David L. Mitchell

THE BIOLOGY OF THE (6–4) PHOTOPRODUCT

The (6-4) photoproduct is an important determinant of the lethal and mutagenic effects of UV irradiation of biological systems. The removal of this lesion appears to correlate closely with the early DNA repair responses of mammalian cells, including DNA incision events, repair synthesis and removal of replication blocks. The processing of (6-4) photoproducts and cyclobutane dimers appears to be enzymatically coupled in bacteria and most mammalian cell lines examined (i.e. a mutation affecting the repair of one lesion also often affects the other), although exceptions exist in which repair capacity may be evident for one photoproduct and not the other (e.g. UV61 and the XP revertant cell line). These differences in the processing of the two photoproducts in some cell lines of human and rodent origin suggest that in mammalian cells, different pathways for the repair of (6-4) photoproducts and cyclobutane dimers may be used. This observation is further supported by pleiotropic repair phenotypes such as those observed in CHO complementation class 2 mutants (e.g., UV5, UVL-1, UVL-13, and V-H1). Indirect data, from HCR of UV irradiated reported genes and the cytotoxic responses of UV61, suggest that the (6-4) photoproduct is cytotoxic in mammalian cells and may account for 20 to 30% of the cell killing after UV irradiation of rodent cells. Cytotoxicity of the (6-4) photoproduct may be important in the etiology of sunlight-induced carcinogenesis, affecting mutagenesis as well as tumorigenesis. The intricate photochemistry of the (6-4) photoproduct, its formation and photoisomerization, is in itself extremely interesting and may also be relevant to sunlight carcinogenesis. The data reviewed in this article support the notion that the (6-4) photoproduct and its Dewar photoisomer are important cytotoxic determinants of UV light. The idea that the (6-4) photoproduct is an important component in the spectrum of UV-induced cytotoxic damage may help clarify our understanding of why rodent cells survive the effects of UV irradiation as well as human cells, without apparent cyclobutane dimer repair in the bulk of their DNA. The preferential repair of cyclobutane dimers in essential genes has been proposed to account for this observation (Bohr et al., 1985, 1986; Mellon et al., 1986). The data reviewed here suggest that understanding the repair of a prominent type of noncyclobutane dimer damage, the (6-4) photoproduct, may also be important in resolving this paradox.

The relative cytotoxicity of (6-4) photoproducts and cyclobutane dimers in mammalian cells.

Abstract— The significance of the pyrimidine(6‐4)pyrimidone photoproduct in mammalian cell killing is considered. Photochemical data indicate that the(6–4) photoproduct is induced at a substantial frequency compared to the cyclobutane dimer and that the action spectra for the induction of both lesions are equivalent. The repair of(6–4) photoproducts in various normal and UV‐hypcrsensitive mammalian cell lines, including several recently derived somatic cell hybrids and transformants, is presented. The sensitivity of these cells to ultraviolet irradiation correlates better with the capacity to repair(6–4) photoproducts than cyclobutane dimers. These data are used to support that idea that the(6–4) photoproduct is one of the major cytotoxic lesions induced in DNA by ultraviolet light.

(6–4)Photoproducts are removed from the DNA of UV-irradiated mammalian cells more efficiently than cyclobutane pyrimidine dimers

A polyclonal antiserum raised against UV-irradiated DNA can be used to assay cyclobutane pyrimidine dimers and Pyr(6-4)Pyo photoproducts specifically by changing the nature of the 32P-labelled antigen. Pyr(6-4)Pyo photoproducts were removed faster than cyclobutane dimers in UV-irradiated human, hamster and mouse cells. Xeroderma pigmentosum cells from complementation groups A, C and D were deficient in the repair of both lesions.

ACTION SPECTRA FOR THE INDUCTION OF PYRIMIDINE(6-4)PYRIMIDONE PHOTOPRODUCTS AND CYCLOBUTANE PYRIMIDINE DIMERS IN NORMAL HUMAN SKIN FIBROBLASTS

Abstract. Normal human skin fibroblasts were exposed to 265–313‐nm monochromatic UV wavelengths and the yield of pyrimidine(6‐4)pyrimidone photoproducts [(6‐4) photoproducts] and cyclobutane pyrimidine dimers (dimers) measured by radioimmunoassay. The action spectra for the induction of these two types of DNA damage were very similar for wavelengths from 254 to 302 nm. However, the action spectrum value for (6‐4) photoproduct production was less than half the value obtained for dimer induction by 313‐nm UV. Cells were also exposed to the UV produced by a broad‐spectrum fluorescent sunlamp (280–360 nm, peak at 313 nm) under conditions in which various wavelength components were removed from the spectrum. For exposures in which the (6‐4) photoproducts and dimers were induced primarily by wavelengths shorter than 310 nm, their rates of induction, relative to 254‐nm‐irradiated cells, were similar. However, the level of (6‐4) photoproduct production was about three‐fold lower than dimer induction for sunlamp irradiations in which the 315–330‐nm component of this source was primarily responsible for the formation of this damage. These results are consistent with the conclusion that for treatments in which (6‐4) photoproducts are produced by wavelengths in the 310–320‐nm range, which represent the region of absorption peaks for these photoproducts, both the induction and photolysis of (6‐4) photoproducts occur simultaneously during irradiation.

Long-term persistence of bacterial DNA

The persistence of bacterial DNA over geological timespans remains a contentious issue. In direct contrast to in vitro based predictions, bacterial DNA and even culturable cells have been reported from various ancient specimens many million years (Ma) old [1–8]. As both ancient DNA studies and the revival of microorganisms are known to be susceptible to contamination [8–10], it is concerning that these results have not been independently replicated to confirm their authenticity. Furthermore, they show no obvious relationship between sample age, and either bacterial composition or DNA persistence, although bacteria are known to differ markedly in hardiness and resistance to DNA degradation [11]. We present the first study of DNA durability and degradation of a broad variety of bacteria preserved under optimal frozen conditions, using rigorous ancient DNA methods [8–10]. The results demonstrate that nonspore-forming gram-positive (GP) Actinobacteria are by far the most durable, out-surviving endosporeformers such as Bacillaceae and Clostridiaceae. The observed DNA degradation rates are close to theoretical calculations [9], indicating a limit of ca. 400 thousand years (kyr) beyond which PCR amplifications are prevented by the formation of DNA interstrand crosslinks (ICLs). The twelve permafrost samples (0-8.1 Ma) investigated were obtained from northeast Siberia and Beacon Valley, Antarctica. DNA preservation at these sites is exceptional due to constant subzero temperatures, largely neutral pH, and anaerobic conditions. Epifluorescence microscopy revealed ~107cells/gram wetweight in the bacterial size range. The cell counts are in agreement with previous results obtained on permafrost [2,3]. 16S rDNA sequences of 120 bp and 600 bp could be reproducibly amplified from samples up to 400–600 kyr, and show an inverse relationship between PCR amplification efficiency and fragment length that is typical of ancient DNA [8–10,12]. Controls for surface contamination during sampling were negative. Chimeric sequences were excluded from analysis, along with sequences that failed a bootstrap test for independent reproducibility [13]. DNA concentrations and taxonomic diversity were found to decrease with age until 400–600 kyr, at which point the percentage of templates with ICLs reached 100% (Figure 1A–C). Sequences from the older samples appear to be a subset of those from younger material, and all identified bacterial taxa are known soil inhabitants, indicating that permafrost is a nonextremophile environment. There were clear age-related patterns in taxon survival across geographically widespread samples (separated up to 1400 km). Sequences of non-sporeforming GP Actinobacteria, affiliated largely to the genus Arthrobacter (99–100% similarity), consistently persisted for the longest time, followed by GP endospore-forming Bacillaceae and Clostridiaceae and finally gram-negative (GN) bacteria, mostly Proteobacteria (Figure 1D).

AMBIENT SOLAR RADIATION-INDUCED PHOTODAMAGE IN MARINE BACTERIOPLANKTON

Abstract— There has been much recent concern about the effects of increased UV radiation at certain locations on the earths surface. There have been extensive studies of ultraviolet radiation effects on phytoplankton and primary production, yet the effects of UVB upon bacterioplankton have been largely overlooked. Bacteria play a central role in the cycling of nutrients and energy flow to higher trophic levels, serving as both mineralizers and secondary producers that are consumed by higher organisms. We have begun to investigate the induction of DNA photodamage by UVB in marine planktonic communities using a highly specific radioimmunoassay to measure cyclobutane pyrimidine dimers in samples collected from the northern Gulf of Mexico. DNA damage in the bacterioplankton size‐fraction (< 0.8 μ.m) was greater than in the larger eukaryotic size fraction (>0.8 μm <120 μm) in 9 of 10 samples. Diel patterns of dimer accumulation and repair were observed in surface waters over a 48 h period in the bacterioplankton size fraction and in the larger eukaryotic plankton size fraction. Depth profiles of DNA damage in the bacterioplankton size fraction appear to be dependent on surface water mixing. Damage was greatest in surface waters, decreased with depth and could be detected to 10 m in calm seas. No net accumulation of damage was observed in moderate seas, even at the surface. Solar radiation was found to inhibit significantly both 3H‐thymidine and 14C‐leucine incorporation. Ultraviolet B was responsible for approximately half of the total inhibition of 3H‐thymidine incorporation, UVA contributing the other half of the inhibition. The vast majority of 14C‐leucine incorporation inhibition was due to UVB, suggesting that protein synthesis is less affected by UVA. The results demonstrate that direct measures of DNA damage can be made of indigenous planktonic communities and that bacterioplankton are highly susceptible to UVB damage and may serve as a more sensitive indicator of UVR stress than other microorganisms.

RELATIVE INDUCTION OF CYCLOBUTANE DIMERS and CYTOSINE PHOTOHYDRATES IN DNA IRRADIATED in vitro and in vivo WITH ULTRAVIOLET‐C and ULTRAVIOLET‐B LIGHT

SV40 DNA was irradiated in vitro and in vivo with UV‐C (240–280nm) and UV‐B (280–320nm) light, and damaged sites sensitive to digestion with Escherichia coli endonuclease III (endo III) and bacteriophage T4 endonuclease V (endo V) were quantified. The frequency of endo Ill‐sensitive sites (primarily cytosine photohydrates) induced was1–2% of the frequency of endo V‐sensitive sites (cyclobutane dimers) in both purified SV40 DNA and intracellular episomal SV40 DNA. Endo III‐ and endo V‐sensitive sites in DNA were induced in the same relative proportion at both UV‐C and UV‐B wavelengths. We found no evidence to support earlier inferences that intracellular conditions enhance the formation of cytosine photohydrates or other monobasic forms of DNA damage.

GCN5 and E2F1 stimulate nucleotide excision repair by promoting H3K9 acetylation at sites of damage

Chromatin structure is known to be a barrier to DNA repair and a large number of studies have now identified various factors that modify histones and remodel nucleosomes to facilitate repair. In response to ultraviolet (UV) radiation several histones are acetylated and this enhances the repair of DNA photoproducts by the nucleotide excision repair (NER) pathway. However, the molecular mechanism by which UV radiation induces histone acetylation to allow for efficient NER is not completely understood. We recently discovered that the E2F1 transcription factor accumulates at sites of UV-induced DNA damage and directly stimulates NER through a non-transcriptional mechanism. Here we demonstrate that E2F1 associates with the GCN5 acetyltransferase in response to UV radiation and recruits GCN5 to sites of damage. UV radiation induces the acetylation of histone H3 lysine 9 (H3K9) and this requires both GCN5 and E2F1. Moreover, as previously observed for E2F1, knock down of GCN5 results in impaired recruitment of NER factors to sites of damage and inefficient DNA repair. These findings demonstrate a direct role for GCN5 and E2F1 in NER involving H3K9 acetylation and increased accessibility to the NER machinery.

The effect of vitamin E acetate on ultraviolet‐induced mouse skin carcinogenesis

Despite the benefits of sunscreens, ultraviolet (UV) exposure can still lead to skin cancer. In this study we investigated the effect of topical application of the antioxidant vitamin E acetate (VEA) on the inhibition of UV‐induced carcinogenesis. Hairless SKH‐1 mice received 5.2 mg of VEA 30 min before (VEA/UV) or after (UV/VEA) a single minimal erythemic dose of UV light. Vehicle‐control animals received acetone 30 min before UV exposure (Ace/UV). After 24 h, cyclobutane dimer repair was twofold and 1.5‐fold greater in the UV/VEA and VEA/UV groups, respectively. Expression of p53 protein in the UV/VEA group was maximum at 12 h after UV exposure, whereas in the Ace/UV‐ and VEA/UV‐treated mice, maximum p53 immunostaining was statistically higher at 15 h (P = 0.03). DNA synthesis as determined by 5‐bromo‐2′‐deoxyuridine incorporation was twofold higher after 15 h in all groups but was not statistically different among treatment groups. Protein levels of cyclin D1 and p21 were increased in both VEA groups by 6 h. In addition, VEA treatments delayed tumor formation and yield for the first 20 wk, although this difference was lost by 30 wk. The telomerase activity of carcinomas from the UV/VEA‐treated mice was statistically lower than that of the Ace/UV‐treated mice (P = 0.05). This study showed that although VEA may mitigate some of the initial events associated with UV irradiation such as DNA damage and p53 expression, it has limited potential in preventing UV‐induced proliferation and tumor formation. Mol. Carcinog. 23:175–184, 1998.

Crosslinks rather than strand breaks determine access to ancient DNA sequences from frozen sediments

Diagenesis was studied in DNA obtained from Siberian permafrost (permanently frozen soil) ranging from 10,000 to 400,000 years in age. Despite optimal preservation conditions, we found the sedimentary DNA to be severely modified by interstrand crosslinks; single- and double-stranded breaks; and freely exposed sugar, phosphate, and hydroxyl groups. Intriguingly, interstrand crosslinks were found to accumulate ∼100 times faster than single-stranded breaks, suggesting that crosslinking rather than depurination is the primary limiting factor for ancient DNA amplification under frozen conditions. The results question the reliability of the commonly used models relying on depurination kinetics for predicting the long-term survival of DNA under permafrost conditions and suggest that new strategies for repair of ancient DNA must be considered if the yield of amplifiable DNA from permafrost sediments is to be significantly increased. Using the obtained rate constant for interstrand crosslinks the maximal survival time of amplifiable 120-bp fragments of bacterial 16S ribosomal DNA was estimated to be ∼400,000 years. Additionally, a clear relationship was found between DNA damage and sample age, contradicting previously raised concerns about the possible leaching of free DNA molecules between permafrost layers.