The phosphatidylinositol 3-kinase-related protein kinases are key regulators controlling a wide range of cellular events. The yeast Tel1 and Mec1·Ddc2 complex (ATM and ATR-ATRIP in humans) play pivotal roles in DNA replication, DNA damage signaling, and repair. Here, we present the first structural insight for dimers of Mec1·Ddc2 and Tel1 using single-particle electron microscopy. Both kinases reveal a head to head dimer with one major dimeric interface through the N-terminal HEAT (named after Huntingtin, elongation factor 3, protein phosphatase 2A, and yeast kinase TOR1) repeat. Their dimeric interface is significantly distinct from the interface of mTOR complex 1 dimer, which oligomerizes through two spatially separate interfaces. We also observe different structural organizations of kinase domains of Mec1 and Tel1. The kinase domains in the Mec1·Ddc2 dimer are located in close proximity to each other. However, in the Tel1 dimer they are fully separated, providing potential access of substrates to this kinase, even in its dimeric form.

In response to DNA damage or replication fork stalling, the basal activity of Saccharomyces cerevisiae Mec1 ATR is stimulated in a cell cycle dependent manner, leading to cell cycle arrest and promoting DNA repair. Mec1 ATR dysfunction leads to cell death in yeast and causes chromosome instability and embryonic lethality in mammals. Thus, ATR is a major target for cancer therapies in homologous recombination-deficient cancers. Here we identify a single mutation in Mec1, conserved in ATR, that results in constitutive activity. Using cryo electron microscopy, we determined the structures of this constitutively active form (Mec1(F2244L)-Ddc2) at 2.8 Å, and the wild-type at 3.8 Å, both in complex with Mg 2+ -AMP-PNP. These structures yield near complete atomic models for Mec1-Ddc2 and uncover the molecular basis for low basal activity and the conformational changes required for activation. Combined with biochemical and genetic data, we discover key regulatory regions and propose a Mec1 activation mechanism.

Several RNases H1 cleave the RNA-DNA junction of Okazaki fragment-like RNA-DNA/DNA substrate. This activity, termed 3’-junction ribonuclease (3’-JRNase) activity, is different from the 5’-JRNase activity of RNase H2 that cleaves the 5’-side of the ribonucleotide of the RNA-DNA junction and is required to initiate the ribonucleotide excision repair pathway. To examine whether RNase H1 exhibits 3’-JRNase activity for dsDNA containing a single ribonucleotide and can remove this ribonucleotide in collaboration with RNase H2, cleavage of a DNA8-RNA1-DNA9/DNA18 substrate with E. coli RNase H1 and H2 was analyzed. This substrate was cleaved by E. coli RNase H1 at the (5’)RNA-DNA(3’) junction, regardless of whether it was cleaved by E. coli RNase H2 at the (5’)DNA-RNA(3’) junction in advance or not. Likewise, this substrate was cleaved by E. coli RNase H2 at the (5’)DNA-RNA(3’) junction, regardless of whether it was cleaved by E. coli RNase H1 at the (5’)RNA-DNA(3’) junction in advance or not. When this substrate was cleaved by a mixture of E. coli RNases H1 and H2, the ribonucleotide was removed from the substrate. We propose that RNase H1 is involved in the excision of single ribonucleotides misincorporated into DNA in collaboration with RNase H2.

RNase H1 from extreme halophilic archaeon Halobacterium sp. NRC-1 (Halo-RNH1) consists of an N-terminal domain with unknown function and a C-terminal RNase H domain. It is characterized by the high content of acidic residues on the protein surface. The far- and near-UV CD spectra of Halo-RNH1 suggested that Halo-RNH1 assumes a partially folded structure in the absence of salt and divalent metal ions. It requires either salt or divalent metal ions for folding. However, thermal denaturation of Halo-RNH1 analyzed in the presence of salt and/or divalent metal ions by CD spectroscopy suggested that salt and divalent metal ions independently stabilize the protein and thereby facilitate folding. Divalent metal ions stabilize the protein probably by binding mainly to the active site and suppressing negative charge repulsions at this site. Salt stabilizes the protein probably by increasing hydrophobic interactions at the protein core and decreasing negative charge repulsions on the protein surface. Halo-RNH1 exhibited activity in the presence of divalent metal ions regardless of the presence or absence of 3 M NaCl. However, higher concentrations of divalent metal ions are required for activity in the absence of salt to facilitate folding. Thus, divalent metal ions play a dual role in catalysis and folding of Halo-RNH1. Construction of the Halo-RNH1 derivatives lacking an N- or C-terminal domain, followed by biochemical characterizations, indicated that an N-terminal domain is dispensable for stability, activity, folding, and substrate binding of Halo-RNH1.