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Human exonuclease 1 (hExo1) is a member of the RAD2/XPG structure-specific 5'-nuclease superfamily. Its dominant, processive 5'-3' exonuclease and secondary 5'-flap endonuclease activities participate in various DNA repair, recombination, and replication processes. A single active site processes both recessed ends and 5'-flap substrates. By initiating enzyme reactions in crystals, we have trapped hExo1 reaction intermediates that reveal structures of these substrates before and after their exo- and endonucleolytic cleavage, as well as structures of uncleaved, unthreaded, and partially threaded 5' flaps. Their distinctive 5' ends are accommodated by a small, mobile arch in the active site that binds recessed ends at its base and threads 5' flaps through a narrow aperture within its interior. A sequence of successive, interlocking conformational changes guides the two substrate types into a shared reaction mechanism that catalyzes their cleavage by an elaborated variant of the two-metal, in-line hydrolysis mechanism. Coupling of substrate-dependent arch motions to transition-state stabilization suppresses inappropriate or premature cleavage, enhancing processing fidelity. The striking reduction in flap conformational entropy is catalyzed, in part, by arch motions and transient binding interactions between the flap and unprocessed DNA strand. At the end of the observed reaction sequence, hExo1 resets without relinquishing DNA binding, suggesting a structural basis for its processivity., Competing Interests: The authors declare no conflict of interest.

Nucleotide excision repair (NER) is responsible for the removal of a large variety of structurally diverse DNA lesions. Mutations of the involved proteins cause the xeroderma pigmentosum (XP) cancer predisposition syndrome. Although the general mechanism of the NER process is well studied, the function of the XPA protein, which is of central importance for successful NER, has remained enigmatic. It is known, that XPA binds kinked DNA structures and that it interacts also with DNA duplexes containing certain lesions, but the mechanism of interactions is unknown. Here we present two crystal structures of the DNA binding domain (DBD) of the yeast XPA homolog Rad14 bound to DNA with either a cisplatin lesion (1,2-GG) or an acetylaminofluorene adduct (AAF-dG). In the structures, we see that two Rad14 molecules bind to the duplex, which induces DNA melting of the duplex remote from the lesion. Each monomer interrogates the duplex with a β-hairpin, which creates a 13mer duplex recognition motif additionally characterized by a sharp 70° DNA kink at the position of the lesion. Although the 1,2-GG lesion stabilizes the kink due to the covalent fixation of the crosslinked dG bases at a 90° angle, the AAF-dG fully intercalates into the duplex to stabilize the kinked structure.

Polynucleotide kinase/phosphatase (PNKP) is a critical mammalian DNA repair enzyme that generates 5'-phosphate and 3'-hydroxyl groups at damaged DNA termini that are required for subsequent processing by DNA ligases and polymerases. The PNKP phosphatase domain recognizes 3'-phosphate termini within DNA nicks, gaps, or at double- or single-strand breaks. Here we present a mechanistic rationale for the recognition of damaged DNA termini by the PNKP phosphatase domain. The crystal structures of PNKP bound to single-stranded DNA substrates reveals a narrow active site cleft that accommodates a single-stranded substrate in a sequence-independent manner. Biochemical studies suggest that the terminal base pairs of double-stranded substrates near the 3'-phosphate are destabilized by PNKP to allow substrate access to the active site. A positively charged surface distinct from the active site specifically facilitates interactions with double-stranded substrates, providing a complex DNA binding surface that enables the recognition of diverse substrates.

High concentration of NaCl increases DNA breaks both in cell culture and in vivo. The breaks remain elevated as long as NaCl concentration remains high and are rapidly repaired when the concentration is lowered. The exact nature of the breaks, and their location, has not been entirely clear, and it has not been evident how cells survive, replicate, and maintain genome integrity in environments like the renal inner medulla in which cells are constantly exposed to high NaCl concentration. Repair of the breaks after NaCl is reduced is accompanied by formation of foci containing phosphorylated H2AX (γH2AX), which occurs around DNA double-strand breaks and contributes to their repair. Here, we confirm by specific comet assay and pulsed-field electrophoresis that cells adapted to high NaCl have increased levels of double-strand breaks. Importantly, γH2AX foci that occur during repair of the breaks are nonrandomly distributed in the mouse genome. By chromatin immunoprecipitation using anti-γH2AX antibody, followed by massive parallel sequencing (ChIP-Seq), we find that during repair of double-strand breaks induced by high NaCl, γH2AX is predominantly localized to regions of the genome devoid of genes ("gene deserts"), indicating that the high NaCl-induced double-strand breaks are located there. Localization to gene deserts helps explain why the DNA breaks are less harmful than are the random breaks induced by genotoxic agents such as UV radiation, ionizing radiation, and oxidants. We propose that the universal presence of NaCl around animal cells has directly influenced the evolution of the structure of their genomes.

Cernunnos/XLF is a core protein of the nonhomologous DNA end-joining (NHEJ) pathway that processes the majority of DNA double-strand breaks in mammals. Cernunnos stimulates the final ligation step catalyzed by the complex between DNA ligase IV and Xrcc4 (X4). Here we present the crystal structure of the X4(1-157)-Cernunnos(1-224) complex at 5.5-Å resolution and identify the relative positions of the two factors and their binding sites. The X-ray structure reveals a filament arrangement for X4(1-157) and Cernunnos(1-224) homodimers mediated by repeated interactions through their N-terminal head domains. A filament arrangement of the X4-Cernunnos complex was confirmed by transmission electron microscopy analyses both with truncated and full-length proteins. We further modeled the interface and used structure-based site-directed mutagenesis and calorimetry to characterize the roles of various residues at the X4-Cernunnos interface. We identified four X4 residues (Glu(55), Asp(58), Met(61), and Phe(106)) essential for the interaction with Cernunnos. These findings provide new insights into the molecular bases for stimulatory and bridging roles of Cernunnos in the final DNA ligation step.

Editing of the pre-mRNA for the DNA repair enzyme NEIL1 causes a lysine to arginine change in the lesion recognition loop of the protein. The two forms of NEIL1 are shown here to have distinct enzymatic properties. The edited form removes thymine glycol from duplex DNA 30 times more slowly than the form encoded in the genome, whereas editing enhances repair of the guanidinohydantoin lesion by NEIL1. In addition, we show that the NEIL1 recoding site is a preferred editing site for the RNA editing adenosine deaminase ADAR1. The edited adenosine resides in an A-C mismatch in a hairpin stem formed by pairing of exon 6 to the immediate upstream intron 5 sequence. As expected for an ADAR1 site, editing at this position is increased in human cells treated with interferon α. These results suggest a unique regulatory mechanism for DNA repair and extend our understanding of the impact of RNA editing.

The DNA ligase D (LigD) 3'-phosphoesterase (PE) module is a conserved component of the bacterial nonhomologous end-joining (NHEJ) apparatus that performs 3' end-healing reactions at DNA double-strand breaks. Here we report the 1.9 A crystal structure of Pseudomonas aeruginosa PE, which reveals that PE exemplifies a unique class of DNA repair enzyme. PE has a distinctive fold in which an eight stranded beta barrel with a hydrophobic interior supports a crescent-shaped hydrophilic active site on its outer surface. Six essential side chains coordinate manganese and a sulfate mimetic of the scissile phosphate. The PE active site and mechanism are unique vis à vis other end-healing enzymes. We find PE homologs in archaeal and eukaryal proteomes, signifying that PEs comprise a DNA repair superfamily.

The error-free repair of double-stranded DNA breaks by homologous recombination requires processing of broken ends. These processed ends are substrates for assembly of DNA strand exchange proteins that mediate DNA strand invasion. Here, we establish that human BLM helicase, a member of the RecQ family, stimulates the nucleolytic activity of human exonuclease 1 (hExo1), a 5'-->3' double-stranded DNA exonuclease. The stimulation is specific because other RecQ homologs fail to stimulate hExo1. Stimulation of DNA resection by hExo1 is independent of BLM helicase activity and is, instead, mediated by an interaction between the 2 proteins. Finally, we show that DNA ends resected by hExo1 and BLM are used by human Rad51, but not its yeast or bacterial counterparts, to promote homologous DNA pairing. This in vitro system recapitulates initial steps of homologous recombination and provides biochemical evidence for a role of BLM and Exo1 in the initiation of recombinational DNA repair.

DNA-protein crosslinks are relatively common DNA lesions that form during the physiological processing of DNA by replication and recombination proteins, by side reactions of base excision repair enzymes, and by cellular exposure to bifunctional DNA-damaging agents such as platinum compounds. The mechanism by which pathological DNA-protein crosslinks are repaired in humans is not known. In this study, we investigated the mechanism of recognition and repair of protein-DNA and oligopeptide-DNA crosslinks by the human excision nuclease. Under our assay conditions, the human nucleotide excision repair system did not remove a 16-kDa protein crosslinked to DNA at a detectable level. However, 4- and 12-aa-long oligopeptides crosslinked to the DNA backbone were recognized by some of the damage recognition factors of the human excision nuclease with moderate selectivity and were excised from DNA at relatively efficient rates. Our data suggest that, if coupled with proteolytic degradation of the crosslinked protein, the human excision nuclease may be the major enzyme system for eliminating protein-DNA crosslinks from the genome.