The Escherichia coli MutS and MutL proteins have been conserved throughout evolution, although their combined functions in mismatch repair (MMR) are poorly understood. We have used biochemical and genetic studies to ascertain a physiologically relevant mechanism for MMR. The MutS protein functions as a regional lesion sensor. ADP-bound MutS specifically recognizes a mismatch. Repetitive rounds of mismatch-provoked ADP-->ATP exchange results in the loading of multiple MutS hydrolysis-independent sliding clamps onto the adjoining duplex DNA. MutL can only associate with ATP-bound MutS sliding clamps. Interaction of the MutS-MutL sliding clamp complex with MutH triggers ATP binding by MutL that enhances the endonuclease activity of MutH. Additionally, MutL promotes ATP binding-independent turnover of idle MutS sliding clamps. These results support a model of MMR that relies on two dynamic and redundant ATP-regulated molecular switches.

An activity has been purified 350-fold from extracts of mouse plasmacytoma cells that forms 5-hydroxymethyluracil (a-hydroxythymine) and apyrimidinic sites with phage SPO1 DNA, which contains this base in place of thymine. This DNA glycosylase presumably functions to eliminate hydroxymethyluracil, a major thymine-derived DNA lesion produced by ionizing radiation and oxidative damage. The enzyme has no cofactor requirement and is active in EDTA. Neither intermediate formation nor hydrolysis of hydroxymethyldeoxyuridine or hydroxymethyldeoxyuridine monophosphate was detected. The enzyme does not cleave apyrimidinic sites in DNA. It does release uracil from the uracil-containing DNA of phage PBS2, but this activity is less than 2% of the predominant uracil DNA glycosylase activity of the cell, which is separated by phosphocellulose chromatography. The major uracil DNA glycosylase does not release hydroxymethyluracil from SPO1 DNA. The hydroxymethyluracil glycosylase is also separated upon phosphocelluose chromatography from a thymine glycol DNA glycosylase activity that is accompanied by an apyrimidinic endonuclease activity.There is an increasing awareness of the importance of oxidative damage to DNA (1) which may be mediated primarily by highly reactive oxygen radicals such as the hydroxyl radical produced by ionizing radiation (2). Hydrated thymine derivatives, including 5,6-dihydroxythymine (thymine glycol), are recognized to be important products of ionizing radiation and are released from bacterial and mammalian DNA in vivo after treatment with various oxidative agents (3). DNA from y-irradiated HeLa cells contains some 5-hydroxymethyluracil (hmU) (a-hydroxythymine) as well as thymine glycol (3, 4), but repair of hmU has not been described.Demple and Linn demonstrated that Escherichia coli endonuclease III (5) has thymine glycol DNA glycosylase activity (6). Endonuclease activities that are specific for DNA damaged by ionizing radiation, high doses of UV irradiation, or OS04 have also been reported from eukaryotes (7-11), and thymine glycol DNA glycosylase activity has been reported to be present in a preparation of calf thymus urea DNA glycosylase (12). We report here that the "DNA repair endonuclease" preparation from mouse MPC-11 cells described by Nes (7) contains a mixture of thymine glycol DNA glycosylase and apyrimidinic endonuclease (AP endonuclease) activities. Moreover, we describe the isolation and initial characterization of a hmU DNA glycosylase activity from these cells. The latter activity, which is separated from any AP endonuclease activity, was identified by using as substrate DNA derived from the Bacillus subtilis phage SPO1, which contains hmU in place of thymine (13). NaCl/2% (vol/vol) glycerol. After 10 min at 0°C, 1.5 ml of the solution was neutralized with 0.5 ml of 0.58 M HCl/80 mM Hepes (Na+), pH 7.9/3.3 M NaCl/4 mM EDTA to form the denatured form 'd DNA (18); then 4 ml of 0.3% OS04 in 0.4 M NaCl was added. After 20 min at 25°C, the DNA was extracted three times with 3 vol of...

Autism is a developmental disorder characterized by impairments in social interaction and communication associated with repetitive patterns of interest or behavior. Autism is highly influenced by genetic factors. Genome-wide linkage and candidate gene association approaches have been used to try and identify autism genes. A few loci have repeatedly been reported linked to autism. Several groups reported evidence for linkage to a region on chromosome 16p. We have applied a direct physical identity-by-descent (IBD) mapping approach to perform a high-density (0.85 megabases) genome-wide linkage scan in 116 families from the AGRE collection. Our results confirm linkage to a region on chromosome 16p with autism. High-resolution single-nucleotide polymorphism (SNP) genotyping and analysis of this region show that haplotypes in the protein kinase c-beta gene are strongly associated with autism. An independent replication of the association in a second set of 167 trio families with autism confirmed our initial findings. Overall, our data provide evidence that the PRKCB1 gene on chromosome 16p may be involved in the etiology of autism. Molecular Psychiatry (2005) 10, 950-960.

The DNA mismatch repair (MMR) system plays a critical role in sensitizing both prokaryotic and eukaryotic cells to the clinically potent anticancer drug cisplatin. It is thought to mediate cytotoxicity through recognition of cisplatin DNA lesions. This drug generates a range of lesions that may also give rise to compound lesions resulting from the misincorporation of a base during translesion synthesis. Using gel mobility shift competition assays and surface plasmon resonance, we have analyzed the interaction of Escherichia coli MutS protein with site-specifically modified DNA oligonucleotides containing each of the four cisplatin cross-links or a set of compound lesions. The major 1,2-d(GpG) cisplatin intrastrand cross-link was recognized with only a 1.5-fold specificity, whereas a 47-fold specificity was found with a natural G/T containing DNA substrate. The rate of association, k on, for binding to the 1,2-d(GpG) adduct was 3.1 ؋ 10 4 M ؊1 s ؊1 and the specificity of binding was essentially dependent on k off . DNA duplexes containing a single 1,2-d(ApG), 1,3-d(GpCpG) adduct, and an interstrand cross-link of cisplatin were not preferentially recognized. Among 12 DNA substrates, each containing a different cisplatin compound lesion derived from replicative misincorporation of one base opposite either of the 1,2-intrastrand adducts, 10 were specifically recognized including those that are more likely formed in vivo based on cisplatin mutation spectra. Moreover, among these lesions, two compound lesions formed when an adenine was misincorporated opposite a 1,2-d(GpG) adduct were not substrates for the MutYdependent mismatch repair pathway. The ability of MutS to sense differentially various platinated DNA substrates suggests that cisplatin compound lesions formed during misincorporation of a base opposite either adducted base of both 1,2-intrastrand cross-links are more plausible critical lesions for MMR-mediated cisplatin cytotoxicity.cis-Diamminedichloroplatinum(II) (cisplatin) 1 is among the most widely used anticancer chemotherapeutic agents in the treatment of many human tumors, particularly testicular and ovarian tumors (1). In the reaction between DNA and cisplatin, different types of bifunctional adducts are formed that are thought to be the key toxic lesions (2-4). Although these adducts are responsible for a variety of cellular responses including replication and/or transcription inhibition, there is not yet a clear understanding of the molecular mechanisms linking the formation of adducts and cisplatin-induced apoptosis (5, 6). Cisplatin reacts preferentially with the N-7 atoms of purine residues in DNA. The major adducts (90%) are 1,2-intrastrand cross-links at d(GpG) and d(ApG) sites and the minor adducts correspond to 1,3-intrastrand cross-links at d(GpNpG) (N being a nucleotide residue) and interstrand cross-links formed at d(GpC/GpC) sites (7-10). Once formed, cisplatin lesions such as the major 1,2-intrastrand cross-links can undergo replication bypass in cells treated with cisplatin (11,12). Be...

Functional interactions of Escherichia coli MutS and MutL in mismatch repair are dependent on ATP. In this study, we show that MutS and MutL associate with immobilised DNA in a manner dependent on ATP hydrolysis and with an ATP concentration near the solution K m of the ATPase of MutS. After removal of MutS, MutL and ATP, much of the protein in this ternary complex is not stably associated, with MutL leaving the complex more rapidly than MutS. The rapid dissociation reveals a dynamic interaction with concurrent rapid association and dissociation of proteins from the DNA. Analysis by surface plasmon resonance showed that the DNA interacting with dynamically bound protein was more resistant to nuclease digestion than the DNA in MutS-DNA complexes. Non-hydrolysable analogs of ATP inhibit the formation of this dynamic complex, but permit formation of a second type of ternary complex with MutS and MutL stably bound to the immobilised DNA.

Background: Autism is a complex, heterogeneous, behaviorally-defined disorder characterized by disruptions of the nervous system and of other systems such as the pituitary-hypothalamic axis. In a previous genome wide screen, we reported linkage of autism with a 1.2 Megabase interval on chromosome 5q31. For the current study, we hypothesized that 3 of the genes in this region could be involved in the development of autism: 1) paired-like homeodomain transcription factor 1 (PITX1), which is a key regulator of hormones within the pituitary-hypothalamic axis, 2) neurogenin 1, a transcription factor involved in neurogenesis, and 3) histone family member Y (H2AFY), which is involved in Xchromosome inactivation in females and could explain the 4:1 male:female gender distortion present in autism.

Background: The monogenic disease osteogenesis imperfecta (OI) is due to single mutations in either of the collagen genes ColA1 or ColA2, but within the same family a given mutation is accompanied by a wide range of disease severity. Although this phenotypic variability implies the existence of modifier gene variants, genome wide scanning of DNA from OI patients has not been reported. Promising genome wide marker-independent physical methods for identifying diseaserelated loci have lacked robustness for widespread applicability. Therefore we sought to improve these methods and demonstrate their performance to identify known and novel loci relevant to OI.

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