John C. Connelly

The sbcC and sbcD genes of Escherichia coli encode a nuclease involved in palindrome inviability and genetic recombination

Background: Long DNA palindromes have the potential to adopt hairpin or cruciform secondary structures that inhibit DNA replication. In Escherichia coli, this palindrome‐mediated inviability results from the activity of the sbcC and sbcD genes, and genetic observations have suggested that they may encode a nuclease. Mutations in these genes also restore the defect in genetic recombination associated with recBC sbcB mutants.

Tethering on the brink: the evolutionarily conserved Mre11–Rad50 complex

Mre11-Rad50 (MR) proteins are encoded by bacteriophage, eubacterial, archeabacterial and eukaryotic genomes, and form a complex with a remarkable protein architecture. This complex is capable of tethering the ends of DNA molecules, possesses a variety of DNA nuclease, helicase, ATPase and annealing activities, and performs a wide range of functions within cells. It is required for meiotic recombination, double-strand break repair, processing of mis-folded DNA structures and maintaining telomere length. This article reviews current knowledge of the structure and enzymatic activities of the MR complex and attempts to integrate biochemical information with the roles of the protein in a cell.

Overexpression, purification, and characterization of the SbcCD protein from Escherichia coli.

The sbcC and sbcD genes mediate palindrome inviability in Escherichia coli. ThesbcCD operon has been cloned into the plasmid pTrc99A under the control of the strong trc promoter and introduced into a strain carrying a chromosomal deletion of sbcCD. The SbcC and SbcD polypeptides were overexpressed to 6% of total cell protein, and both polypeptides copurified in a four-step purification procedure. Purified SbcCD is a processive double-strand exonuclease that has an absolute requirement for Mn2+ and uses ATP as a preferred energy source. Gel filtration chromatography and sedimentation equilibrium analyses were used to show that the SbcC and SbcD polypeptides dissociate at some stage after purification and that this dissociation is reversed by the addition of Mn2+. We demonstrate that SbcD has the potential to form a secondary structural motif found in a number of protein phosphatases and suggest that it is a metalloprotein that contains the catalytic center of the SbcCD exonuclease.

Repair of DNA Covalently Linked to Protein

A potentially lethal form of DNA/RNA modification, a cleavage complex, occurs when a nucleic acid-processing enzyme that acts via a transient covalent intermediate becomes trapped at its site of action. A number of overlapping pathways act to repair these lesions and many of the enzymes involved are those that catalyze recombinational-repair processes. A protein, Tdp1, has been identified that reverses cleavage-complex formation by specifically hydrolyzing a tyrosyl-DNA phosphodiester bond. The study of these pathways is both interesting and pertinent as they modulate the effectiveness of many antitumor/antibacterial drugs that act by stabilizing cleavage-complexes in vivo.

Nucleolytic processing of a protein-bound DNA end by the E. coli SbcCD (MR) complex

SbcCD and other Mre11/Rad50 (MR) complexes are implicated in the metabolism of DNA ends. They cleave ends sealed by hairpin structures and have been postulated to play roles in removing protein bound to DNA termini. Here we provide direct evidence that the Escherichia coli MR complex (SbcCD) removes protein from a protein-bound DNA end by inserting a double-strand break (DSB). These observations indicate a more complex biochemical action than has been assumed previously and argue that this class of protein has the potential to play a direct role in deprotecting protein-bound DNA ends in vivo.

The molecular basis of variable phenotypic severity among common missense mutations causing Rett syndrome

Rett syndrome is caused by mutations in the X-linked MECP2 gene, which encodes a chromosomal protein that binds to methylated DNA. Mouse models mirror the human disorder and therefore allow investigation of phenotypes at a molecular level. We describe an Mecp2 allelic series representing the three most common missense Rett syndrome (RTT) mutations, including first reports of Mecp2[R133C] and Mecp2[T158M] knock-in mice, in addition to Mecp2[R306C] mutant mice. Together these three alleles comprise ∼25% of all RTT mutations in humans, but they vary significantly in average severity. This spectrum is mimicked in the mouse models; R133C being least severe, T158M most severe and R306C of intermediate severity. Both R133C and T158M mutations cause compound phenotypes at the molecular level, combining compromised DNA binding with reduced stability, the destabilizing effect of T158M being more severe. Our findings contradict the hypothesis that the R133C mutation exclusively abolishes binding to hydroxymethylated DNA, as interactions with DNA containing methyl-CG, methyl-CA and hydroxymethyl-CA are all reduced in vivo. We find that MeCP2[T158M] is significantly less stable than MeCP2[R133C], which may account for the divergent clinical impact of the mutations. Overall, this allelic series recapitulates human RTT severity, reveals compound molecular aetiologies and provides a valuable resource in the search for personalized therapeutic interventions.

MeCP2 recognizes cytosine methylated tri-nucleotide and di-nucleotide sequences to tune transcription in the mammalian brain

Mutations in the gene encoding the methyl-CG binding protein MeCP2 cause several neurological disorders including Rett syndrome. The di-nucleotide methyl-CG (mCG) is the classical MeCP2 DNA recognition sequence, but additional methylated sequence targets have been reported. Here we show by in vitro and in vivo analyses that MeCP2 binding to non-CG methylated sites in brain is largely confined to the tri-nucleotide sequence mCAC. MeCP2 binding to chromosomal DNA in mouse brain is proportional to mCAC + mCG density and unexpectedly defines large genomic domains within which transcription is sensitive to MeCP2 occupancy. Our results suggest that MeCP2 integrates patterns of mCAC and mCG in the brain to restrain transcription of genes critical for neuronal function.

Sequence‐specific DNA binding by AT‐hook motifs in MeCP2

MeCP2 is a chromatin‐associated protein that is mutated in Rett syndrome. Its methyl‐CpG‐binding domain interacts with DNA containing methylated cytosine, but other modes of recruitment to the genome have also been proposed. Here, we use in vitro and in vivo assays to investigate the DNA binding specificity of two AT‐hook motifs in MeCP2. One exhibits robust sequence‐specific DNA binding, whereas the other is a much weaker AT‐hook. Our data indicate that these motifs are secondary contributors to DNA binding by MeCP2, and this view is supported by the absence of disease‐causing missense mutations at these sites.

Domains of methylated CAC and CG target MeCP2 to tune transcription in the brain

Mutations in the gene encoding the methyl-CG binding protein MeCP2 cause neurological disorders including Rett syndrome. The di-nucleotide methyl-CG (mCG) is the canonical MeCP2 DNA recognition sequence, but additional targets including non-methylated sequences have been reported. Here we use brain-specific depletion of DNA methyltransferase to show that DNA methylation is the primary determinant of MeCP2 binding in mouse brain. In vitro and in vivo analyses reveal that MeCP2 binding to non-CG methylated sites in brain is largely confined to the tri-nucleotide sequence mCAC. Structural modeling suggests that mCG and mCAC may be interchangeable as minimal structural perturbation of MeCP2 accompanies binding. MeCP2 binding to chromosomal DNA in mouse brain is proportional to mCG + mCAC density and defines domains within which transcription is sensitive to MeCP2 occupancy. The results suggest that MeCP2 interprets patterns of mCAC and mCG in the brain to negatively modulate transcription of genes critical for neuronal function.

PDGF-Mediated Regulation of Liver Fibrosis

The activation of mesenchymal cells to matrix secreting myofibroblasts is central to the initiation and development of liver fibrosis. Understanding the mechanisms that govern this process is a vital step in the rational development of new anti-fibrotic therapies. Platelet-derived growth factors (PDGFs) and their cognate receptors play a key role in hepatic stellate cell and liver myofibroblast biology. In this review, we outline the major hepatic cellular sources of PDGFs and their modes of action during liver fibrogenesis. Furthermore, we examine how the PDGF pathway may represent a tractable anti-fibrotic target, and how the PDGF receptors may be harnessed to allow cell-specific delivery of therapeutics to help treat patients with liver fibrosis.