Solution NMR structure of the C-terminal DNA binding domain of Mcm10 reveals a conserved MCM motif.

The eukaryotic DNA replication protein Mcm10 associates with chromatin in early S-phase and is required for assembly and function of the replication fork protein machinery. Xenopus laevis (X) Mcm10 binds DNA via a highly conserved internal domain (ID) and a C-terminal domain (CTD) that is unique to higher eukaryotes. Although the structural basis of the interactions of the ID with DNA and polymerase alpha is known, little information is available for the CTD. We have identified the minimal DNA binding region of the XMcm10-CTD and determined its three-dimensional structure by solution NMR. The CTD contains a globular domain composed of two zinc binding motifs. NMR chemical shift perturbation and mutational analysis show that ssDNA binds only to the N-terminal (CCCH-type) zinc motif, whose structure is unique to Mcm10. The second (CCCC-type) zinc motif is not involved in DNA binding. However, it is structurally similar to the CCCC zinc ribbon in the N-terminal oligomerization domain of eukaryotic and archaeal MCM helicases. NMR analysis of a construct spanning both the ID and CTD reveals that the two DNA binding domains are structurally independent in solution, supporting a modular architecture for vertebrate Mcm10. Our results provide insight in the action of Mcm10 in the replisome and support a model in which it serves as a central scaffold through coupling of interactions with partner proteins and the DNA.

Tetrameric Ctp1 coordinates DNA binding and DNA bridging in DNA double-strand-break repair.

Ctp1 (also known as CtIP or Sae2) collaborates with Mre11-Rad50-Nbs1 to initiate repair of DNA double-strand breaks (DSBs), but its functions remain enigmatic. We report that tetrameric Schizosaccharomyces pombe Ctp1 contains multivalent DNA-binding and DNA-bridging activities. Through structural and biophysical analyses of the Ctp1 tetramer, we define the salient features of Ctp1 architecture: an N-terminal interlocking tetrameric helical dimer-of-dimers (THDD) domain and a central intrinsically disordered region (IDR) linked to C-terminal 'RHR' DNA-interaction motifs. The THDD, IDR and RHR are required for Ctp1 DNA-bridging activity in vitro, and both the THDD and RHR are required for efficient DSB repair in S. pombe. Our results establish non-nucleolytic roles of Ctp1 in binding and coordination of DSB-repair intermediates and suggest that ablation of human CtIP DNA binding by truncating mutations underlie the CtIP-linked Seckel and Jawad syndromes.

Mechanism of repair of 5'-topoisomerase II-DNA adducts by mammalian tyrosyl-DNA phosphodiesterase 2.

The topoisomerase II (topo II) DNA incision-and-ligation cycle can be poisoned (for example following treatment with cancer chemotherapeutics) to generate cytotoxic DNA double-strand breaks (DSBs) with topo II covalently conjugated to DNA. Tyrosyl-DNA phosphodiesterase 2 (Tdp2) protects genomic integrity by reversing 5'-phosphotyrosyl-linked topo II-DNA adducts. Here, X-ray structures of mouse Tdp2-DNA complexes reveal that Tdp2 ß-2-helix-ß DNA damage-binding 'grasp', helical 'cap' and DNA lesion-binding elements fuse to form an elongated protein-DNA conjugate substrate-interaction groove. The Tdp2 DNA-binding surface is highly tailored for engagement of 5'-adducted single-stranded DNA ends and restricts nonspecific endonucleolytic or exonucleolytic processing. Structural, mutational and functional analyses support a single-metal ion catalytic mechanism for the exonuclease-endonuclease-phosphatase (EEP) nuclease superfamily and establish a molecular framework for targeted small-molecule blockade of Tdp2-mediated resistance to anticancer topoisomerase drugs.

Structure of an aprataxin-DNA complex with insights into AOA1 neurodegenerative disease.

DNA ligases finalize DNA replication and repair through DNA nick-sealing reactions that can abort to generate cytotoxic 5'-adenylation DNA damage. Aprataxin (Aptx) catalyzes direct reversal of 5'-adenylate adducts to protect genome integrity. Here the structure of a Schizosaccharomyces pombe Aptx-DNA-AMP-Zn(2+) complex reveals active site and DNA interaction clefts formed by fusing a histidine triad (HIT) nucleotide hydrolase with a DNA minor groove-binding C(2)HE zinc finger (Znf). An Aptx helical 'wedge' interrogates the base stack for sensing DNA ends or DNA nicks. The HIT-Znf, the wedge and an '[F/Y]PK' pivot motif cooperate to distort terminal DNA base-pairing and direct 5'-adenylate into the active site pocket. Structural and mutational data support a wedge-pivot-cut HIT-Znf catalytic mechanism for 5'-adenylate adduct recognition and removal and suggest that mutations affecting protein folding, the active site pocket and the pivot motif underlie Aptx dysfunction in the neurodegenerative disorder ataxia with oculomotor apraxia 1 (AOA1).

Domain architecture and biochemical characterization of vertebrate Mcm10.

Mcm10 plays a key role in initiation and elongation of eukaryotic chromosomal DNA replication. As a first step to better understand the structure and function of vertebrate Mcm10, we have determined the structural architecture of Xenopus laevis Mcm10 (xMcm10) and characterized each domain biochemically. Limited proteolytic digestion of the full-length protein revealed N-terminal-, internal (ID)-, and C-terminal (CTD)-structured domains. Analytical ultracentrifugation revealed that xMcm10 self-associates and that the N-terminal domain forms homodimeric assemblies. DNA binding activity of xMcm10 was mapped to the ID and CTD, each of which binds to single- and double-stranded DNA with low micromolar affinity. The structural integrity of xMcm10-ID and CTD is dependent on the presence of bound zinc, which was experimentally verified by atomic absorption spectroscopy and proteolysis protection assays. The ID and CTD also bind independently to the N-terminal 323 residues of the p180 subunit of DNA polymerase alpha-primase. We propose that the modularity of the protein architecture, with discrete domains for dimerization and for binding to DNA and DNA polymerase alpha-primase, provides an effective means for coordinating the biochemical activities of Mcm10 within the replisome.