The Arg-62 residues of the TREX1 exonuclease act across the dimer interface contributing to catalysis in the opposing protomers.

TREX1 is a 3'-deoxyribonuclease that degrades single- and double-stranded DNA (ssDNA and dsDNA) to prevent inappropriate nucleic acid-mediated immune activation. More than 40 different disease-causing TREX1 mutations have been identified exhibiting dominant and recessive genetic phenotypes in a spectrum of autoimmune disorders. Mutations in TREX1 at positions Asp-18 and Asp-200 to His and Asn exhibit dominant autoimmune phenotypes associated with the clinical disorders familial chilblain lupus and Aicardi-Goutières syndrome. Our previous biochemical studies showed that the TREX1 dominant autoimmune disease phenotype depends upon an intact DNA-binding process coupled with dysfunctional active site chemistry. Studies here show that the TREX1 Arg-62 residues extend across the dimer interface into the active site of the opposing protomer to coordinate substrate DNA and to affect catalysis in the opposing protomer. The TREX1(R62A/R62A) homodimer exhibits ∼50-fold reduced ssDNA and dsDNA degradation activities relative to TREX1(WT). The TREX1 D18H, D18N, D200H, and D200N dominant mutant enzymes were prepared as compound heterodimers with the TREX1 R62A substitution in the opposing protomer. The TREX1(D18H/R62A), TREX1(D18N/R62A), TREX1(D200H/R62A), and TREX1(D200N/R62A) compound heterodimers exhibit higher levels of ss- and dsDNA degradation activities than the homodimers demonstrating the requirement for TREX1 Arg-62 residues to provide necessary structural elements for full catalytic activity in the opposing TREX1 protomer. This concept is further supported by the loss of dominant negative effects in the TREX1 D18H, D18N, D200H, and D200N compound heterodimers. These data provide compelling evidence for the required TREX1 dimeric structure for full catalytic function.

Synonymous mutations in RNASEH2A create cryptic splice sites impairing RNase H2 enzyme function in Aicardi-Goutières syndrome.

Aicardi-Goutières syndrome is an inflammatory disorder resulting from mutations in TREX1, RNASEH2A/2B/2C, SAMHD1, or ADAR1. Here, we provide molecular, biochemical, and cellular evidence for the pathogenicity of two synonymous variants in RNASEH2A. Firstly, the c.69G>A (p.Val23Val) mutation causes the formation of a splice donor site within exon 1, resulting in an out of frame deletion at the end of exon 1, leading to reduced RNase H2 protein levels. The second mutation, c.75C>T (p.Arg25Arg), also introduces a splice donor site within exon 1, and the internal deletion of 18 amino acids. The truncated protein still forms a heterotrimeric RNase H2 complex, but lacks catalytic activity. However, as a likely result of leaky splicing, a small amount of full-length active protein is apparently produced in an individual homozygous for this mutation. Recognition of the disease causing status of these variants allows for diagnostic testing in relevant families.

Dominant mutation of the TREX1 exonuclease gene in lupus and Aicardi-Goutieres syndrome.

TREX1 is a potent 3' → 5' exonuclease that degrades single- and double-stranded DNA (ssDNA and dsDNA). TREX1 mutations at amino acid positions Asp-18 and Asp-200 in familial chilblain lupus and Aicardi-Goutières syndrome elicit dominant immune dysfunction phenotypes. Failure to appropriately disassemble genomic DNA during normal cell death processes could lead to persistent DNA signals that trigger the innate immune response and autoimmunity. We tested this concept using dsDNA plasmid and chromatin and show that the TREX1 exonuclease locates 3' termini generated by endonucleases and degrades the nicked DNA polynucleotide. A competition assay was designed using TREX1 dominant mutants and variants to demonstrate that an intact DNA binding process, coupled with dysfunctional chemistry in the active sites, explains the dominant phenotypes in TREX1 D18N, D200N, and D200H alleles. The TREX1 residues Arg-174 and Lys-175 positioned adjacent to the active sites act with the Arg-128 residues positioned in the catalytic cores to facilitate melting of dsDNA and generate ssDNA for entry into the active sites. Metal-dependent ssDNA binding in the active sites of the catalytically inactive dominant TREX1 mutants contributes to DNA retention and precludes access to DNA 3' termini by active TREX1 enzyme. Thus, the dominant disease genetics exhibited by the TREX1 D18N, D200N, and D200H alleles parallel precisely the biochemical properties of these TREX1 dimers during dsDNA degradation of plasmid and chromatin DNA in vitro. These results support the concept that failure to degrade genomic dsDNA is a principal pathway of immune activation in TREX1-mediated autoimmune disease.

Functional consequences of the RNase H2A subunit mutations that cause Aicardi-Goutieres syndrome.

Mutations in the three genes encoding the heterotrimeric RNase H2 complex cause Aicardi-Goutières Syndrome (AGS). Our mouse RNase H2 structure revealed that the catalytic RNase H2A subunit interfaces mostly with the RNase H2C subunit that is intricately interwoven with the RNase H2B subunit. We mapped the positions of AGS-causing RNase H2A mutations using the mouse RNase H2 structure and proposed that these mutations cause varied effects on catalytic potential. To determine the functional consequences of these mutations, heterotrimeric human RNase H2 complexes containing the RNase H2A subunit mutations were prepared, and catalytic efficiencies and nucleic acid binding properties were compared with the wild-type (WT) complex. These analyses reveal a dramatic range of effects with mutations at conserved positions G37S, R186W, and R235Q, reducing enzymatic activities and substrate binding affinities by as much as a 1000-fold, whereas mutations at non-conserved positions R108W, N212I, F230L, T240M, and R291H reduced activities and binding modestly or not at all. All mutants purify as three-subunit complexes, further supporting the required heterotrimeric structure in eukaryotic RNase H2. These kinetic properties reveal varied functional consequences of AGS-causing mutations in the catalytic RNase H2A subunit and reflect the complex mechanisms of nuclease dysfunction that include catalytic deficiencies and altered protein-nucleic acid interactions relevant in AGS.

Escherichia coli DNA adenine methyltransferase: intrasite processivity and substrate-induced dimerization and activation.

Methylation of GATC sites in Escherichia coli by DNA adenine methyltransferase (EcoDam) is essential for proper DNA replication timing, gene regulation, and mismatch repair. The low cellular concentration of EcoDam and the high number of GATC sites in the genome (approximately 20000) support the reliance on methylation efficiency-enhancing strategies such as extensive intersite processivity. Here, we present evidence that EcoDam has evolved other unique mechanisms of activation not commonly observed with restriction-modification methyltransferases. EcoDam dimerizes on short, synthetic DNA, resulting in enhanced catalysis; however, dimerization is not observed on large genomic DNA where the potential for intersite processive methylation precludes any dimerization-dependent activation. An activated form of the enzyme is apparent on large genomic DNA and can also be achieved with high concentrations of short, synthetic substrates. We suggest that this activation is inherent on polymeric DNA where either multiple GATC sites are available for methylation or the partitioning of the enzyme onto nonspecific DNA is favored. Unlike other restriction-modification methyltransferases, EcoDam carries out intrasite processive catalysis whereby the enzyme-DNA complex methylates both strands of an unmethylated GATC site prior to dissociation from the DNA. This occurs with short 21 bp oligonucleotides and is highly dependent upon salt concentrations. Kinetic modeling which invokes enzyme activation by both dimerization and excess substrate provides mechanistic insights into key regulatory checkpoints for an enzyme involved in multiple, diverse biological pathways.

Escherichia coli DNA adenine methyltransferase: the structural basis of processive catalysis and indirect read-out.

We have investigated the structural basis of processive GATC methylation by the Escherichia coli DNA adenine methyltransferase, which is critical in chromosome replication and mismatch repair. We determined the contribution of the orthologically conserved phosphate interactions involving residues Arg(95), Asn(126), Asn(132), Arg(116), and Lys(139), which directly contact the DNA outside the cognate recognition site (GATC) to processive catalysis, and that of residue Arg(137), which is not conserved and contacts the DNA backbone within the GATC sequence. Alanine substitutions at the conserved positions have large impacts on processivity yet do not impact k(cat)/K(m)(DNA) or DNA affinity (K(D)(DNA)). However, these mutants cause large preferences for GATC sites varying in flanking sequences when considering the pre-steady state efficiency constant k(chem)/K(D)(DNA). These changes occur mainly at the level of the methylation rate constant, which results in the observed decreases in processive catalysis. Thus, processivity and catalytic efficiency (k(cat)/K(m)(DNA)) are uncoupled in these mutants. These results reveal that the binding energy involved in DNA recognition contributes to the assembly of the active site rather than tight binding. Furthermore, the conserved residues (Arg(95), Asn(126), Asn(132), and Arg(116)) repress the modulation of the response of the enzyme to flanking sequence effects. Processivity impacted mutants do not show substrate-induced dimerization as is observed for the wild type enzyme. This study describes the structural means by which an enzyme that does not completely enclose its substrate has evolved to achieve processive catalysis, and how interactions with DNA flanking the recognition site alter this processivity.

Modulation of Escherichia coli DNA methyltransferase activity by biologically derived GATC-flanking sequences.

Escherichia coli DNA adenine methyltransferase (EcoDam) methylates the N-6 position of the adenine in the sequence 5'-GATC-3' and plays vital roles in gene regulation, mismatch repair, and DNA replication. It remains unclear how the small number of critical GATC sites involved in the regulation of replication and gene expression are differentially methylated, whereas the approximately 20,000 GATCs important for mismatch repair and dispersed throughout the genome are extensively methylated. Our prior work, limited to the pap regulon, showed that methylation efficiency is controlled by sequences immediately flanking the GATC sites. We extend these studies to include GATC sites involved in diverse gene regulatory and DNA replication pathways as well as sites previously shown to undergo differential in vivo methylation but whose function remains to be assigned. EcoDam shows no change in affinity with variations in flanking sequences derived from these sources, but methylation kinetics varied 12-fold. A-tracts immediately adjacent to the GATC site contribute significantly to these differences in methylation kinetics. Interestingly, only when the poly(A) is located 5' of the GATC are the changes in methylation kinetics revealed. Preferential methylation is obscured when two GATC sites are positioned on the same DNA molecule, unless both sites are surrounded by large amounts of nonspecific DNA. Thus, facilitated diffusion and sequences immediately flanking target sites contribute to higher order specificity for EcoDam; we suggest that the diverse biological roles of the enzyme are in part regulated by these two factors, which may be important for other enzymes that sequence-specifically modify DNA.