The variant form of the human syndrome xeroderma pigmentosum (XPV) is caused by a deficiency in DNA polymerase η (Polη), a DNA polymerase that enables replication through ultraviolet-induced pyrimidine dimers. Here we report high-resolution crystal structures of human Polη at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. Polη 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 Polη orthologues form specific hydrogen bonds with the lesion and the incoming nucleotide to assist translesion synthesis. On the basis of the structures, eight Polη missense mutations causing XPV can be rationalized as undermining the molecular splint or perturbing the active-site alignment. The structures also provide an insight into the role of Polη in replicating through D loop and DNA fragile sites.

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 chromatin-dependent 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.

EMBO Press is an editorially independent publishing platform for the development of EMBO scientific publications.