Human flap endonuclease structures, DNA double-base flipping, and a unified understanding of the FEN1 superfamily.

Flap endonuclease (FEN1), essential for DNA replication and repair, removes RNA and DNA 5' flaps. FEN1 5' nuclease superfamily members acting in nucleotide excision repair (XPG), mismatch repair (EXO1), and homologous recombination (GEN1) paradoxically incise structurally distinct bubbles, ends, or Holliday junctions, respectively. Here, structural and functional analyses of human FEN1:DNA complexes show structure-specific, sequence-independent recognition for nicked dsDNA bent 100° with unpaired 3' and 5' flaps. Above the active site, a helical cap over a gateway formed by two helices enforces ssDNA threading and specificity for free 5' ends. Crystallographic analyses of product and substrate complexes reveal that dsDNA binding and bending, the ssDNA gateway, and double-base unpairing flanking the scissile phosphate control precise flap incision by the two-metal-ion active site. Superfamily conserved motifs bind and open dsDNA; direct the target region into the helical gateway, permitting only nonbase-paired oligonucleotides active site access; and support a unified understanding of superfamily substrate specificity.

Uracil DNA N-glycosylase promotes assembly of human centromere protein A.

Uracil is removed from DNA by the conserved enzyme uracil DNA N-glycosylase (UNG). Previously, we observed that inhibiting UNG in Xenopus egg extracts blocked assembly of CENP-A, a histone H3 variant. CENP-A is an essential protein in all species, since it is required for chromosome segregation during mitosis. Thus, the implication of UNG in CENP-A assembly implies that UNG would also be essential, but UNG mutants lacking catalytic activity are viable in all species. In this paper, we present evidence that UNG2 colocalizes with CENP-A and H2AX phosphorylation at centromeres in normally cycling cells. Reduction of UNG2 in human cells blocks CENP-A assembly, and results in reduced cell proliferation, associated with increased frequencies of mitotic abnormalities and rapid cell death. Overexpression of UNG2 induces high levels of CENP-A assembly in human cells. Using a multiphoton laser approach, we demonstrate that UNG2 is rapidly recruited to sites of DNA damage. Taken together, our data are consistent with a model in which the N-terminus of UNG2 interacts with the active site of the enzyme and with chromatin.

Double-strand DNA breaks recruit the centromeric histone CENP-A.

The histone H3 variant CENP-A is required for epigenetic specification of centromere identity through a loading mechanism independent of DNA sequence. Using multiphoton absorption and DNA cleavage at unique sites by I-SceI endonuclease, we demonstrate that CENP-A is rapidly recruited to double-strand breaks in DNA, along with three components (CENP-N, CENP-T, and CENP-U) associated with CENP-A at centromeres. The centromere-targeting domain of CENP-A is both necessary and sufficient for recruitment to double-strand breaks. CENP-A accumulation at DNA breaks is enhanced by active non-homologous end-joining but does not require DNA-PKcs or Ligase IV, and is independent of H2AX. Thus, induction of a double-strand break is sufficient to recruit CENP-A in human and mouse cells. Finally, since cell survival after radiation-induced DNA damage correlates with CENP-A expression level, we propose that CENP-A may have a function in DNA repair.

Three metal ions participate in the reaction catalyzed by T5 flap endonuclease.

Protein nucleases and RNA enzymes depend on divalent metal ions to catalyze the rapid hydrolysis of phosphate diester linkages of nucleic acids during DNA replication, DNA repair, RNA processing, and RNA degradation. These enzymes are widely proposed to catalyze phosphate diester hydrolysis using a "two-metal-ion mechanism." Yet, analyses of flap endonuclease (FEN) family members, which occur in all domains of life and act in DNA replication and repair, exemplify controversies regarding the classical two-metal-ion mechanism for phosphate diester hydrolysis. Whereas substrate-free structures of FENs identify two active site metal ions, their typical separation of > 4 A appears incompatible with this mechanism. To clarify the roles played by FEN metal ions, we report here a detailed evaluation of the magnesium ion response of T5FEN. Kinetic investigations reveal that overall the T5FEN-catalyzed reaction requires at least three magnesium ions, implying that an additional metal ion is bound. The presence of at least two ions bound with differing affinity is required to catalyze phosphate diester hydrolysis. Analysis of the inhibition of reactions by calcium ions is consistent with a requirement for two viable cofactors (Mg2+ or Mn2+). The apparent substrate association constant is maximized by binding two magnesium ions. This may reflect a metal-dependent unpairing of duplex substrate required to position the scissile phosphate in contact with metal ion(s). The combined results suggest that T5FEN primarily uses a two-metal-ion mechanism for chemical catalysis, but that its overall metallobiochemistry is more complex and requires three ions.

Comparison of the catalytic parameters and reaction specificities of a phage and an archaeal flap endonuclease.

Flap endonucleases (FENs) catalyse the exonucleolytic hydrolysis of blunt-ended duplex DNA substrates and the endonucleolytic cleavage of 5'-bifurcated nucleic acids at the junction formed between single and double-stranded DNA. The specificity and catalytic parameters of FENs derived from T5 bacteriophage and Archaeoglobus fulgidus were studied with a range of single oligonucleotide DNA substrates. These substrates contained one or more hairpin turns and mimic duplex, 5'-overhanging duplex, pseudo-Y, nicked DNA, and flap structures. The FEN-catalysed reaction properties of nicked DNA and flap structures possessing an extrahelical 3'-nucleotide (nt) were also characterised. The phage enzyme produced multiple reaction products of differing length with all the substrates tested, except when the length of duplex DNA downstream of the reaction site was truncated. Only larger DNAs containing two duplex regions are effective substrates for the archaeal enzyme and undergo reaction at multiple sites when they lack a 3'-extrahelical nucleotide. However, a single product corresponding to reaction 1 nt into the double-stranded region occurred with A. fulgidus FEN when substrates possessed a 3'-extrahelical nt. Steady-state and pre-steady-state catalytic parameters reveal that the phage enzyme is rate-limited by product release with all the substrates tested. Single-turnover maximal rates of reaction are similar with most substrates. In contrast, turnover numbers for T5FEN decrease as the size of the DNA substrate is increased. Comparison of the catalytic parameters of the A. fulgidus FEN employing flap and double-flap substrates indicates that binding interactions with the 3'-extrahelical nucleotide stabilise the ground state FEN-DNA interaction, leading to stimulation of comparative reactions at DNA concentrations below saturation with the single flap substrate. Maximal multiple turnover rates of the archaeal enzyme with flap and double flap substrates are similar. A model is proposed to account for the varying specificities of the two enzymes with regard to cleavage patterns and substrate preferences.

The C-terminal domain of yeast PCNA is required for physical and functional interactions with Cdc9 DNA ligase.

There is compelling evidence that proliferating cell nuclear antigen (PCNA), a DNA sliding clamp, co-ordinates the processing and joining of Okazaki fragments during eukaryotic DNA replication. However, a detailed mechanistic understanding of functional PCNA:ligase I interactions has been incomplete. Here we present the co-crystal structure of yeast PCNA with a peptide encompassing the conserved PCNA interaction motif of Cdc9, yeast DNA ligase I. The Cdc9 peptide contacts both the inter-domain connector loop (IDCL) and residues near the C-terminus of PCNA. Complementary mutational and biochemical results demonstrate that these two interaction interfaces are required for complex formation both in the absence of DNA and when PCNA is topologically linked to DNA. Similar to the functionally homologous human proteins, yeast RFC interacts with and inhibits Cdc9 DNA ligase whereas the addition of PCNA alleviates inhibition by RFC. Here we show that the ability of PCNA to overcome RFC-mediated inhibition of Cdc9 is dependent upon both the IDCL and the C-terminal interaction interfaces of PCNA. Together these results demonstrate the functional significance of the beta-zipper structure formed between the C-terminal domain of PCNA and Cdc9 and reveal differences in the interactions of FEN-1 and Cdc9 with the two PCNA interfaces that may contribute to the co-ordinated, sequential action of these enzymes.

Proliferating cell nuclear antigen loaded onto double-stranded DNA: dynamics, minor groove interactions and functional implications.

Proliferating cell nuclear antigen (PCNA) acts as a biologically essential processivity factor that encircles DNA and provides binding sites for polymerase, flap endonuclease-1 (FEN-1) and ligase during DNA replication and repair. We have computationally characterized the interactions of human and Archaeoglobus fulgidus PCNA trimer with double-stranded DNA (ds DNA) using multi-nanosecond classical molecular dynamics simulations. The results reveal the interactions of DNA passing through the PCNA trimeric ring including the contacts formed, overall orientation and motion with respect to the sliding clamp. Notably, we observe pronounced tilting of the axis of dsDNA with respect to the PCNA ring plane reflecting interactions between the DNA phosphodiester backbone and positively charged arginine and lysine residues lining the PCNA inner surface. Covariance matrix analysis revealed a pattern of correlated motions within and between the three equivalent subunits involving the PCNA C-terminal region and linker strand associated with partner protein binding sites. Additionally, principal component analysis identified low frequency global PCNA subunit motions suitable for translocation along duplex DNA. The PCNA motions and interactions with the DNA minor groove, identified here computationally, provide an unexpected basis for PCNA to act in the coordinated handoff of intermediates from polymerase to FEN-1 to ligase during DNA replication and repair.

Dual modes of RNA-silencing suppression by Flock House virus protein B2.

As a counter-defense against antiviral RNA silencing during infection, the insect Flock House virus (FHV) expresses the silencing suppressor protein B2. Biochemical experiments show that B2 binds to double-stranded RNA (dsRNA) without regard to length and inhibits cleavage of dsRNA by Dicer in vitro. A cocrystal structure reveals that a B2 dimer forms a four-helix bundle that binds to one face of an A-form RNA duplex independently of sequence. These results suggest that B2 blocks both cleavage of the FHV genome by Dicer and incorporation of FHV small interfering RNAs into the RNA-induced silencing complex.

Interaction interface of human flap endonuclease-1 with its DNA substrates.

Flap endonuclease-1 or FEN-1 is a structure-specific and multifunctional nuclease critical for DNA replication, repair, and recombination; however, its interaction with DNA substrates has not been fully understood. In the current study, we have defined the borders of the interaction between the FEN-1 protein and its DNA substrates and identified six clusters of conserved positively charged amino acid residues, which are in direct contact with DNA substrate. To map further the corresponding interactions between FEN-1 residues and DNA substrates, we performed biochemical assays employing a series of flap DNA substrates lacking some structural components and a series of binding-deficient point mutants of FEN-1. It was revealed that Arg(47), Arg(70), and Lys(326)-Arg(327) of FEN-1 interact with the upstream duplex of DNA substrates, whereas Lys(244)-Arg(245) interact with the downstream duplex. This result indicates the orientation of the FEN-1-DNA interaction. Moreover, Arg(70) and Arg(47) were determined to interact with the sites around the 2nd nucleotide (Arg(70)) or the 5th/6th nucleotide (Arg(47)) of the template strand in the upstream duplex portion counting from the nick point of the flap substrate. Together with previously published data and the crystallographic ainformation from the FEN-1.DNA complex that we published recently (Chapados, B. R., Hosfield, D. J., Han, S., Qiu, J., Yelent, B., Shen, B., Tainer, J. A. (2004) Cell 116, 39-50) we are able to propose a reasonable model for how the human FEN-1 protein interacts with its DNA substrates.

Structural basis for FEN-1 substrate specificity and PCNA-mediated activation in DNA replication and repair.

Flap EndoNuclease-1 (FEN-1) and the processivity factor proliferating cell nuclear antigen (PCNA) are central to DNA replication and repair. To clarify the molecular basis of FEN-1 specificity and PCNA activation, we report here structures of FEN-1:DNA and PCNA:FEN-1-peptide complexes, along with fluorescence resonance energy transfer (FRET) and mutational results. FEN-1 binds the unpaired 3' DNA end (3' flap), opens and kinks the DNA, and promotes conformational closing of a flexible helical clamp to facilitate 5' cleavage specificity. Ordering of unstructured C-terminal regions in FEN-1 and PCNA creates an intermolecular beta sheet interface that directly links adjacent PCNA and DNA binding regions of FEN-1 and suggests how PCNA stimulates FEN-1 activity. The DNA and protein conformational changes, composite complex structures, FRET, and mutational results support enzyme-PCNA alignments and a kinked DNA pivot point that appear suitable to coordinate rotary handoffs of kinked DNA intermediates among enzymes localized by the three PCNA binding sites.