-mortality, as well as stochastic processes including fire, a cyclone, predation and ineffective breeding, and so small sample sizes hamper our results. Such establishment problems would not occur were we to attempt targeted gene flow into already established quoll populations, and our observation of successful hybridisation suggests targeted gene flow could be a viable strategy in established quoll populations soon to be impacted by toads.

Antibody‐mediated immunity is highly protective against disease. The majority of current vaccines confer protection through humoral immunity, but there is high variability in responsiveness across populations. Identifying immune mechanisms that mediate low antibody responsiveness may provide potential strategies to boost vaccine efficacy. Here, we report diverse antibody responsiveness to unadjuvanted as well as adjuvanted immunization in substrains of BALB/c mice, resulting in high and low antibody response phenotypes. Furthermore, these antibody phenotypes were not affected by changes in environmental factors such as the gut microbiota composition. Antigen‐specific B cells following immunization had a marked difference in capability to class switch, resulting in perturbed IgG isotype antibody production. In vitro, a B‐cell intrinsic defect in the regulation of class‐switch recombination was identified in mice with low IgG antibody production. Whole genome sequencing identified polymorphisms associated with the magnitude of antibody produced, and we propose candidate genes that may regulate isotype class‐switching capability. This study highlights that mice sourced from different vendors can have significantly altered humoral immune response profiles, and provides a resource to interrogate genetic regulators of antibody responsiveness. Together these results further our understanding of immune heterogeneity and suggest additional research on the genetic influences of adjuvanted vaccine strategies is warranted for enhancing vaccine efficacy.

© 2019, Springer-Verlag GmbH Germany, part of Springer Nature. Abstract: If bold animals are more likely to be trapped than shy animals, we take a biased sample of personalities—a problem for behavioural research. Such a bias is problematic, also, for population estimation using mark-recapture models that assume homogeneity in detection probabilities. In this study, we investigated whether differences in boldness result in differences in detection probability in a native Australian rodent, the grassland melomys (Melomys burtoni). During a mark-recapture study of this species, we used modified open field tests to assess the boldness (via emergence, and interaction with a novel object) of melomys trapped on the last night of four trapping nights in each of two trapping sessions. Despite melomys showing repeatable variation in these behavioural traits, neither boldness nor emergence latency had an effect on detection probability, and we found no evidence that detection probability varied between individuals. This result suggests that any neophobia is experienced and resolved in individuals of this species on a scale of minutes, rather than the hours across which traps are made available each night. Our work demonstrates that personality-caused sampling bias may not be inevitable, even in situations where animals are required to respond to novelty to be detected, such as in baited traps. Heterogeneity in personality does not inevitably lead to heterogeneity in detection probability. Significance statement: Historically, passive traps were assumed a non-biased means of sampling animal populations. Increasingly behavioural ecologists suggest that personality traits, particularly individual boldness, may influence behaviour and, as a consequence, could result in sampling bias. Here, we present a comprehensive example of when animal personality has no effect on detection probability. Despite having distinct personalities, detection probabilities of a native Australian roden

Invasive species are a leading cause of native biodiversity loss. In Australia, the toxic, invasive cane toad Rhinella marina has caused massive and widespread declines of northern quolls Dasyurus hallucatus. Quolls are fatally poisoned if they mistakenly prey on adult toads. To prevent the extinction of this native dasyurid from the Top End, an insurance population was set up in 2003 on two toad‐free islands in Arnhem Land. In 2015, quolls were collected from one of these islands (Astell) for reintroduction. We used conditioned taste aversion to render 22 of these toad‐naïve quolls toad averse. Seven quolls received no taste aversion training. The source island was also predator‐free, so all quolls received very basic predator‐aversion training. In an attempt to re‐establish the mainland population, we reintroduced these 29 northern quolls into Kakadu National Park in northern Australia where cane toads have been established for 13 years. The difference in survival between toad‐averse and toad‐naive quolls was immediately apparent. Toad‐naive quolls were almost all killed by toads within 3 days. Toad‐averse quolls, on the other hand, not only survived longer but also were recorded mating. Our predator training, however, was far less effective. Dingo predation accounted for a significant proportion of toad‐smart quoll mortality. In Kakadu, dingoes have been responsible for high levels of quoll predation in the past and reintroduced animals are often vulnerable to predation‐mediated population extinction. Dingoes may also be more effective predators in fire degraded landscapes. Together, these factors could explain the extreme predation mortality that we witnessed. In addition, predator aversion may have been lost from the predator‐free island populations. These possibilities are not mutually exclusive but need to be investigated because they have clear bearing on the long‐term recovery of the endangered northern quoll.

© 2018 Elsevier Inc. The occurrence of a spontaneous nephropathy with intranuclear inclusions in laboratory mice has puzzled pathologists for over 4 decades, because its etiology remains elusive. The condition is more severe in immunodeficient animals, suggesting an infectious cause. Using metagenomics, we identify the causative agent as an atypical virus, termed “mouse kidney parvovirus” (MKPV), belonging to a divergent genus of Parvoviridae. MKPV was identified in animal facilities in Australia and North America, is transmitted via a fecal-oral or urinary-oral route, and is controlled by the adaptive immune system. Detailed analysis of the clinical course and histopathological features demonstrated a stepwise progression of pathology ranging from sporadic tubular inclusions to tubular degeneration and interstitial fibrosis and culminating in renal failure. In summary, we identify a widely distributed pathogen in laboratory mice and establish MKPV-induced nephropathy as a new tool for elucidating mechanisms of tubulointerstitial fibrosis that shares molecular features with chronic kidney disease in humans. A kidney parvovirus found in multiple laboratory mouse colonies causes spontaneous nephropathy and represents a new tool for studying chronic kidney disease.

© 2018 The Author(s) Published by the Royal Society. All rights reserved. When imperilled by a threatening process, the choice is often made to conserve threatened species on offshore islands that typically lack the full suite of mainland predators. While keeping the species extant, this releases the conserved population from predator-driven natural selection. Antipredator traits are no longer maintained by natural selection and may be lost. It is implicitly assumed that such trait loss will happen slowly, but there are few empirical tests. In Australia, northern quolls (Dasyurus hallucatus) were moved onto a predator-free offshore island in 2003 to protect the species from the arrival of invasive cane toads on the mainland. We compared the antipredator behaviours of wild-caught quolls from the predator-rich mainland with those from this predator-free island. We compared the responses of both wild-caught animals and their captive-born offspring, to olfactory cues of two of their major predators (feral cats and dingoes). Wild-caught, mainland quolls recognized and avoided predator scents, as did their captive-born offspring. Island quolls, isolated from these predators for only 13 generations, showed no recognition or aversion to these predators. This study suggests that predator aversion behaviours can be lost very rapidly, and that this may make a population unsuitable for reintroduction to a predator-rich mainland.

AID deaminates C to U in either strand of Ig genes, exclusively producing C:G/G:C to T:A/A:T transition mutations if U is left unrepaired. Error-prone processing by UNG2 or mismatch repair diversifies mutation, predominantly at C:G or A:T base pairs, respectively. Here, we show that transversions at C:G base pairs occur by two distinct processing pathways that are dictated by sequence context. Within and near AGCT mutation hotspots, transversion mutation at C:G was driven by UNG2 without requirement for mismatch repair. Deaminations in AGCT were refractive both to processing by UNG2 and to high-fidelity base excision repair (BER) downstream of UNG2, regardless of mismatch repair activity. We propose that AGCT sequences resist faithful BER because they bind BER-inhibitory protein(s) and/or because hemi-deaminated AGCT motifs innately form a BERresistant DNA structure. Distal to AGCT sequences, transversions at G were largely co-dependent on UNG2 and mismatch repair. We propose that AGCTdistal transversions are produced when apyrimidinic sites are exposed in mismatch excision patches, because completion of mismatch repair would require bypass of these sites.

Immunoglobulin (Ig) affinity maturation requires the enzyme AID, which converts cytosines (C) in Ig genes into uracils (U). This alone produces C:G to T:A transition mutations. Processing of U:G base pairs via U N-glycosylase 2 (UNG2) or MutSα generates further point mutations, predominantly at G:C or A:T base pairs, respectively, but it is unclear why processing is mutagenic. We aimed to test whether the cell cycle phase of U processing determines fidelity. Accordingly, we ectopically restricted UNG2 activity in vivo to predefined cell cycle phases by fusing a UNG2 inhibitor peptide to cell cycle-regulated degradation motifs. We found that excision of AID-induced U by UNG2 occurs predominantly during G1 phase, inducing faithful repair, mutagenic processing, and class switching. Surprisingly, UNG2 does not appear to process U:G base pairs at all in Ig genes outside G1 phase. © 2012 Sharbeen et al.