Paul Hasty - Academia.edu (original) (raw)

Papers by Paul Hasty

BioTechniques, 1999

A lacZ transgene, expressed by the myogenin promoter, was introduced into the mouse hypoxanthine ... more A lacZ transgene, expressed by the myogenin promoter, was introduced into the mouse hypoxanthine phosphoribosyltransferase (Hprt) locus by gene targeting in embryonic stem cells. Embryos between E10.5—E18.5 days were analyzed for expression of the transgene after staining for β-galactosidase activity. Transgene expression was restricted to the skeletal muscle lineages reflecting a similar temporal and spatial pattern previously demonstrated for the endogenous myogenin gene. Additionally, a second transgene, MC1tk, showed expression in 87% of the clones when targeted to Hprt. This strategy, called targeted transgenesis, provides control for analyzing promoter sequences and for comparing various transgenes expressed by the same promoter.

Mutation research, 2011

Fanconi anemia (FA) is a rare autosomal recessive disease characterized by bone marrow failure, d... more Fanconi anemia (FA) is a rare autosomal recessive disease characterized by bone marrow failure, developmental defects and cancer. There are multiple FA genes that enable the repair of interstrand crosslinks (ICLs) in coordination with a variety of other DNA repair pathways in a way that is poorly understood. Here we present the phenotype of mouse embryonic stem (ES) cells mutated for FancB. We found FancB-mutant cells exhibited reduced cellular proliferation, hypersensitivity to the crosslinking agent mitomycin C (MMC), increased spontaneous and MMC-induced chromosomal abnormalities, reduced spontaneous sister chromatid exchanges (SCEs), reduced gene targeting, reduced MMC-induced Rad51 foci and absent MMC-induced FancD2 foci. Since FancB is on the X chromosome and since ES cells are typically XY, FancB is an excellent target for an epistatic analysis to elucidate FA's role in ICL repair.

Mutation research

Ku80 forms a heterodimer with Ku70, called Ku, that repairs DNA double-strand breaks (DSBs) via t... more Ku80 forms a heterodimer with Ku70, called Ku, that repairs DNA double-strand breaks (DSBs) via the nonhomologous end joining (NHEJ) pathway. As a consequence of deleting NHEJ, Ku80-mutant cells are hypersensitive to agents that cause DNA DSBs like ionizing radiation. Here we show that Ku80 deletion also decreased resistance to ROS and alkylating agents that typically cause base lesions and single-strand breaks (SSBs). This is unusual since base excision repair (BER), not NHEJ, typically repairs these types of lesions. However, we show that deletion of another NHEJ protein, DNA ligase IV (Lig4), did not cause hypersensitivity to these agents. In addition, the ROS and alkylating agents did not induce γ-H2AX foci that are diagnostic of DSBs. Furthermore, deletion of Ku80, but not Lig4 or Ku70, reduced BER capacity. Ku80 deletion also impaired BER at the initial lesion recognition/strand scission step; thus, involvement of a DSB is unlikely. Therefore, our data suggests that Ku80 delet...

Mutation research, Jan 4, 2004

SNP analysis has come to the forefront of genomics since the mouse and human genomes have been se... more SNP analysis has come to the forefront of genomics since the mouse and human genomes have been sequenced. High throughput functional screens are necessary to evaluate these sequence databases. Described here is a genotoxic screen: a rapid method that determines the cellular dose-response to a wide range of agents that either damage DNA or alter basic cellular pathways important for maintaining genomic integrity. Importantly, a single person utilizing standard tissue culture equipment may perform these assays composed of 20 agents that attack genomic integrity or maintenance at many different levels. Thus, a small lab may perform this screen to determine the integrity of a wide range of DNA repair, chromatin metabolism, and response pathways without the limitations of investigator bias. A genotoxic screen will be useful when analyzing cells with either known genetic alterations (generated directly by the investigator or derived from individuals with known mutations) or unknown geneti...

PLoS ONE, 2014

Ku80 and DNA-PK CS are both involved in the repair of double strand DNA breaks via the nonhomolog... more Ku80 and DNA-PK CS are both involved in the repair of double strand DNA breaks via the nonhomologous end joining (NHEJ) pathway. While ku80 2/2 mice exhibit a severely reduced lifespan and size, this phenotype is less pronounced in dna-pk cs 2/2 mice. However, these observations are based on independent studies with varying genetic backgrounds. Here, we generated ku80 2/2 , dna-pk cs 2/2 and double knock out mice in a C57Bl6/J*FVB F1 hybrid background and compared their lifespan, end of life pathology and mutation frequency in liver and spleen using a lacZ reporter. Our data confirm that inactivation of Ku80 and DNA-PK CS causes reduced lifespan and bodyweights, which is most severe in ku80 2/2 mice. All mutant mice exhibited a strong increase in lymphoma incidence as well as other aging-related pathology (skin epidermal and adnexal atrophy, trabacular bone reduction, kidney tubular anisokaryosis, and cortical and medullar atrophy) and severe lymphoid depletion. LacZ mutation frequency analysis did not show strong differences in mutation frequencies between knock out and wild type mice. The ku80 2/2 mice had the most severe phenotype and the Ku80-mutation was dominant over the DNA-PK CS-mutation. Presumably, the more severe degenerative effect of Ku80 inactivation on lifespan compared to DNA-PK CS inactivation is caused by additional functions of Ku80 or activity of free Ku70 since both Ku80 and DNA-PK CS are essential for NHEJ.

Science, 2003

Recent progress in the science of aging is driven largely by the use of model systems, ranging fr... more Recent progress in the science of aging is driven largely by the use of model systems, ranging from yeast and nematodes to mice. These models have revealed conservation in genetic pathways that balance energy production and its damaging by-products with pathways that preserve somatic maintenance. Maintaining genome integrity has emerged as a major factor in longevity and cell viability. Here we discuss the use of mouse models with defects in genome maintenance for understanding the molecular basis of aging in humans.

PLoS ONE, 2009

Background: Negative elongation factor (NELF) is a four-subunit protein complex conserved from Dr... more Background: Negative elongation factor (NELF) is a four-subunit protein complex conserved from Drosophila to humans. In vitro biochemical and tissue culture-based studies have demonstrated an important role of NELF in controlling RNA polymerase II (Pol II) pausing in transcription. However, the physiological significance of NELF function is not clear due to the lack of any genetic systems for studying NELF.

PLoS Genetics, 2009

To characterize the repair pathways of chromosome double-strand breaks (DSBs), one approach invol... more To characterize the repair pathways of chromosome double-strand breaks (DSBs), one approach involves monitoring the repair of site-specific DSBs generated by rare-cutting endonucleases, such as I-SceI. Using this method, we first describe the roles of Ercc1, Msh2, Nbs1, Xrcc4, and Brca1 in a set of distinct repair events. Subsequently, we considered that the outcome of such assays could be influenced by the persistent nature of I-SceI-induced DSBs, in that end-joining (EJ) products that restore the I-SceI site are prone to repeated cutting. To address this aspect of repair, we modified I-SceI-induced DSBs by coexpressing I-SceI with a non-processive 39 exonuclease, Trex2, which we predicted would cause partial degradation of I-SceI 39 overhangs. We find that Trex2 expression facilitates the formation of I-SceI-resistant EJ products, which reduces the potential for repeated cutting by I-SceI and, hence, limits the persistence of I-SceI-induced DSBs. Using this approach, we find that Trex2 expression causes a significant reduction in the frequency of repair pathways that result in substantial deletion mutations: EJ between distal ends of two tandem DSBs, single-strand annealing, and alternative-NHEJ. In contrast, Trex2 expression does not inhibit homology-directed repair. These results indicate that limiting the persistence of a DSB causes a reduction in the frequency of repair pathways that lead to significant genetic loss. Furthermore, we find that individual genetic factors play distinct roles during repair of non-cohesive DSB ends that are generated via co-expression of I-SceI with Trex2.

PLoS Genetics, 2006

How congenital defects causing genome instability can result in the pleiotropic symptoms reminisc... more How congenital defects causing genome instability can result in the pleiotropic symptoms reminiscent of aging but in a segmental and accelerated fashion remains largely unknown. Most segmental progerias are associated with accelerated fibroblast senescence, suggesting that cellular senescence is a likely contributing mechanism. Contrary to expectations, neither accelerated senescence nor acute oxidative stress hypersensitivity was detected in primary fibroblast or erythroblast cultures from multiple progeroid mouse models for defects in the nucleotide excision DNA repair pathway, which share premature aging features including postnatal growth retardation, cerebellar ataxia, and death before weaning. Instead, we report a prominent phenotypic overlap with long-lived dwarfism and calorie restriction during postnatal development (2 wk of age), including reduced size, reduced body temperature, hypoglycemia, and perturbation of the growth hormone/insulin-like growth factor 1 neuroendocrine axis. These symptoms were also present at 2 wk of age in a novel progeroid nucleotide excision repair-deficient mouse model (XPD G602D/R722W /XPA À/À) that survived weaning with high penetrance. However, despite persistent cachectic dwarfism, blood glucose and serum insulin-like growth factor 1 levels returned to normal by 10 wk, with hypoglycemia reappearing near premature death at 5 mo of age. These data strongly suggest changes in energy metabolism as part of an adaptive response during the stressful period of postnatal growth. Interestingly, a similar perturbation of the postnatal growth axis was not detected in another progeroid mouse model, the double-strand DNA break repair deficient Ku80 À/À mouse. Specific (but not all) types of genome instability may thus engage a conserved response to stress that evolved to cope with environmental pressures such as food shortage.

Nature, 2010

Mammalian cells require Nonhomologous end joining (NHEJ) for efficient repair of chromosomal DNA ... more Mammalian cells require Nonhomologous end joining (NHEJ) for efficient repair of chromosomal DNA double-strand breaks 1. A key feature of biological sources of strand breaks is associated nucleotide damage, including base loss (abasic or AP sites) 2. At single strand breaks, 5' terminal abasic sites are excised by pol β's 5'dRP lyase activity 3 , 4 , 5 , 6 : we show here in vitro and in cells that accurate and efficient repair by NHEJ of double-strand breaks with such damage similarly requires 5'dRP/AP lyase activity (Figure 1a). Classically defined NHEJ is moreover uniquely effective at coupling this end-cleaning step to joining in cells, helping distinguish this pathway from otherwise robust alternate NHEJ pathways. Surprisingly, the NHEJ factor Ku can be identified as an effective 5'dRP/AP lyase. Similar to other lyases 7 , Ku nicks DNA 3' of an abasic site by a mechanism involving a Schiff base covalent intermediate with the abasic site. We demonstrate using cell extracts that Ku is essential for efficient removal of AP sites near doublestrand breaks and, consistent with this result, joining of such breaks is specifically reduced in cells complemented with a lyase-attenuated Ku mutant. Ku had previously been presumed only to recognize ends and recruit other factors that processed ends; our data supports an unexpected direct role for Ku in end processing steps as well. Abasic sites are frequently associated with double-strand breaks generated by ionizing radiation, treatment with radiomimetic drugs 2 , 8 , after aborted base excision repair 9 , 10 , or as an intermediate in class switch recombination 11 , 12. We first addressed how NHEJ resolves such damage in vitro, using 250 bp DNA substrates with abasic sites located near their 5' Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Molecular and Cellular Biology, 2008

ERCC1-XPF endonuclease is required for nucleotide excision repair (NER) of helix-distorting DNA l... more ERCC1-XPF endonuclease is required for nucleotide excision repair (NER) of helix-distorting DNA lesions. However, mutations in ERCC1 or XPF in humans or mice cause a more severe phenotype than absence of NER, prompting a search for novel repair activities of the nuclease. In Saccharomyces cerevisiae , orthologs of ERCC1-XPF (Rad10-Rad1) participate in the repair of double-strand breaks (DSBs). Rad10-Rad1 contributes to two error-prone DSB repair pathways: microhomology-mediated end joining (a Ku86-independent mechanism) and single-strand annealing. To determine if ERCC1-XPF participates in DSB repair in mammals, mutant cells and mice were screened for sensitivity to gamma irradiation. ERCC1-XPF-deficient fibroblasts were hypersensitive to gamma irradiation, and γH2AX foci, a marker of DSBs, persisted in irradiated mutant cells, consistent with a defect in DSB repair. Mutant mice were also hypersensitive to irradiation, establishing an essential role for ERCC1-XPF in protecting again...

Molecular and Cellular Biology, 2000

Absence of Ku80 results in increased sensitivity to ionizing radiation, defective lymphocyte deve... more Absence of Ku80 results in increased sensitivity to ionizing radiation, defective lymphocyte development, early onset of an age-related phenotype, and premature replicative senescence. Here we investigate the role of p53 on the phenotype of ku80 -mutant mice and cells. Reducing levels of p53 increased the cancer incidence for ku80 −/− mice. About 20% of ku80 −/− p53 +/− mice developed a broad spectrum of cancer by 40 weeks and all ku80 −/− p53 −/− mice developed pro-B-cell lymphoma by 16 weeks. Reducing levels of p53 rescued populations of ku80 −/− cells from replicative senescence by enabling spontaneous immortalization. The double-mutant cells are impaired for the G 1 /S checkpoint due to the p53 mutation and are hypersensitive to γ-radiation and reactive oxygen species due to the Ku80 mutation. These data show that replicative senescence is caused by a p53-dependent cell cycle response to damaged DNA in ku80 −/− cells and that p53 is essential for preventing very early onset of p...

Mechanisms of Ageing and Development, 2001

The phenomenon that caloric restriction increases life span in a variety of species from yeast to... more The phenomenon that caloric restriction increases life span in a variety of species from yeast to mice has been the focus of much interest. Recent observations suggest that a protein important for heterochromatin formation, Sir2, is central for caloric restriction-induced longevity in lower organisms. Interestingly, Sir2 is also capable of repairing DNA doublestrand breaks by nonhomologous end joining which may be important, along with proteins that repair breaks by recombinational repair, for minimizing the age-related deleterious effects of DNA damage induced by oxygen by-products of metabolism. I propose that competition between these two distinct functions could influence longevity and the onset of senescence. In addition, sequence and functional similarities between Sir2 and other chromatin metabolism proteins present the possibility that genetic components for longevity and senescence are conserved from yeast to mammals.

Mechanisms of Ageing and Development, 2007

Semantic distinctions between "normal" aging, "pathological" aging (or age-related disease) and "... more Semantic distinctions between "normal" aging, "pathological" aging (or age-related disease) and "premature" aging (otherwise known as segmental progeria) potentially confound important insights into the nature of each of the complex processes. Here we review a recent, unexpected discovery: the presence of longevity-associated characteristics typical of long-lived endocrine-mutant and dietary-restricted animals in short-lived progeroid mice. These data suggest that a subset of symptoms observed in premature aging, and possibly normal aging as well, may be indirect manifestations of a beneficial adaptive stress response to endogenous oxidative damage, rather than a detrimental result of the damage itself.

Journal of Animal Physiology and Animal Nutrition, 2009

Osteoarthritis (OA) is the most common form of arthritis, which causes severe inflammation and lo... more Osteoarthritis (OA) is the most common form of arthritis, which causes severe inflammation and loss of cartilage. It is a debilitating disease that commonly affects thousands of horses each year. Recently, we assessed the anti-arthritic and anti-inflammatory potential of undenatured type II collagen (UC-II) in horses. This comparative investigation evaluated arthritic pain in horses receiving daily placebo, UC-II 320 mg/day (providing 80 mg active UC-II), 480 mg/day (providing 120 mg active UC-II), or 640 mg/day (providing 160 mg active UC-II), and glucosamine and chondroitin (5.4 g/ day and 1.8 g/day, respectively, bid for the first month, and thereafter once daily) for 150 days. Pain in each leg was evaluated using the flexion test and the lameness-grading system after the initial two strides. Average pain of all four legs represented the pain for each horse. Horses receiving placebo showed no change in arthritic condition, while those receiving 320, 480, or 640 mg UC-II exhibited significant reduction in arthritic pain (P < 0.05). UC-II at 480 mg dose provided optimal effects. With this dose, reduction in overall pain was from 5.7 ± 0.0.42 (100%) to 0.7 ± 0.42 (12%); and in pain upon limb manipulation from 2.35 ± 0.37 (100%) to 0.52 ± 0.18 (22%). In regards to glucosamine and chondroitin treated group, although reduction in pain was significant compared to pretreated values, the efficacy was significantly less compared with that observed with UC-II. UC-II was found to be twice as effective as glucosamine and chondroitin in arthritic horses. Clinically, physical condition, and liver (ALP, GGT, and bilirubin), kidney (BUN and creatinine), and heart (CK) functions remained unchanged, suggesting that these supplements were well tolerated. Overall, these results demonstrate that UC-II was significantly more efficacious than glucosamine and chondroitin in arthritic horses.

genesis, 2011

Transgenesis enables the elucidation of gene function; however, constant transgene expression is ... more Transgenesis enables the elucidation of gene function; however, constant transgene expression is not always desired. The tetracycline responsive system was devised to turn on and off transgene expression at will. It has two components: a doxycycline controlled transactivator (TA) and an inducible expression cassette. Integration of these transgenes requires two transfection steps usually accomplished by sequential random integration. Unfortunately, random integration can be problematic due to chromatin position effects, integration of variable transgene units and mutation at the integration site. Therefore, targeted transgenesis and knockin were developed to target the TA and the inducible expression cassette to a specific location, but these approaches can be costly in time, labor and money. Here we describe a one-step Cre-mediated knockin system in mouse embryonic stem (ES) cells that positions the TA and inducible expression cassette to a single location. Using this system, we show doxycycline-dependent regulation of eGFP at the DNA topoisomerase 3β (Top3β) promoter. Since Cre-mediated recombination is used in lieu of gene targeting, this system is fast and efficient.

genesis, 2008

Single nucleotide polymorphisms (SNPs) may influence protein function possibly contributing to ph... more Single nucleotide polymorphisms (SNPs) may influence protein function possibly contributing to phenotype; yet, for most SNPs their potential influence is unknown. Here, we present a technique in mouse embryonic stem cells that enables high-throughput knock-in (the placement of coding sequences adjacent to a specific endogenous promoter). Our methodology utilizes gene targeting with a combination of two selection cassettes (SAbetageo and the HPRT minigene) along with site-specific recombinases (Cre/loxP and FLP/FRT) to efficiently introduce multiple DNA sequences, including enhanced green fluorescent protein (eGFP), adjacent to the DNA topoisomerase 3beta (Top3beta) promoter. This technology enables rapid and efficient introduction of DNA sequences to a specific location and advances high-throughput analysis of many SNPs with control for expression and genetic background.