Holly Miller

The novel DNA glycosylase, NEIL1, protects mammalian cells from radiation-mediated cell death

DNA damage mediated by reactive oxygen species generates miscoding and blocking lesions that may lead to mutations or cell death. Base excision repair (BER) constitutes a universal mechanism for removing oxidatively damaged bases and restoring the integrity of genomic DNA. In Escherichia coli, the DNA glycosylases Nei, Fpg, and Nth initiate BER of oxidative lesions; OGG1 and NTH1 proteins fulfill a similar function in mammalian cells. Three human genes, designated NEIL1, NEIL2 and NEIL3, encode proteins that contain sequence homologies to Nei and Fpg. We have cloned the corresponding mouse genes and have overexpressed and purified mNeil1, a DNA glycosylase that efficiently removes a wide spectrum of mutagenic and cytotoxic DNA lesions. These lesions include the two cis-thymineglycol(Tg) stereoisomers, guanine- and adenine-derived formamidopyrimidines, and 5,6-dihydrouracil. Two of these lesions, fapyA and 5S,6R thymine glycol, are not excised by mOgg1 or mNth1. We have also used RNA interference technology to establish embryonic stem cell lines deficient in Neil1 protein and showed them to be sensitive to low levels of gamma-irradiation. The results of these studies suggest that Neil1 is an essential component of base excision repair in mammalian cells; its presence may contribute to the redundant repair capacity observed in Ogg1 -/- and Nth1 -/- mice.

Lesion (in)tolerance reveals insights into DNA replication fidelity

The initial encounter of an unrepaired DNA lesion is likely to be with a replicative DNA polymerase, and the outcome of this event determines whether an error‐prone or error‐free damage avoidance pathway is taken. To understand the atomic details of this critical encounter, we have determined the crystal structures of the pol α family RB69 DNA polymerase with DNA containing the two most prevalent, spontaneously generated premutagenic lesions, an abasic site and 2′‐deoxy‐7,8‐dihydro‐8‐oxoguanosine (8‐oxodG). Identification of the interactions between these damaged nucleotides and the active site provides insight into the capacity of the polymerase to incorporate a base opposite the lesion. A novel open, catalytically inactive conformation of the DNA polymerase has been identified in the complex with a primed abasic site template. This structure provides the first molecular characterization of the DNA synthesis barrier caused by an abasic site and suggests a general mechanism for polymerase fidelity. In contrast, the structure of the ternary 8‐oxodG:dCTP complex is almost identical to the replicating complex containing unmodified DNA, explaining the relative ease and fidelity by which this lesion is bypassed.

Responses to the major acrolein-derived deoxyguanosine adduct in Escherichia coli.

Acrolein, a reactive α,β-unsaturated aldehyde found ubiquitously in the environment and formed endogenously in mammalian cells, reacts with DNA to form an exocyclic DNA adduct, 3H-8-hydroxy-3-(β-d-2′-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purine-9-one (γ-OH-PdG). The cellular processing and mutagenic potential of γ-OH-PdG have been examined, using a site-specific approach in which a single adduct is embedded in double-strand plasmid DNA. Analysis of progeny plasmid reveals that this adduct is excised by nucleotide excision repair. The apparent level of inhibition of DNA synthesis is ∼70% in Escherichia coli ΔrecA, uvrA. The block to DNA synthesis can be overcome partially byrecA-dependent recombination repair. Targeted G → T transversions were observed at a frequency of 7 × 10−4/translesion synthesis. Inactivation ofpolB, dinB, and umuD,C genes coding for “SOS” DNA polymerases did not affect significantly the efficiency or fidelity of translesion synthesis. In vitroprimer extension experiments revealed that the Klenow fragment of polymerase I catalyzes error-prone synthesis, preferentially incorporating dAMP and dGMP opposite γ-OH-PdG. We conclude from this study that DNA polymerase III catalyzes translesion synthesis across γ-OH-PdG in an error-free manner. Nucleotide excision repair, recombination repair, and highly accurate translesion synthesis combine to protect E. coli from the potential genotoxicity of this DNA adduct.

The thermodynamics of template-directed DNA synthesis: Base insertion and extension enthalpies

We used stopped-flow calorimetry to measure the overall enthalpy change associated with template-directed nucleotide insertion and DNA extension. Specifically, we used families of hairpin self-priming templates in conjunction with an exonuclease-free DNA polymerase to study primer extension by one or more dA or dT residues. Our results reveal exothermic heats between –9.8 and –16.0 kcal/bp for template-directed enzymatic polymerization. These extension enthalpies depend on the identity of the inserting base, the primer terminus, and/or the preceding base. Despite the complexity of the overall process, the sign, magnitude, and sequence dependence of these insertion and extension enthalpies are consistent with nearest-neighbor data derived from DNA melting studies. We recognize that the overall process studied here involves contributions from a multitude of events, including dNTP to dNMP hydrolysis, phosphodiester bond formation, and enzyme conformational changes. It is therefore noteworthy that the overall enthalpic driving force per base pair is of a magnitude similar to that expected for addition of one base pair or base stack per insertion event, rather than that associated with the rupture and/or formation of covalent bonds, as occurs during this catalytic process. Our data suggest a constant sequence-independent background of compensating enthalpic contributions to the overall process of DNA synthesis, with discrimination expressed by differences in noncovalent interactions at the template–primer level. Such enthalpic discrimination underscores a model in which complex biological events are regulated by relatively modest energy balances involving weak interactions, thereby allowing subtle mechanisms of regulation.

Questioning new answers regarding Holocene chicken domestication in China

Xiang et al. (1) assert that chickens were domesticated on the North China plain 10,000 y ago. Although a great deal remains unknown about the temporal and geographic origins of poultry husbandry, this claim is extraordinary. We welcome the increasing application of modern bioarcheological techniques to questions pertaining to animal domestication in China, but we are skeptical about these conclusions for several reasons.

RNA aptamers selected against DNA polymerase β inhibit the polymerase activities of DNA polymerases β and κ

DNA polymerase β (polβ), a member of the X family of DNA polymerases, is the major polymerase in the base excision repair pathway. Using in vitro selection, we obtained RNA aptamers for polβ from a variable pool of 8 × 1012 individual RNA sequences containing 30 random nucleotides. A total of 60 individual clones selected after seven rounds were screened for the ability to inhibit polβ activity. All of the inhibitory aptamers analyzed have a predicted tri-lobed structure. Gel mobility shift assays demonstrate that the aptamers can displace the DNA substrate from the polβ active site. Inhibition by the aptamers is not polymerase specific; inhibitors of polβ also inhibited DNA polymerase κ, a Y-family DNA polymerase. However, the RNA aptamers did not inhibit the Klenow fragment of DNA polymerase I and only had a minor effect on RB69 DNA polymerase activity. Polβ and κ, despite sharing little sequence similarity and belonging to different DNA polymerase families, have similarly open active sites and relatively few interactions with their DNA substrates. This may allow the aptamers to bind and inhibit polymerase activity. RNA aptamers with inhibitory properties may be useful in modulating DNA polymerase actvity in cells.

Fallow deer (Dama dama dama) management in Roman South-East Britain

This paper presents new carbon, nitrogen and sulphur isotope data for European fallow deer (Dama dama dama) in Roman Britain and discusses results in light of evidence from classical texts, landscape archaeology, zooarchaeology and the limited available samples of metric data. The new isotope data presented here are from Fishbourne Roman Palace (Sussex), two sites on the Isle of Thanet (Kent) and a further two sites in London. In spite of small sample sizes the data make an important contribution to the very limited corpus of scientific research on the species and provide new resolution to the nature of fallow deer movement and management in Roman Britain.

Disentangling the effect of farming practice from aridity on crop stable isotope values: A present-day model from Morocco and its application to early farming sites in the eastern Mediterranean

Agriculture has played a pivotal role in shaping landscapes, soils and vegetation. Developing a better understanding of early farming practices can contribute to wider questions regarding the long-term impact of farming and its nature in comparison with present-day traditional agrosystems. In this study we determine stable carbon and nitrogen isotope values of barley grains from a series of present-day traditionally managed farming plots in Morocco, capturing a range of annual rainfall and farming practices. This allows a framework to be developed to refine current isotopic approaches used to infer manuring intensity and crop water status in (semi-)arid regions. This method has been applied to charred crop remains from two early farming sites in the eastern Mediterranean: Abu Hureyra and ‘Ain Ghazal. In this way, our study enhances knowledge of agricultural practice in the past, adding to understanding of how people have shaped and adapted to their environment over thousands of years.

Dead or alive? Investigating long-distance transport of live fallow deer and their body parts in antiquity

The extent to which breeding populations of fallow deer were established in Roman Europe has been obscured by the possibility that the skeletal remains of the species, in particular Dama foot bones and antlers, were traded over long distances as objects in their own right. This paper sets out to refine our understanding of the evidence for the transportation of living and dead fallow deer in Iron Age and Roman Europe. To achieve this, museum archives containing purportedly early examples of Dama antler were searched, with available specimens sampled for carbon, nitrogen and strontium isotope analyses, and compared with data for archaeological fallow deer from across Europe. Importantly, the resulting isotope values can be interpreted in light of new modern baseline data for fallow deer presented here. Together these multi-isotope results for modern and archaeological fallow deer provide a more critical perspective on the transportation of fallow deer and their body parts in antiquity.

DNA repair investigations using siRNA.

Small interfering RNA (siRNA) is a revolutionary tool for the experimental modulation of gene expression, in many cases making redundant the need for specific gene mutations and allowing examination of the effect of modulating essential genes. It has now been shown that siRNA phenotypes resulting from stable transfection with short hairpin RNA (shRNA) can be transmitted through the mouse germ line and Rosenquist and his colleagues have used shRNA, which is processed in vivo to siRNA, to create germline transgenic mice in which a target DNA repair gene has been silenced. Here, Holly Miller and Arthur P. Grollman give the background of these discoveries, provide an overview of current uses, and look at future applications of this research.