No abstract

Noonan syndrome (NS) and related disorders are autosomal dominant disorders characterized by heart defects, facial dysmorphism, ectodermal abnormalities, and mental retardation. The dysregulation of the RAS/MAPK pathway appears to be a common molecular pathogenesis of these disorders: mutations in PTPN11, KRAS, and SOS1 have been identified in patients with NS, those in KRAS, BRAF, MAP2K1, and MAP2K2 in patients with CFC syndrome, and those in HRAS mutations in Costello syndrome patients. Recently, mutations in RAF1 have been also identified in patients with NS and two patients with LEOPARD (multiple lentigines, electrocardiographic conduction abnormalities, ocular hypertelorism, pulmonary stenosis, abnormal genitalia, retardation of growth, and sensorineural deafness) syndrome. In the current study, we identified eight RAF1 mutations in 18 of 119 patients with NS and related conditions without mutations in known genes. We summarized clinical manifestations in patients with RAF1 mutations as well as those in NS patients withPTPN11, SOS1, or KRAS mutations previously reported. Hypertrophic cardiomyopathy and short stature were found to be more frequently observed in patients with RAF1 mutations. Mutations in RAF1 were clustered in the conserved region 2 (CR2) domain, which carries an inhibitory phosphorylation site (serine at position 259; S259). Functional studies revealed that the RAF1 mutants located in the CR2 domain resulted in the decreased phosphorylation of S259, and that mutant RAF1 then dissociated from 14-3-3, leading to a partial ERK activation. Our results suggest that the dephosphorylation of S259 is the primary pathogenic mechanism in the activation of RAF1 mutants located in the CR2 domain as well as of downstream ERK.

In living cells, a great amount of DNA damage arises as a result of errors during DNA replication, genetic recombination, and other processes (1). Because the accumulation of such damage can result in various genetic diseases, many DNA repair systems have evolved to remove any lesions. One of these systems is the mismatch repair (MMR), 2 which is conserved throughout all organisms (2). It is known that inactivation of MMR in humans has been implicated in over 90% of hereditary nonpolyposis colorectal cancers (3). The mechanism of MMR has been well characterized in Escherichia coli, and the reconstituted system has been established (4). The early reactions in the E. coli MMR are performed by the MutHLS system (5, 6), which consists of three proteins, MutS, MutL, and MutH. In this system, a mismatched base in double-stranded DNA (dsDNA) is recognized by a MutS dimer. A MutL dimer interacts with the MutS-mismatched DNA complex and stabilizes its complex, and then the MutH endonuclease is activated by MutL. MutH nicks the unmethylated strand at the hemimethylated GATC site to provide an entry point for the excision and to direct the repair to the new DNA strand. To complete the repair, the strand containing the error is removed by helicases and exonucleases, and a new strand is synthesized by DNA polymerase III holoenzyme and ligase. Homologues of E. coli MutS and MutL exist in the majority of organisms (Fig. 1A), suggesting that MMR is a common repair mechanism among those species. However, despite the prevalence of the MMR system, no homologue of E. coli MutH has been identified in the majority of organisms (7; Fig. 1A). Therefore, in organisms lacking mutH, the MMR had not been well understood.The research for the primary reactions in the eukaryotic MMR relies on homologues of bacterial MutS and MutL. Many studies on the mammalian MMR have shown that a strand discontinuity serves as a signal that directs the repair to one strand of the mismatched heteroduplex (2, 4). The discontinuities in the newly synthesized strands such as the 3Ј ends or termini of Okazaki fragments may designate which strand is to be repaired. For biochemical characterization of MMR, the nicked circular heteroduplex has been used as a substrate containing a strand discontinuity. It has been shown that the excision system in MMR selects the shorter path from a nick to a mismatch (8). Therefore, the distinction between 5Ј-and 3Ј-directed MMR should exist. Interestingly, 5Ј-to 3Ј-exonuclease activity of Exo I is required for both 5Ј-and 3Ј-directed removal (9, 10). Recently, Modrich and co-workers (11, 12) explained how 3Ј-directed excision is performed by the 5Ј-to 3Ј-exonuclease activity of ExoI. They demonstrated that a human MutL homologue, MutL␣ (MLH1-PMS2 heterodimer), is a latent endonuclease that incises on both sides of a mismatch, and the 5Ј-to 3Ј-exonuclease activity of ExoI removes the DNA segment spanning the mismatch. They also showed that MutL␣, in the presence of manganese ions, incises a supercoiled homoduplex without other MMR...

RecJ is a single-stranded DNA (ssDNA)-specific 5-3 exonuclease that plays an important role in DNA repair and recombination. To elucidate how RecJ achieves its high specificity for ssDNA, we determined the entire structures of RecJ both in a ligand-free form and in a complex with Mn 2؉ or Mg 2؉ by x-ray crystallography. The entire RecJ consists of four domains that form a molecule with an O-like structure. One of two newly identified domains had structural similarities to an oligonucleotide/oligosaccharide-binding (OB) fold. The OB fold domain alone could bind to DNA, indicating that this domain is a novel member of the OB fold superfamily. The truncated RecJ containing only the core domain exhibited much lower affinity for the ssDNA substrate compared with intact RecJ. These results support the hypothesis that these structural features allow specific binding of RecJ to ssDNA. In addition, the structure of the RecJ-Mn 2؉ complex suggests that the hydrolysis reaction catalyzed by RecJ proceeds through a two-metal ion mechanism.All of the organisms possess numerous pathways to repair DNA damages to maintain the integrity of the information contained in the genome. The single-stranded DNA (ssDNA) 2 processing exonuclease RecJ has been implicated in many of these repair pathways. RecJ was originally identified as an essential gene for the RecF pathway of homologous recombination (1). RecJ produces 3Ј-ssDNA tails, which are required to initiate recombination from a double-stranded break (2). RecJ is also an exonuclease that mediates the excision step during mismatch repair (3, 4) and degrades abasic residues generated during base excision repair (5). Almost all eubacteria and archaea contain RecJ orthologs (6). These observations suggest that RecJ plays an important role in DNA repair.RecJ is the only known ssDNA-specific 5Ј exonuclease. It specifically hydrolyzes ssDNA in the 5Ј to 3Ј direction, which is dependent on metal cations (7). It processively degrades its substrate at ϳ700 (8) or 1,000 (9) nucleotides/single binding event and appears to have a site that can bind seven nucleotides (9).The crystal structure of a truncated Thermus thermophilus RecJ (ttRecJ) shows that the core domain of ttRecJ (cd-ttRecJ) (residues 40 -463) consists of two domains that are interconnected by a long helix, forming a central cleft (10). Mn 2ϩ in the active site is located on the wall of the cleft and is coordinated by conserved residues characteristic of a family of phosphoesterases that includes RecJ proteins. Although cd-ttRecJ contains the conserved residues of DHH (motifs I-IV) and DHHA1 motifs (11) (supplemental Fig. S1), the entire structure including the N-and C-terminal regions has not been determined.To elucidate how RecJ achieves its high specificity for ssDNA, we determined the intact ttRecJ structures (666 residues) both in a ligand-free form and in a complex with Mn 2ϩ or Mg 2ϩ at 2.15-2.50 Å resolution. Intact ttRecJ consists of four domains that form a molecule with an O-like structure. Interestingly, one of two ne...