ALBERT

Description: Genomic integrity is compromised by DNA polymerase replication errors, which occur in a sequence-dependent manner across the genome. Accurate and complete quantification of a DNA polymerase's error spectrum is challenging because errors are rare and difficult to detect. We report a high-throughput sequencing assay to map in vitro DNA replication errors at the single-molecule level. Unlike previous methods, our assay is able to rapidly detect a large number of polymerase errors at base resolution over any template substrate without quantification bias. To overcome the high error rate of high-throughput sequencing, our assay uses a barcoding strategy in which each replication product is tagged with a unique nucleotide sequence before amplification. This allows multiple sequencing reads of the same product to be compared so that sequencing errors can be found and removed. We demonstrate the ability of our assay to characterize the average error rate, error hotspots and lesion bypass fidelity of several DNA polymerases.

Description: Mammalian splicing regulatory protein RNA-binding motif protein 4 (RBM4) has an alanine repeat-containing C-terminal domain (CAD) that confers both nuclear- and splicing speckle-targeting activities. Alanine-repeat expansion has pathological potential. Here we show that the alanine-repeat tracts influence the subnuclear targeting properties of the RBM4 CAD in cultured human cells. Notably, truncation of the alanine tracts redistributed a portion of RBM4 to paraspeckles. The alanine-deficient CAD was sufficient for paraspeckle targeting. On the other hand, alanine-repeat expansion reduced the mobility of RBM4 and impaired its splicing activity. We further took advantage of the putative coactivator activator (CoAA)-RBM4 conjoined splicing factor, CoAZ, to investigate the function of the CAD in subnuclear targeting. Transiently expressed CoAZ formed discrete nuclear foci that emerged and subsequently separated—fully or partially—from paraspeckles. Alanine-repeat expansion appeared to prevent CoAZ separation from paraspeckles, resulting in their complete colocalization. CoAZ foci were dynamic but, unlike paraspeckles, were resistant to RNase treatment. Our results indicate that the alanine-rich CAD, in conjunction with its conjoined RNA-binding domain(s), differentially influences the subnuclear localization and biogenesis of RBM4 and CoAZ.

Description: Etheno DNA adducts are a prevalent type of DNA damage caused by vinyl chloride (VC) exposure and oxidative stress. Etheno adducts are mutagenic and may contribute to the initiation of several pathologies; thus, elucidating the pathways by which they induce cellular transformation is critical. Although N 2 ,3-ethenoguanine ( N 2 ,3-G) is the most abundant etheno adduct, its biological consequences have not been well characterized in cells due to its labile glycosidic bond. Here, a stabilized 2'-fluoro-2'-deoxyribose analog of N 2 ,3-G was used to quantify directly its genotoxicity and mutagenicity. A multiplex method involving next-generation sequencing enabled a large-scale in vivo analysis, in which both N 2 ,3-G and its isomer 1, N 2 -ethenoguanine (1, N 2 -G) were evaluated in various repair and replication backgrounds. We found that N 2 ,3-G potently induces G to A transitions, the same mutation previously observed in VC-associated tumors. By contrast, 1, N 2 -G induces various substitutions and frameshifts. We also found that N 2 ,3-G is the only etheno lesion that cannot be repaired by AlkB, which partially explains its persistence. Both G lesions are strong replication blocks and DinB, a translesion polymerase, facilitates the mutagenic bypass of both lesions. Collectively, our results indicate that N 2 ,3-G is a biologically important lesion and may have a functional role in VC-induced or inflammation-driven carcinogenesis.

Description: Seismic ambient noise tomography is applied to central and southern Mozambique, located in the tip of the East African Rift (EAR). The deployment of MOZART seismic network, with a total of 30 broad-band stations continuously recording for 26 months, allowed us to carry out the first tomographic study of the crust under this region, which until now remained largely unexplored at this scale. From cross-correlations extracted from coherent noise we obtained Rayleigh wave group velocity dispersion curves for the period range 5–40 s. These dispersion relations were inverted to produce group velocity maps, and 1-D shear wave velocity profiles at selected points. High group velocities are observed at all periods on the eastern edge of the Kaapvaal and Zimbabwe cratons, in agreement with the findings of previous studies. Further east, a pronounced slow anomaly is observed in central and southern Mozambique, where the rifting between southern Africa and Antarctica created a passive margin in the Mesozoic, and further rifting is currently happening as a result of the southward propagation of the EAR. In this study, we also addressed the question concerning the nature of the crust (continental versus oceanic) in the Mozambique Coastal Plains (MCP), still in debate. Our data do not support previous suggestions that the MCP are floored by oceanic crust since a shallow Moho could not be detected, and we discuss an alternative explanation for its ocean-like magnetic signature. Our velocity maps suggest that the crystalline basement of the Zimbabwe craton may extend further east well into Mozambique underneath the sediment cover, contrary to what is usually assumed, while further south the Kaapval craton passes into slow rifted crust at the Lebombo monocline as expected. The sharp passage from fast crust to slow crust on the northern part of the study area coincides with the seismically active NNE-SSW Urema rift, while further south the Mazenga graben adopts an N-S direction parallel to the eastern limit of the Kaapvaal craton. We conclude that these two extensional structures herald the southward continuation of the EAR, and infer a structural control of the transition between the two types of crust on the ongoing deformation.

Description: Escherichia coli has three DNA polymerases implicated in the bypass of DNA damage, a process called translesion synthesis (TLS) that alleviates replication stalling. Although these polymerases are specialized for different DNA lesions, it is unclear if they interact differently with the replication machinery. Of the three, DNA polymerase (Pol) II remains the most enigmatic. Here we report a stable ternary complex of Pol II, the replicative polymerase Pol III core complex and the dimeric processivity clamp, β. Single-molecule experiments reveal that the interactions of Pol II and Pol III with β allow for rapid exchange during DNA synthesis. As with another TLS polymerase, Pol IV, increasing concentrations of Pol II displace the Pol III core during DNA synthesis in a minimal reconstitution of primer extension. However, in contrast to Pol IV, Pol II is inefficient at disrupting rolling-circle synthesis by the fully reconstituted Pol III replisome. Together, these data suggest a β-mediated mechanism of exchange between Pol II and Pol III that occurs outside the replication fork.

Description: Mutations in GATA4 and TBX5 are associated with congenital heart defects in humans. Interaction between GATA4 and TBX5 is important for normal cardiac septation, but the underlying molecular mechanisms are not well understood. Here, we show that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle, but after E15.5, ventricular expression of Tbx5 decreases. Co-localization and co-immunoprecipitation studies demonstrate an interaction of Gata4 and Tbx5 in the developing atria and ventricles, but the ventricular interaction declines after E14.5. Gata4 +/– ;Tbx5 +/– mouse embryos display decreased atrial and ventricular myocardial thickness at E11.5, prior to cardiac septation. To determine the cell lineage in which the interaction was functionally significant in vivo , mice heterozygous for Gata4 in the myocardium or endocardium and heterozygous for Tbx5 ( Gata4 MyoDel/wt ;Tbx5 +/– and Gata4 EndoDel/wt ;Tbx5 +/– , respectively) were generated. Gata4 MyoDel/wt ;Tbx5 +/– mice displayed embryonic lethality, thin myocardium with reduced cell proliferation, and atrioventricular septation defects similar to Gata4;Tbx5 compound heterozygotes while Gata4 EndoDel/wt ;Tbx5 +/– embryos were normal. Cdk4 and Cdk2 , cyclin-dependent kinases required for myocardial development and septation were reduced in Gata4 +/– ;Tbx5 +/– hearts. Cdk4 is a known direct target of Gata4 and the regulation of Cdk2 in the developing heart has not been studied. Chromatin immunoprecipitation and transactivation studies demonstrate that Gata4 and Tbx5 directly regulate Cdk4 while only Tbx5 activates Cdk2 expression. These findings highlight the mechanisms by which disruption of the Gata4 and Tbx5 interaction in the myocardium contributes to cardiac septation defects in humans.

Description: Antedependence models, also known as transition models, have proven to be useful for longitudinal data exhibiting serial correlation, especially when the variances and/or same-lag correlations are time-varying. Statistical inference procedures associated with normal antedependence models are well-developed and have many nice properties, but they are not appropriate for longitudinal data that exhibit considerable skewness. We propose two direct extensions of normal antedependence models to skew-normal antedependence models. The first is obtained by imposing antedependence on a multivariate skew-normal distribution, and the second is a sequential autoregressive model with skew-normal innovations. For both models, necessary and sufficient conditions for $p$ th-order antedependence are established, and likelihood-based estimation and testing procedures for models satisfying those conditions are developed. The procedures are applied to simulated data and to real data from a study of cattle growth.

Description: Scaling relationships among twig size, leaf size and leafing intensity fundamentally influence the twig–leaf deployment pattern, a property that affects the architecture and functioning of plants. However, our understanding of how these relationships change within a species or between species as a function of forest succession is unclear. We determined log–log scaling relationships between twig cross-sectional area (twig size) and each of total and individual leaf area, and leafing intensity (the number of leaves per twig volume) for 78 woody species along a successional series in subtropical evergreen forests in eastern China. The series included four stages: secondary shrub (S1), young (S2), sub-climax (S3) and climax evergreen broadleaved forests (S4). The scaling slopes in each of the three relationships did not differ among the four stages. The y -intercept did not shift among the successional stages in the relationship between twig cross-sectional area and total leaf area; however, the y -intercept was greatest in S4, intermediate in S3 and lowest in S2 and S1 for the relationship between twig size and individual leaf area, while the opposite pattern was found for the twig size-leafing intensity relationship. This indicates that late successional trees have few but large leaves while early successional trees have more small leaves per unit twig size. For the relationship between twig cross-sectional area and total leaf area, there was no difference in the regression slope between recurrent (appear in more than one stages) and non-recurrent species (appear in only one stage) for each of the S1–S2, S2–S3 and S3–S4 pairs. A significant difference in the y -intercept was found in the S2–S3 pair only. In the relationship between twig cross-sectional area and individual leaf area, the regression slope between recurrent and non-recurrent species was homogeneous in the S1–S2 and S3–S4 pairs, but heterogeneous in the S2–S3 pair. We conclude that forest succession caused the shift in the intercept, but did not affect scaling slopes for relationships among twig size, leaf size and leaf intensity. For recurrent species, the invariant scaling slope in the twig–leaf size relationship between adjacent pairs of successional stages may be related to their phenotypic plasticity by adjusting their twig and leaf deployment strategy to similar to what the non-recurrent species display.

Description: The unification scheme of active galactic nuclei invokes an optically thick molecular torus component hiding the broad emission line region. Assuming the presence of a thick neutral component in the molecular torus characterized by a H  i column density 〉10 22 cm –2 , we propose that far-UV radiation around Lyα can be significantly polarized through Rayleigh scattering. Adopting a Monte Carlo technique, we compute polarization of Rayleigh scattered radiation near Lyα in a thick neutral region in the shape of a slab and a cylindrical shell. It is found that radiation near Lyα Rayleigh reflected from a very thick slab can be significantly polarized in a fairly large range of wavelength  ~ 50 Å exhibiting a flux profile similar to the incident one. Rayleigh transmitted radiation in a slab is characterized by the central dip with a complicated polarization behaviour. The optically thick part near Lyα centre is polarized in the direction perpendicular to the slab normal, which is in contrast to weakly polarized wing parts in the direction parallel to the slab normal. A similar polarization flip phenomenon is also found in the case of a tall cylindrical shell, in which the spatial diffusion along the vertical direction near the inner cylinder wall for core photons leads to a tendency of the electric field aligned to the direction perpendicular to the vertical axis. Observational implications are briefly discussed including spectropolarimetry of the quasar PG 1630+377 by Koratkar et al. in 1990 where Lyα is strongly polarized with no other emission lines polarized.