The variant form of human xeroderma pigmentosum syndrome (XPV) is caused by a deficiency in DNA polymerase η (Pol η) that enables replication through sunlight-induced pyrimidine dimers. We report high-resolution crystal structures of human Pol η at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. Pol η acts like a molecular splint to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for two-metal ion catalysis. Two residues conserved among Pol η orthologs form specific hydrogen bonds with the lesion and the incoming nucleotide to assist translesion synthesis. Based on the structures, eight Pol η missense mutations causing XPV can be rationalized as undermining the “molecular splint” or perturbing the active-site alignment. The structures also shed light on the role of Pol η in replicating through D loop and DNA fragile sites.

Nuclear processes such as transcription, DNA replication, and recombination are dynamically regulated by chromatin structure. Transcription is known to be regulated by chromatin-associated proteins containing conserved protein domains that specifically recognize distinct covalent posttranslational modifications on histones. However, it has been unclear whether similar mechanisms are involved in mammalian DNA recombination. Here, we show that RAG2 -an essential component of the RAG1/2 V(D)J recombinase, that mediates antigen receptor gene assembly 1 -contains a plant homeodomain (PHD) finger that specifically recognizes histone H3 trimethylated at lysine 4 (H3K4me3). The high-resolution crystal structure of the RAG2 PHD finger bound to H3K4me3 reveals the molecular basis of H3K4me3-recognition by RAG2. Mutations that abrogate RAG2's recognition of H3K4me3 severely impair V(D)J recombination in vivo. Reducing the level of H3K4me3 similarly leads to a decrease in V(D)J recombination in vivo. Notably, a conserved tryptophan residue (W453) that constitutes a key structural component of the K4me3-binding surface and is essential for RAG2's recognition of H3K4me3 is mutated in patients with immunodeficiency syndromes. Together our results identify a novel function for histone methylation in mammalian DNA recombination. Furthermore, our results provide the first evidence suggesting that disrupting the read-out of histone modifications can cause an inherited human disease. +To whom correspondence should be addressed: oettinger@frodo.mgh.harvard.edu; ogozani@stanford.edu. * These authors contributed equally to the work Note added in proof: While this work was under review, another study also reported that the RAG2 PHD finger binds to methylated H3K4 30 .Atomic coordinates and structure factors of the RAG2 PHD -H3K4me3 peptide complex have been deposited in the Protein Data Bank with the accession code of 2v89. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions.Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Since RAG2 contains a noncanonical plant homeodomain (PHD) finger 6,7 -a module that can mediate interactions with chromatin 8-10 -we asked whether a polypeptide encompassing the RAG2 PHD finger (RAG2 PHD : aa 414-527) can recognize modified histone proteins. In an in vitro screen of peptide microarrays containing ~70 distinct modified histone peptides, we found that RAG2 PHD specifically binds to histone H3 trimethylated at lysine 4 (H3K4me3) ( Fig. 1a ; Fig. S1; data not shown). The specificity of this interaction was confirmed by peptide pulldown assays ( Fig. 1b ; Fig. S2; Fig. S3). RAG2 has a C-terminal extension of 40 aa that is essential for phosphoinositide (PtdInsP)-binding 7 (aa 488-527), but this region is dispensable for H3K4me3-binding as the minimal PHD finger alone (aa 414-487) is sufficient for H3K4me3-recognition (Fig. 1c). In addition, the acidic hinge region of RAG2 (aa 388-412), previously implicated in...

MutL assists the mismatch recognition protein MutS to initiate and coordinate mismatch repair in species ranging from bacteria to humans. The MutL N-terminal ATPase domain is highly conserved, but the C-terminal region shares little sequence similarity among MutL homologs. We report here the crystal structure of the Escherichia coli MutL C-terminal dimerization domain and the likelihood of its conservation among MutL homologs. A 100-residue proline-rich linker between the ATPase and dimerization domains, which generates a large central cavity in MutL dimers, tolerates sequence substitutions and deletions of one-third of its length with no functional consequences in vivo or in vitro. Along the surface of the central cavity, residues essential for DNA binding are located in both the N- and C-terminal domains. Each domain of MutL interacts with UvrD helicase and is required for activating the helicase activity. The DNA-binding capacity of MutL is correlated with the level of UvrD activation. A model of how MutL utilizes its ATPase and DNA-binding activities to mediate mismatch-dependent activation of MutH endonuclease and UvrD helicase is proposed

Recombination activating gene (RAG) 1 and RAG2 together catalyze V(D)J gene rearrangement in lymphocytes as the first step in the assembly and maturation of antigen receptors. RAG2 contains a plant homeodomain (PHD) near its C terminus (RAG2-PHD) that recognizes histone H3 methylated at lysine 4 (H3K4me) and influences V(D)J recombination. We report here crystal structures of RAG2-PHD alone and complexed with five modified H3 peptides. Two aspects of RAG2-PHD are unique. First, in the absence of the modified peptide, a peptide N-terminal to RAG2-PHD occupies the substrate-binding site, which may reflect an autoregulatory mechanism. Second, in contrast to other H3K4me3-binding PHD domains, RAG2-PHD substitutes a carboxylate that interacts with arginine 2 (R2) with a Tyr, resulting in binding to H3K4me3 that is enhanced rather than inhibited by dimethylation of R2. Five residues involved in histone H3 recognition were found mutated in severe combined immunodeficiency (SCID) patients. Disruption of the RAG2-PHD structure appears to lead to the absence of T and B lymphocytes, whereas failure to bind H3K4me3 is linked to Omenn Syndrome. This work provides a molecular basis for chromatindependent gene recombination and presents a single protein domain that simultaneously recognizes two distinct histone modifications, revealing added complexity in the read-out of combinatorial histone modifications.J recombination is the site-specific DNA rearrangement that assembles antigen receptor genes from dispersed arrays of V, D, and J gene segments. Recombination is initiated by the lymphoid-specific recombination activating gene (RAG) 1 and RAG2 recombinase, which recognizes and cleaves the recombination signal sequences (1). V(D)J recombination is tightly regulated, occurring in a preferred temporal order and only in specific cell types and developmental stages. Ig heavy chain rearrangement precedes light chain rearrangement and Ig heavy-chain D to J joining precedes V to DJ recombination. In addition, Ig genes are fully rearranged only in B cells (not T cells), and T cell receptor genes are assembled in T but not B cells (1). Overexpression of RAG1 and RAG2 in nonlymphoid cells is sufficient to induce recombination of an artificial extrachromosomal substrate but does not support V(D)J recombination of endogenous loci (2). Therefore, the accessibility of these loci to the recombinase must be regulated (3). A large body of evidence suggests that the regulation of chromatin structure is involved in the regulation of V(D)J recombination (4-6).

N-Acetyl-L-glutamate kinase (NAGK), a member of the amino acid kinase family, catalyzes the second and frequently controlling step of arginine synthesis. The Escherichia coli NAGK crystal structure to 1.5 A resolution reveals a 258-residue subunit homodimer nucleated by a central 16-stranded molecular open beta sheet sandwiched between alpha helices. In each subunit, AMPPNP, as an alphabetagamma-phosphate-Mg2+ complex, binds along the sheet C edge, and N-acetyl-L-glutamate binds near the dyadic axis with its gamma-COO- aligned at short distance from the gamma-phosphoryl, indicating associative phosphoryl transfer assisted by: (1) Mg2+ complexation; (2) the positive charges on Lys8, Lys217, and on two helix dipoles; and (3) by hydrogen bonding with the y-phosphate. The structural resemblance with carbamate kinase and the alignment of the sequences suggest that NAGK is a structural and functional prototype for the amino acid kinase family, which differs from other acylphosphate-making devices represented by phosphoglycerate kinase, acetate kinase, and biotin carboxylase.

Upregulation of CAD, the multifunctional protein that initiates and controls the de novo biosynthesis of pyrimidines in animals, is essential for cell proliferation. Deciphering the architecture and functioning of CAD is of interest for its potential usage as an antitumoral target. However, there is no detailed structural information about CAD other than that it self-assembles into hexamers of ∼1.5 MDa. Here we report the crystal structure and functional characterization of the dihydroorotase domain of human CAD. Contradicting all assumptions, the structure reveals an active site enclosed by a flexible loop with two Zn²⁺ ions bridged by a carboxylated lysine and a third Zn coordinating a rare histidinate ion. Site-directed mutagenesis and functional assays prove the involvement of the Zn and flexible loop in catalysis. Comparison with homologous bacterial enzymes supports a reclassification of the DHOase family and provides strong evidence against current models of the architecture of CAD.

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