Heterozygous mutations in DNA mismatch repair (MMR) genes result in predisposition to colorectal cancer (hereditary nonpolyposis colorectal cancer or Lynch syndrome). Patients with biallelic mutations in these genes, however, present earlier, with constitutional mismatch repair deficiency cancer syndrome (CMMRD), which is characterized by a spectrum of rare childhood malignancies and café-au-lait skin patches. The hallmark of MMR deficiency, microsatellite instability (MSI), is readily detectable in tumor DNA in Lynch syndrome, but is also present in constitutional DNA of CMMRD patients. However, detection of constitutional or germline MSI (gMSI) has hitherto relied on technically difficult assays that are not routinely applicable for clinical diagnosis. Consequently, we have developed a simple high-throughput screening methodology to detect gMSI in CMMRD patients based on the presence of stutter peaks flanking a dinucleotide repeat allele when amplified from patient blood DNA samples. Using the three different microsatellite markers, the gMSI ratio was determined in a cohort of normal individuals and 10 CMMRD patients, with biallelic germline mutations in PMS2 (seven patients), MSH2 (one patient), or MSH6 (two patients). Subjects with either PMS2 or MSH2 mutations were easily identified; however, this measure was not altered in patients with CMMRD due to MSH6 mutation.

In this report, we show that yccV, a gene of unknown function, encodes a protein having an affinity for a hemimethylated oriC DNA and that the protein negatively controls dnaA gene expression in vivo.The minimum replication origin of Escherichia coli (oriC) has an elevated number of DNA adenine methylation (Dam) sites (18) that become hemimethylated immediately after initiation of replication and stay in this state for one-quarter of the generation time; these sites differ from most Dam sites located elsewhere on the chromosome (6). The finding that the hemimethylated oriC interacts with an outer membrane preparation of E. coli (20) prompted a search for hemimethylated DNA binding proteins in the membrane fraction. The existence of such a category of proteins in the membrane has been reported previously; in fact, Southwestern blot analysis of the membrane preparation demonstrated the presence of 25-and 16-kDa peptides reacting with a hemimethylated oriC probe (9). These proteins might participate in the hemimethylated oriC membrane sequestration that prevents initiation of chromosome replication in E. coli (16). Lu et al. (17) isolated the seqA gene that codes for a 21-kDa peptide and has an affinity for the hemimethylated oriC DNA (24). In a seqA mutant, the duration of the hemimethylation period of oriC is shortened, and the reinitiation of replication occurs at oriC repeatedly in a single replication cycle (17). These phenotypes may be explained by a lack of hemimethylated oriC sequestration by either SeqA or a SeqA membrane complex; the membrane obtained from the seqA mutant failed to bind the hemimethylated oriC DNA in vitro (2, 24). Although these results show the predominant role of the SeqA protein in oriC sequestration, the auxiliary role played by the membrane in the process should not be disregarded. Shakibai et al. (23) observed that the hemimethylated oriC binding activity of SeqA is stimulated by addition of a membrane protein preparation designated SeqB. It has also been found that the membrane obtained from a seqA mutant stimulates the protective activity of a His-tagged SeqA protein against DNase I attack of hemimethylated oriC DNA (7).In order to isolate auxiliary factors of SeqA, we adopted a strategy of searching for factors among the dnaA46 suppressor gene products. The rationale behind this approach was based on the observation that the seqA mutation partially suppresses the temperature sensitivity of dnaA46 (17). Likewise, mutation of the auxiliary factor gene should also result in suppression of dnaA46. Previously, this type of work has been undertaken by Katayama's group, who used a system involving random insertion of Tn10-Tet r into the chromosome. In this way, they found one new suppressor mutation for dnaA46, hslU (12).Isolation of dnaA46 suppressors by mini-Tn10 insertion. Briefly, random insertion of mini-Tn10 into the chromosome containing dnaA46 (KA413⌬H) ( Table 1) was accomplished by infection of the mutant with a lambda phage (1098) carrying mini-Tn10 (27), followed by plat...

Identification of de novo minisatellite mutations in the offspring of parents exposed to mutagenic agents offers a potentially sensitive measure of germ line genetic events induced by ionizing radiation and genotoxic chemicals. Germ line minisatellite mutations (GMM) are usually detected by hybridizing Southern blots of unamplified size-fractionated genomic DNA with minisatellite probes. However, this consumes a relatively large amount of DNA, requires several steps and may lack sensitivity. We have developed a polymerase chain reaction (PCR)-based GMM assay, which we applied to the hypermutable minisatellite, CEB1. Here, we compare the sensitivity and specificity of this assay with the conventional Southern hybridization method using DNA from 10 spouse pairs, one parent of each pair being a survivor of cancer in childhood, and their 20 offspring. We report that both methods have similar specificity but that the PCR method uses 250 times less DNA, has fewer steps and is better at detecting GMM with single repeats provided that specific guidelines for allele sizing are followed. The PCR GMM method is easier to apply to families where the amount of offspring DNA sample is limited.