The roles of specific glycosylases in determining the mutagenic consequences of clustered DNA base damage

The potential for genetic change arising from specific single types of DNA lesion has been thoroughly explored, but much less is known about the mutagenic effects of DNA lesions present in clustered damage sites. Localized clustering of damage is a hallmark of certain DNA-damaging agents, particularly ionizing radiation. We have investigated the potential of a non-mutagenic DNA base lesion, 5,6-dihydrothymine (DHT), to influence the mutagenicity of 8-oxo-7,8-dihydroguanine (8-oxoG) when the two lesions are closely opposed. Using a bacterial plasmid-based assay we present the first report of a significantly higher mutation frequency for the clustered DHT and 8-oxoG lesions than for single 8-oxoG in wild-type and in glycosylase-deficient strains. We propose that endonuclease III has an important role in the initial stages of processing DHT/8-oxoG clusters, removing DHT to give an intermediate with an abasic site or single-strand break opposing 8-oxoG. We suggest that this mutagenic intermediate is common to several different combinations of base lesions forming clustered DNA damage sites. The MutY glycosylase, acting post-replication, is most important for reducing mutation formation. Recovered plasmids commonly gave rise to both wild-type and mutant progeny, suggesting that there is differential replication of the two DNA strands carrying specific forms of base damage

ホウレンソウ雌性間性株における突然変異誘発ならびに低シュウ酸個体の選抜

This study was conducted to evalute mutagenesis in gynomonoecious spinach (Spinacia oleracea L.) plants for inducing low oxalate variants.Gamma-ray and ion beams of 220 MeV12C5+ and 50MeV 4He2+ ware used as mutagen in seed irradiation. Optimum dosages for irradiation were determined to be about 100Gy, 15-20Gy and 150-200Gy in gamma-ray, 12C5+ and 4He2+, respectively. In M2 generation, there was one line segregating albino seedlings, one line segregating xantha seedlings and two lines segregating dioesious spinach. To save on labor and time for analysis, selection of low oxalate variants in M2generation was conducted by a two-step selebtion which consisted of the first snalysis of bulked leaves from 2 plants as one specimen followed by the second analysis of selected individual plants. In the first analysis of 813 specimens, we selected 13 specimens as low and 9 specimens as high in oxalate content. In the second analysus, there was consistency in the distribution of low and high oxalate content corresponding to the first screening, indicating that selebtion of low oxalate variants could be achived by this two-step selebtion with half the labor and time for analysis as compares to non-bulked method. There were no clear differences in distribution of oxalate content between M3progenies of plants selected as low or high oxalate content, suggesting that the low oxalate content in plants isolated in M2generation was not of a genetic origin. From these results, it seems to be necessary to explore a variant with obvious deviation from the bontinuous variation of oxalate content in the M 2 generation.本実験では，ホウレンソウ雌性間性株における突然変異誘発ならびに低シュウ酸個体の選抜を試みた．種子照射の変異原としては，γ線と220MeV 12

Processing of thymine glycol in a clustered DNA damage site: mutagenic or cytotoxic

Localized clustering of damage is a hallmark of certain DNA-damaging agents, particularly ionizing radiation. The potential for genetic change arising from the effects of clustered damage sites containing combinations of AP sites, 8-oxo-7,8-dihydroguanine (8-oxoG) or 5,6-dihydrothymine is high. To date clusters containing a DNA base lesion that is a strong block to replicative polymerases, have not been explored. Since thymine glycol (Tg) is non-mutagenic but a strong block to replicative polymerases, we have investigated whether clusters containing Tg are highly mutagenic or lead to potentially cytotoxic lesions, when closely opposed to either 8-oxoG or an AP site. Using a bacterial plasmid-based assay and repair assays using cell extracts or purified proteins, we have shown that DNA double-strand breaks (DSBs) arise when Tg is opposite to an AP site, either through attempted base excision repair or at replication. In contrast, 8-oxoG opposite to Tg in a cluster ‘protects’ against DSB formation but does enhance the mutation frequency at the site of 8-oxoG relative to that at a single 8-oxoG, due to the decisive role of endonucleases in the initial stages of processing Tg/8-oxoG clusters, removing Tg to give an intermediate with an abasic site or single-strand break

Study of radiation-induced clustered DNA damage by fluorescence anisotropy measurement based on Förster resonance energy transfer

高LET放射線の飛跡周辺や二次電子の飛跡末端で生じやすいとされているクラスターＤＮＡ損傷（複数の損傷がＤＮＡ上の狭い領域に集中的に生じている）は修復が困難とされているが、その化学構造、線質・エネルギーの違いとの関係についてはほとんど明らかにされていない。本特別講演ではクラスターＤＮＡ損傷の概要説明、及び発表者が開発した損傷局在性評価法（ホモFRET）の開発経緯、及びそれを利用して得られた結果について報告する。第3回QST国際シンポジウムに参

Strand with mutagenic lesion is preferentially used as a template in the region of a bi-stranded clustered DNA damage site in Escherichia coli

The damaging potential of ionizing radiation arises largely from the generation of clustered DNA damage sites within cells. Previous studies using synthetic DNA lesions have demonstrated that models of clustered DNA damage exhibit enhanced mutagenic potential of the comprising lesions. However, little is known regarding the processes that lead to mutations in these sites, apart from the fact that base excision repair of lesions within the cluster is compromised. Unique features of the mutation frequencies within bi-stranded clusters have led researchers to speculate that the strand containing the mutagenic lesion is preferentially used as the template for DNA synthesis. To gain further insights into the processing of clustered DNA damage sites, we used a plasmid-based assay in E. coli cells. Our findings revealed that the strand containing a mutagenic lesion within a bi-stranded clustered DNA damage site is frequently used as the template. This suggests the presence of an, as yet unknown, strand synthesis process that is unrelated to base excision repair, and that this process plays an important role in mutagenesis. The length of the region of strand preference was found to be determined by DNA polymerase I

Study of radiation-induced clustered DNA damage by fluorescence anisotropy measurement based on Förster resonance energy transfer

It is well-known that DNA lesions induced by ionizing radiation and chemicals can cause mutation and carcinogenesis. In particular, “clustered damage" site, that is a DNA region with multiple lesions within a few helical turns, is believed to hardly be repaired. This type of damage is considered to be induced around high-LET radiation tracks and at a track-end of secondary electron. However, chemical and spatial details of them are not known. We have developed a methodology for estimating localization of the lesions using fluorescence anisotropy. The fluorescence anisotropy generally decreases with increasing Förster resonance energy transfer (FRET) efficiency. We found that experimentally-obtained anisotropy for the heat-treated DNA correspond to theoretical ones calculated on the basis of exponential distribution. Now we are applying the methodology based on fluorescence anisotropy to liner-formed pUC19 irradiated with radiation such as Co-60 gamma-rays, helium, carbon, and Fe ion beams. The results and the prospective will be discussed.ICRR(International Congress on Radiation Research) 201

Study of radiation-induced clustered DNA damage by fluorescence anisotropy measurement based on Förster resonance energy transfer

高LET放射線の飛跡周辺や二次電子の飛跡末端で生じやすいとされているクラスターＤＮＡ損傷（複数の損傷がＤＮＡ上の狭い領域に集中的に生じている）は修復が困難とされているが、その化学構造、線質・エネルギーの違いとの関係についてはほとんど明らかにされていない。本特別講演ではクラスターＤＮＡ損傷の概要説明、及び発表者が開発した損傷局在性評価法（ホモFRET）の開発経緯、及びそれを利用して得られた結果について報告する。第3回QST国際シンポジウムに参

Fluorescence anisotropy study of radiation-induced DNA damage clustering based on FRET

DNA lesion induced by ionizing radiation and chemicals can cause mutation and carcinogenesis. In particular, clustered DNA damage site (cluster), that is a DNA region with multiple lesions within a few helical turns, is believed to hardly be repaired. However, little is known about the structural details of a cluster. We develop a method to characterize a cluster by fluorescence anisotropy measurements based on Förster resonance energy transfer (FRET) using an irradiated plasmid DNA sample labeled with fluorescent dyes on the lesions. We find that proximity of the lesions and/or probability of cluster formation increase with increasing linear energy transfer (LET) of radiation