Incubation of DNA with S‐adenosyl‐L‐methionine (SAM) in neutral aqueous solution leads to base modification, with formation of small amounts of 7‐methylguanine and 3‐methyladenine. The products have been identified by high performance liquid chromatography of DNA hydrolysates and by the selective release of free 3‐methyladenine from SAM‐treated DNA by a specific DNA glycosylase. We conclude that SAM acts as a weak DNA‐alkylating agent. Several control experiments including extensive purification of [3H‐methyl]SAM preparations and elimination of the alkylating activity by pretreatment of SAM with a phage T3‐induced SAM cleaving enzyme, have been performed to determine that the activity observed was due to SAM itself and not to a contaminating substance. We estimate that SAM, at an intracellular concentration of 4 X 10(‐5) M, causes DNA alkylation at a level similar to that expected from continuous exposure of cells to 2 X 10(‐8) M methyl methane‐sulphonate. This ability of SAM to act as a methyl donor in a nonenzymatic reaction could result in a background of mutagenesis and carcinogenesis. The data provide an explanation for the apparently universal occurrence of multiple DNA repair enzymes specific for methylation damage.

Homologous recombination (HR) repairs chromosome damage and is indispensable for tumor suppression in humans. RAD51 mediates the DNA strand-pairing step in HR. RAD51 associated protein 1 (RAD51AP1) is a RAD51-interacting protein whose function has remained elusive. Knockdown of RAD51AP1 in human cells by RNA interference engenders sensitivity to different types of genotoxic stress, and RAD51AP1 is epistatic to the HR protein XRCC3. Moreover, RAD51AP1-depleted cells are impaired for the recombinational repair of a DNA double-strand break and exhibit chromatid breaks both spontaneously and upon DNA-damaging treatment. Purified RAD51AP1 binds both dsDNA and a D loop structure and, only when able to interact with RAD51, greatly stimulates the RAD51-mediated D loop reaction. Biochemical and cytological results show that RAD51AP1 functions at a step subsequent to the assembly of the RAD51-ssDNA nucleoprotein filament. Our findings provide evidence that RAD51AP1 helps maintain genomic integrity via RAD51 recombinase enhancement.

Saliency is an important perceptual cue that occurs at different levels of resolution. Important attributes of saliency are symmetry, continuity, and closure. Detection of these attributes is often hindered by noise, variation in scale, and incomplete information. This paper introduces the iterative voting method, which uses oriented kernels for inferring saliency as it relates to symmetry. A unique aspect of the technique is the kernel topography, which is refined and reoriented iteratively. The technique can cluster and group nonconvex perceptual circular symmetries along the radial line of an object's shape. It has an excellent noise immunity and is shown to be tolerant to perturbation in scale. The application of this technique to images obtained through various modes of microscopy is demonstrated. Furthermore, as a case example, the method has been applied to quantify kinetics of nuclear foci formation that are formed by phosphorylation of histone gammaH2AX following ionizing radiation. Iterative voting has been implemented in both 2-D and 3-D for multi image analysis.

Misincorporated ribonucleotides in DNA will cause DNA backbone distortion and may be targeted by DNA repair enzymes. Using double-stranded oligonucleotide probes containing a single ribose, we demonstrate a robust activity in human, yeast, and Escherichia coli cell-free extracts that nicks 5 of the ribose. The human and yeast extracts also make a subsequent cut 3 of the ribonucleotide releasing a ribonucleotide monophosphate. The resulting 1-nt gap is an ideal substrate for polymerase and ligase to complete a proposed repair sequence that effectively replaces the ribose with deoxyribose. T here is presently a nearly total lack of information about repair of deoxyribose modifications in DNA. Such modifications can be caused by external agents, such as oxidizing agents and ionizing radiation (1-3), and can also occur naturally by misincorporation of ribonucleotides into DNA during DNA replication (4). The presence of ribose in DNA is a hindrance to formation of normal B form DNA as evidenced by the structure of RNA͞DNA hybrid molecules (5), and consequently a single ribose in DNA will result in a local DNA backbone distortion (6). Other bulky modified sugars are also likely to cause backbone distortions, and it can be hypothesized that they pose a hindrance for DNA polymerases and can be mutagenic.Progressive DNA and RNA polymerases are similar in structure and belong to the same class of proteins (4), probably with a common evolutionary origin (7). The specificity toward deoxyribonucleoside triphosphates (dNTPs) or ribonucleoside triphosphates (rNTPs) has been found to be determined by subtle differences at the active site (4). Gao et al. (8) could largely eliminate the discrimination between the rNTPs and dNTPs by introducing a single amino acid change in a reverse transcriptase, and similar observations in mutant polymerases have recently been made by several investigators (7, 9-13). On the basis of such observations, it has been suggested that the discrimination against ribonucleotides by DNA polymerases is largely accomplished by a ''steric gate'' that will not give enough space for the 2Ј hydroxyl group present in rNTPs (4). However, the discrimination against rNTPs is not 100%, and detectable incorporation of rNTPs has been found in vitro by using purified DNA polymerases with a wide variety of discrimination factors ranging from a few thousand-fold (7, 11) to several million-fold (13). However, it is presently not known to what extent ribonucleotides are misincorporated into DNA during normal in vivo DNA replication. The intranuclear milieu contains both ribonucleotides and deoxyribonucleotides, with the ribonucleotide concentration generally higher than the deoxyribonucleotide concentration (14). The deoxyribonucleotides are produced from the ribonucleotide pool by the enzyme ribonucleotide diphosphate reductase. This enzyme can be inhibited in eukaryotic cells by hydroxyurea (HU), which blocks DNA replication when given to cell cultures in sufficient concentration.Gao and Goff (15) mutagenized a viral p...

Stenerlöw, B., Karlsson, K. H., Cooper, B. and Rydberg, B. Measurement of Prompt DNA Double-Strand Breaks in Mammalian Cells Without Including Heat-Labile Sites. Results for Cells Deficient in Non-homologous End Joining. Radiat. Res. Ionizing radiation induces prompt single-strand breaks and double-strand breaks in DNA. In addition, labile sites are induced that can convert to breaks by heat or mild alkali. When such labile lesions are present within multiply damaged sites, additional double-strand breaks can form. Current protocols for measurement of DNA double-strand breaks involve a lysis step at elevated temperature, and consequently breaks from heat-labile sites will be generated during lysis and will be included in the measurement. However, such sites may not develop into breaks within the cell and may not need DNA double-strand break repair processes for elimination. We present here a new lysis and pulsed-field gel electrophoresis protocol that is carried out entirely at 0-4°C and thus avoids inclusion of heat-labile sites into the measurement. The new recommended lysis procedure involves two steps: the first step includes proteinase-K, which has sufficient activity at 0°C to support lysis, and the second step includes a high salt buffer to strip the DNA from histones and other proteins. By various tests we conclude that lysis is sufficient with this procedure to allow accurate determination of double-strand breaks by pulsed-field gel electrophoresis. Using the new protocol, it was found that heat-labile sites account for 30% of the initial number of double-strand breaks measured by conventional protocols after low LET radiation. In addition we show that heat-labile sites that can convert to double-strand breaks are repaired with fast kinetics and are nearly completely eliminated after 1 hr at 37°C. A study of cells deficient in non-homologous end joining reveals that the residual fast repair response typically seen in such cells is solely due to repair at heat-labile sites and is not due to repair of prompt DSBs. 2