Greg A. Freyer

RMI1/NCE4, a suppressor of genome instability, encodes a member of the RecQ helicase/Topo III complex

SGS1 encodes a DNA helicase whose homologues in human cells include the BLM, WRN, and RECQ4 genes, mutations in which lead to cancer‐predisposition syndromes. Clustering of synthetic genetic interactions identified by large‐scale genetic network analysis revealed that the genetic interaction profile of the gene RMI1 (RecQ‐mediated genome instability, also known as NCE4 and YPL024W) was highly similar to that of SGS1 and TOP3, suggesting a functional relationship between Rmi1 and the Sgs1/Top3 complex. We show that Rmi1 physically interacts with Sgs1 and Top3 and is a third member of this complex. Cells lacking RMI1 activate the Rad53 checkpoint kinase, undergo a mitotic delay, and display increased relocalization of the recombination repair protein Rad52, indicating the presence of spontaneous DNA damage. Consistent with a role for RMI1 in maintaining genome integrity, rmi1Δ cells exhibit increased recombination frequency and increased frequency of gross chromosomal rearrangements. In addition, rmi1Δ strains fail to fully activate Rad53 upon exposure to DNA‐damaging agents, suggesting that Rmi1 is also an important part of the Rad53‐dependent DNA damage response.

Association of Childhood Obesity With Maternal Exposure to Ambient Air Polycyclic Aromatic Hydrocarbons During Pregnancy

There are concerns that prenatal exposure to endocrine-disrupting chemicals increases childrens risk of obesity. African-American and Hispanic children born in the Bronx or Northern Manhattan, New York (1998-2006), whose mothers underwent personal air monitoring for polycyclic aromatic hydrocarbon (PAH) exposure during pregnancy, were followed up to ages 5 (n = 422) and 7 (n = 341) years. At age 5 years, 21% of the children were obese, as were 25% of those followed to age 7 years. After adjustment for childs sex, age at measurement, ethnicity, and birth weight and maternal receipt of public assistance and prepregnancy obesity, higher prenatal PAH exposures were significantly associated with higher childhood body size. In adjusted analyses, compared with children of mothers in the lowest tertile of PAH exposure, children of mothers in the highest exposure tertile had a 0.39-unit higher body mass index z score (95% confidence interval (CI): 0.08, 0.70) and a relative risk of 1.79 (95% CI: 1.09, 2.96) for obesity at age 5 years, and they had a 0.30-unit higher body mass index z score (95% CI: 0.01, 0.59), a 1.93-unit higher percentage of body fat (95% CI: 0.33, 3.54), and a relative risk of 2.26 (95% CI: 1.28, 4.00) for obesity at age 7 years. The data indicate that prenatal exposure to PAHs is associated with obesity in childhood.

Fission Yeast rad12+ Regulates Cell Cycle Checkpoint Control and Is Homologous to the Bloom’s Syndrome Disease Gene

ABSTRACT The human BLM gene is a member of the Escherichia coli recQ helicase family, which includes the Saccharomyces cerevisiae SGS1 and human WRN genes. Defects inBLM are responsible for the human disease Bloom’s syndrome, which is characterized in part by genomic instability and a high incidence of cancer. Here we describe the cloning ofrad12+, which is the fission yeast homolog ofBLM and is identical to the recently reportedrhq1 + gene. We showed that rad12null cells are sensitive to DNA damage induced by UV light and γ radiation, as well as to the DNA synthesis inhibitor hydroxyurea. Overexpression of the wild-type rad12 + gene also leads to sensitivity to these agents and to defects associated with the loss of the S-phase and G2-phase checkpoint control. We showed genetically and biochemically thatrad12 + acts upstream fromrad9 +, one of the fission yeast G2checkpoint control genes, in regulating exit from the S-phase checkpoint. The physical chromosome segregation defects seen inrad12 null cells combined with the checkpoint regulation defect seen in the rad12 + overproducer implicate rad12 + as a key coupler of chromosomal integrity with cell cycle progression.

Microbial mineral weathering for nutrient acquisition releases arsenic.

ABSTRACT Tens of millions of people in Southeast Asia drink groundwater contaminated with naturally occurring arsenic. How arsenic is released from the sediment into the water remains poorly understood. Here, we show in laboratory experiments that phosphate-limited cells of Burkholderia fungorum mobilize ancillary arsenic from apatite. We hypothesize that arsenic mobilization is a by-product of mineral weathering for nutrient acquisition. The released arsenic does not undergo a redox transformation but appears to be solubilized from the apatite mineral lattice during weathering. Analysis of apatite from the source area in the Himalayan basin indicates the presence of elevated levels of arsenic, with an average concentration of 210 mg/kg. The rate of arsenic release is independent of the initial dissolved arsenic concentration and occurs at phosphate levels observed in Bangladesh aquifers. We also demonstrate the presence of the microbial phenotype that releases arsenic from apatite in Bangladesh aquifer sediments and groundwater. These results suggest that microbial mineral weathering for nutrient acquisition could be an important mechanism for arsenic mobilization.

A single unbranched S-phase DNA damage and replication fork blockage checkpoint pathway

The eukaryotic intra-S-phase checkpoint, which slows DNA synthesis in response to DNA damage, is poorly understood. Is DNA damage recognized directly, or indirectly through its effects on replication forks? Is the slowing of S phase in part because of competition between DNA synthesis and recombination/repair processes? The results of our genetic analyses of the intra-S-phase checkpoint in the fission yeast, Schizosaccharomyces pombe, suggest that the slowing of S phase depends weakly on the helicases Rqh1 and Srs2 but not on other recombination/repair pathways. The slowing of S phase depends strongly on the six checkpoint-Rad proteins, on Cds1, and on Rad4/Cut5 (similar to budding yeast Dpb11, which interacts with DNA polymerase ɛ) but not on Rhp9 (similar to budding yeast Rad9, necessary for direct damage recognition). These results suggest that, in fission yeast, the signal activating the intra-S-phase checkpoint is generated only when replication forks encounter DNA damage.

An alternative eukaryotic DNA excision repair pathway.

DNA lesions induced by UV light, cyclobutane pyrimidine dimers, and (6-4)pyrimidine pyrimidones are known to be repaired by the process of nucleotide excision repair (NER). However, in the fission yeast Schizosaccharomyces pombe, studies have demonstrated that at least two mechanisms for excising UV photo-products exist; NER and a second, previously unidentified process. Recently we reported that S. pombe contains a DNA endonuclease, SPDE, which recognizes and cleaves at a position immediately adjacent to cyclobutane pyrimidine dimers and (6-4)pyrimidine pyrimidones. Here we report that the UV-sensitive S. pombe rad12-502 mutant lacks SPDE activity. In addition, extracts prepared from the rad12-502 mutant are deficient in DNA excision repair, as demonstrated in an in vitro excision repair assay. DNA repair activity was restored to wild-type levels in extracts prepared from rad12-502 cells by the addition of partially purified SPDE to in vitro repair reaction mixtures. When the rad12-502 mutant was crossed with the NER rad13-A mutant, the resulting double mutant was much more sensitive to UV radiation than either single mutant, demonstrating that the rad12 gene product functions in a DNA repair pathway distinct from NER. These data directly link SPDE to this alternative excision repair process. We propose that the SPDE-dependent DNA repair pathway is the second DNA excision repair process present in S. pombe.

Advection of surface-derived organic carbon fuels microbial reduction in Bangladesh groundwater

Chronic exposure to arsenic (As) by drinking shallow groundwater causes widespread disease in Bangladesh and neighboring countries. The release of As naturally present in sediment to groundwater has been linked to the reductive dissolution of iron oxides coupled to the microbial respiration of organic carbon (OC). The source of OC driving this microbial reduction—carbon deposited with the sediments or exogenous carbon transported by groundwater—is still debated despite its importance in regulating aquifer redox status and groundwater As levels. Here, we used the radiocarbon (14C) signature of microbial DNA isolated from groundwater samples to determine the relative importance of surface and sediment-derived OC. Three DNA samples collected from the shallow, high-As aquifer and one sample from the underlying, low-As aquifer were consistently younger than the total sediment carbon, by as much as several thousand years. This difference and the dominance of heterotrophic microorganisms implies that younger, surface-derived OC is advected within the aquifer, albeit more slowly than groundwater, and represents a critical pool of OC for aquifer microbial communities. The vertical profile shows that downward transport of dissolved OC is occurring on anthropogenic timescales, but bomb 14C-labeled dissolved OC has not yet accumulated in DNA and is not fueling reduction. These results indicate that advected OC controls aquifer redox status and confirm that As release is a natural process that predates human perturbations to groundwater flow. Anthropogenic perturbations, however, could affect groundwater redox conditions and As levels in the future.

Prenatal Exposure to Phthalates and Childhood Body Size in an Urban Cohort

Background: Phthalate exposures are hypothesized to increase obesity; however, prior research has been largely cross-sectional. Objective: We evaluated associations between prenatal phthalate exposures and body mass index (BMI) at child ages 5 and 7 years. Methods: Nine metabolites of six phthalates—di(2-ethylhexyl) phthalate (DEHP), di-n-octyl-, di-iso-butyl-, di-n-butyl-, butylbenzyl-, and diethyl phthalates—were measured in spot urine samples collected from pregnant African-American and Dominican women during their third trimester, and from their children at ages 3 and 5 years. To reduce multiple comparison issues, we initially used principal component analysis (PCA) to identify major patterns of natural log (ln)-transformed metabolite concentrations. Height and weight were assessed at ages 5 and 7 years, and fat mass and waist circumference at age 7. Linearized generalized estimating equation analyses related maternal component scores to child anthropometric outcomes at ages 5 (n = 326) and 7 (n = 330) years. Results: PCA identified a DEHP component and a non-DEHP component. In boys, higher maternal non-DEHP, but not DEHP, component scores were associated with lower BMI z-score (β = –0.30; 95% CI: –0.50, –0.10, n = 156), lower fat percentage (β = –1.62; 95% CI: –2.91, –0.34, n = 142), and smaller waist circumference (β = –2.02; 95% CI: –3.71, –0.32, n = 124). No significant associations with anthropometric outcomes were seen in girls (for BMI z-score, β = 0.07; 95% CI: –0.18, 0.31, n = 181). Interactions between sex and non-DEHP component association with outcomes were statistically significant (p < 0.01). Conclusions: Contrary to hypotheses, prenatal non-DEHP phthalate exposures were associated with lower BMI z-score, waist circumference, and fat mass in boys during early childhood. Citation: Maresca MM, Hoepner LA, Hassoun A, Oberfield SE, Mooney SJ, Calafat AM, Ramirez J, Freyer G, Perera FP, Whyatt RM, Rundle AG. 2016. Prenatal exposure to phthalates and childhood body size in an urban cohort. Environ Health Perspect 124:514–520; http://dx.doi.org/10.1289/ehp.1408750

XRCC1 polymorphisms and breast cancer risk from the New York Site of the Breast Cancer Family Registry: A family-based case-control study

Background: XRCC1 is a scaffold protein involved in the early and late stages of Base Excision Repair (BER). Three DNA polymorphisms occur in XRCC1, resulting in non-synonymous amino acid changes, which could alter the binding or regulatory activities of XRCC1. Materials and Methods: We used a family-based case-control study design to evaluate the association between XRCC1 polymorphisms and breast cancer risk. Participants were breast cancer cases and their unaffected sisters enrolled in the New York Site of the Breast Cancer Family Registry. Conditional logistic regression was used to assess associations between genotype and breast cancer. XRCC1 mRNA levels and DNA nicking activity were measured in lymphoblastoid cell lines from unaffected sisters to determine whether the XRCC1 R399Q polymorphism has a functional effect on expression or protein activity. Results: XRCC1 194W was associated with a non-significant increase in breast cancer, while XRCC1 280H and XRCC1 399Q were associated with a non-significant decrease in breast cancer. We found a significant increase in XRCC1 expression in 399Q/Q lymphoblastoid cell lines from unaffected sisters (n=28, P=0.03). An increase in median nicking activity was not statistically significant. Conclusions: Our results suggest that XRCC1 399Q may alter mRNA expression and DNA repair phenotype, although the main effects of the genotype were not significantly associated with familial cancer risk. Additional research on the regulation of XRCC1 expression will contribute to an understanding of how this polymorphism may impact disease risk.

Mus81-Eme1-Dependent and -Independent Crossovers Form in Mitotic Cells during Double-Strand Break Repair in Schizosaccharomyces pombe

ABSTRACT During meiosis, double-strand breaks (DSBs) lead to crossovers, thought to arise from the resolution of double Holliday junctions (HJs) by an HJ resolvase. In Schizosaccharomyces pombe, meiotic crossovers are produced primarily through a mechanism requiring the Mus81-Eme1 endonuclease complex. Less is known about the processes that produces crossovers during the repair of DSBs in mitotic cells. We employed an inducible DSB system to determine the role of Rqh1-Top3 and Mus81-Eme1 in mitotic DSB repair and crossover formation in S. pombe. In agreement with the meiotic data, crossovers are suppressed in cells lacking Mus81-Eme1. And relative to the wild type, rqh1Δ cells show a fourfold increase in crossover frequency. This suppression of crossover formation by Rqh1 is dependent on its helicase activity. We found that the synthetic lethality of cells lacking both Rqh1 and Eme1 is suppressed by loss of swi5+, which allowed us to show that the excess crossovers formed in an rqh1Δ background are independent of Mus81-Eme1. This result suggests that a second process for crossover formation exists in S. pombe and is consistent with our finding that deletion of swi5+ restored meiotic crossovers in eme1Δ cells. Evidence suggesting that Rqh1 also acts downstream of Swi5 in crossover formation was uncovered in these studies. Our results suggest that during Rhp51-dependent repair of DSBs, Rqh1-Top3 suppresses crossovers in the Rhp57-dependent pathway while Mus81-Eme1 and possibly Rqh1 promote crossovers in the Swi5-dependent pathway.