Molecular analysis of base damage clustering associated with a site-specific radiation-induced DNA double-strand break

Base damage flanking a radiation-induced DNA double-strand break (DSB) may contribute to DSB complexity and affect break repair. However, to date, an isolated radiation-induced DSB has not been assessed for such structures at the molecular level. In this study, an authentic site-specific radiation-induced DSB was produced in plasmid DNA by triplex forming oligonucleotide-targeted (125)I decay. A restriction fragment terminated by the DSB was isolated and probed for base damage with the E. coli DNA repair enzymes endonuclease III and formamidopyrimidine-DNA glycosylase. Our results demonstrate base damage clustering within 8 bases of the (125)I-targeted base in the DNA duplex. An increased yield of base damage (purine > pyrimidine) was observed for DSBs formed by irradiation in the absence of DMSO. An internal control fragment 1354 bp upstream from the targeted base was insensitive to enzymatic probing, indicating that the damage detected proximal to the DSB was produced by the (125)I decay that formed the DSB. Gas chromatography-mass spectrometry identified three types of damaged bases in the approximately 32-bp region proximal to the DSB. These base lesions were 8-hydroxyguanine, 8-hydroxyadenine and 5-hydroxycytosine. Finally, evidence is presented for base damage >24 bp upstream from the (125)I-decay site that may form via a charge migration mechanism.     

SOS repair and DNA supercoiling influence the genetic stability of DNA triplet repeats in Escherichia coli

Molecular mechanisms responsible for the genetic instability of DNA trinucleotide sequences (TRS) account for at least 20 human hereditary disorders. Many aspects of DNA metabolism influence the frequency of length changes in such repeats. Herein, we demonstrate that expression of Escherichia coli SOS repair proteins dramatically decreases the genetic stability of long (CTG/CAG)n tracts contained in plasmids. Furthermore, the growth characteristics of the bacteria are affected by the (CTG/CAG)n tract, with the effect dependent on the length of the TRS. In an E. coli host strain with constitutive expression of the SOS regulon, the frequency of deletions to the repeat is substantially higher than that in a strain with no SOS response. Analyses of the topology of reporter plasmids isolated from the SOS+ and SOS- strains revealed higher levels of negative supercoiling in strains with the constitutively expressed SOS network. Hence, we used strains with mutations in topoisomerases to examine the effect of DNA topology upon the TRS instability. Higher levels of negative DNA supercoiling correlated with increased deletions in long (CTG/CAG)n, (CGG/CCG)n and (GAA/TTC)n. These observations suggest a link between the induction of bacterial SOS repair, changes in DNA topology and the mechanisms leading to genetic instability of repetitive DNA sequences.     

Structure of the N-terminal domain of the FOP (FGFR1OP) protein and implications for its dimerization and centrosomal localization

The fibroblast growth factor receptor 1 (FGFR1) oncogene partner, FOP, is a centrosomal protein that is involved in the anchoring of microtubules (MTS) to subcellular structures. The protein was originally discovered as a fusion partner with FGFR1 in oncoproteins that give rise to stem cell myeloproliferative disorders. A subsequent proteomics screen identified FOP as a component of the centrosome. FOP contains a Lis-homology (LisH) motif found in more than 100 eukaryotic proteins. LisH motifs are believed to be involved in microtubule dynamics and organization, cell migration, and chromosome segregation; several of them are associated with genetic diseases. We report here a 1.6A resolution crystal structure of the N-terminal dimerization domain of FOP. The structure comprises an alpha-helical bundle composed of two antiparallel chains, each of them having five alpha-helices. The central part of the dimer contains the LisH domain. We further determined that the FOP LisH domain is part of a longer N-terminal segment that is required, albeit not sufficient, for dimerization and centrosomal localization of FOP.     

The ability of DNA polymerase beta to synthesize DNA beyond the gap with displacement of the non-replicated strand

Study was made of the ability of calf thymus DNA polymerases alpha and beta to replicate templates containing a small gap. It was found that during extensive replication of activated DNA or synthetic template.primers or specially prepared circular DNA containing a small gap, catalyzed by DNA polymerase alpha, the levels of incorporated nucleotides corresponded to the amounts of the single-stranded fraction of these templates. In contrast, in the reaction catalyzed by DNA polymerase beta the amounts of products were several times greater. The ability to synthesize the product in a great excess was a specific feature of the latter enzyme. An analysis of the gap-filling products by sucrose gradient centrifugation, gel electrophoresis and Southern hybridization showed that, contrary to DNA polymerase alpha, DNA polymerase beta exhibited the ability to synthesize DNA not only within but also beyond the gap. The "net" DNA product is complementary to the template strand. It is suggested that DNA was synthesized beyond the gap by displacement of the non-replicated strand.     

Characterization of a prokaryotic topoisomerase I activity in chloroplast extracts from spinach.

A topoisomerase I activity has been partially purified from crude extracts of spinach chloroplasts. This activity relaxes the supercoiled covalently closed circular DNA of pBR322. The enzyme requires Mg++, but not ATP, and has an apparent molecular weight of about 115,000. It catalyzes a unit change in the linkage number of supercoiled DNA but cannot relax positive supercoiled DNA. These characteristics of the topoisomerase suggest it is of the prokaryotic type and would tend to support the endosymbiotic theory of plastid origin and evolution.     

Inhibition of calf thymus DNA polymerase α and of normal and cancer cell growth by butylanilinouracil and butylphenylguanine

6-(p-n-Butylanilino)uracil and N²-(p-butylphenyl)guanine inhibited the activity of DNA polymerase α from calf thymus but had no effect on other eukaryotic polymerases (DNA polymerases β and γ) or Escherichia coli DNA polymerase I. Inhibition was competitive with deoxyguanosine 5′-triphosphate and did not occur in the reaction of DNA polymerase α with a template that did not contain cytosine residues. The results support a mechanism which involves hydrogen bonding of inhibitors with cytosines in the DNA template and binding with an inhibitor specific site on the enzyme. A screen of inhibitor effects on normal and cancer cell growth in culture showed that cells were not uniformly sensitive to these compounds, a mouse lymphoma line being least sensitive and a human lung cancer line being most sensitive. It is suggested that these inhibitors may be useful to probe possible structural differences among DNA polymerases α.