The L1-ORF1p coiled coil enables formation of a tightly compacted nucleic acid-bound complex that is associated with retrotransposition.

Long interspersed nuclear element 1 (L1) parasitized most vertebrates and constitutes ∼20% of the human genome. It encodes ORF1p and ORF2p which form an L1-ribonucleoprotein (RNP) with their encoding transcript that is copied into genomic DNA (retrotransposition). ORF1p binds single-stranded nucleic acid (ssNA) and exhibits NA chaperone activity. All vertebrate ORF1ps contain a coiled coil (CC) domain and we previously showed that a CC-retrotransposition null mutant prevented formation of stably bound ORF1p complexes on ssNA. Here, we compared CC variants using our recently improved method that measures ORF1p binding to ssDNA at different forces. Bound proteins decrease ssDNA contour length and at low force, retrotransposition-competent ORF1ps (111p and m14p) exhibit two shortening phases: the first is rapid, coincident with ORF1p binding; the second is slower, consistent with formation of tightly compacted complexes by NA-bound ORF1p. In contrast, two retrotransposition-null CC variants (151p and m15p) did not attain the second tightly compacted state. The C-terminal half of the ORF1p trimer (not the CC) contains the residues that mediate NA-binding. Our demonstrating that the CC governs the ability of NA-bound retrotransposition-competent trimers to form tightly compacted complexes reveals the biochemical phenotype of these coiled coil mutants.

Perturbation of base excision repair sensitizes breast cancer cells to APOBEC3 deaminase-mediated mutations.

Abundant APOBEC3 (A3) deaminase-mediated mutations can dominate the mutational landscape ('mutator phenotype') of some cancers, however, the basis of this sporadic vulnerability is unknown. We show here that elevated expression of the bifunctional DNA glycosylase, NEIL2, sensitizes breast cancer cells to A3B-mediated mutations and double-strand breaks (DSBs) by perturbing canonical base excision repair (BER). NEIL2 usurps the canonical lyase, APE1, at abasic sites in a purified BER system, rendering them poor substrates for polymerase ß. However, the nicked NEIL2 product can serve as an entry site for Exo1 in vitro to generate single-stranded DNA, which would be susceptible to both A3B and DSBs. As NEIL2 or Exo1 depletion mitigates the DNA damage caused by A3B expression, we suggest that aberrant NEIL2 expression can explain certain instances of A3B-mediated mutations.

Phosphorylation of ORF1p is required for L1 retrotransposition.

Although members of the L1 (LINE-1) clade of non-LTR retrotransposons can be deleterious, the L1 clade has remained active in most mammals for ∼100 million years and generated almost 40% of the human genome. The details of L1-host interaction are largely unknown, however. Here we report that L1 activity requires phosphorylation of the protein encoded by the L1 ORF1 (ORF1p). Critical phospho-acceptor residues (two serines and two threonines) reside in four conserved proline-directed protein kinase (PDPK) target sites. The PDPK family includes mitogen-activated protein kinases and cyclin-dependent kinases. Mutation of any PDPK phospho-acceptor inhibits L1 retrotransposition. The phosphomimetic aspartic acid can restore activity at the two serine sites, but not at either threonine site, where it is strongly inhibitory. ORF1p also contains conserved PDPK docking sites, which promote specific interaction of PDPKs with their targets. As expected, mutations in these sites also inhibit L1 activity. PDPK mutations in ORF1p that inactivate L1 have no significant effect on the ability of ORF1p to anneal RNA in vitro, an important biochemical property of the protein. We show that phosphorylated PDPK sites in ORF1p are required for an interaction with the peptidyl prolyl isomerase 1 (Pin1), a critical component of PDPK-mediated regulation. Pin1 acts via isomerization of proline side chains at phosphorylated PDPK motifs, thereby affecting substrate conformation and activity. Our demonstration that L1 activity is dependent on and integrated with cellular phosphorylation regulatory cascades significantly increases our understanding of interactions between L1 and its host.

Mutational analysis of the T4 gp59 helicase loader reveals its sites for interaction with helicase, single-stranded binding protein, and DNA.

Efficient DNA replication involves coordinated interactions among DNA polymerase, multiple factors, and the DNA. From bacteriophage T4 to eukaryotes, these factors include a helicase to unwind the DNA ahead of the replication fork, a single-stranded binding protein (SSB) to bind to the ssDNA on the lagging strand, and a helicase loader that associates with the fork, helicase, and SSB. The previously reported structure of the helicase loader in the T4 system, gene product (gp)59, has revealed an N-terminal domain, which shares structural homology with the high mobility group (HMG) proteins from eukaryotic organisms. Modeling of this structure with fork DNA has suggested that the HMG-like domain could bind to the duplex DNA ahead of the fork, whereas the C-terminal portion of gp59 would provide the docking sites for helicase (T4 gp41), SSB (T4 gp32), and the ssDNA fork arms. To test this model, we have used random and targeted mutagenesis to generate mutations throughout gp59. We have assayed the ability of the mutant proteins to bind to fork, primed fork, and ssDNAs, to interact with SSB, to stimulate helicase activity, and to function in leading and lagging strand DNA synthesis. Our results provide strong biochemical support for the role of the N-terminal gp59 HMG motif in fork binding and the interaction of the C-terminal portion of gp59 with helicase and SSB. Our results also suggest that processive replication may involve the switching of gp59 between its interactions with helicase and SSB.

Polymerization and nucleic acid-binding properties of human L1 ORF1 protein.

The L1 (LINE 1) retrotransposable element encodes two proteins, ORF1p and ORF2p. ORF2p is the L1 replicase, but the role of ORF1p is unknown. Mouse ORF1p, a coiled-coil-mediated trimer of ∼42-kDa monomers, binds nucleic acids and has nucleic acid chaperone activity. We purified human L1 ORF1p expressed in insect cells and made two findings that significantly advance our knowledge of the protein. First, in the absence of nucleic acids, the protein polymerizes under the very conditions (0.05 M NaCl) that are optimal for high (∼1 nM)-affinity nucleic acid binding. The non-coiled-coil C-terminal half mediates formation of the polymer, an active conformer that is instantly resolved to trimers, or multimers thereof, by nucleic acid. Second, the protein has a biphasic effect on mismatched double-stranded DNA, a proxy chaperone substrate. It protects the duplex from dissociation at 37°C before eventually melting it when largely polymeric. Therefore, polymerization of ORF1p seemingly affects its interaction with nucleic acids. Additionally, polymerization of ORF1p at its translation site could explain the heretofore-inexplicable phenomenon of cis preference-the favored retrotransposition of the actively translated L1 transcript, which is essential for L1 survival.

Combined experimental and theoretical study of Al(n)X (n = 1-6; X = As, Sb) clusters: evidence of aromaticity and the Jellium model.

The electronic structure of Al(n)X (n = 1-6; X = As, Sb) clusters has been investigated using a synergistic approach combining negative ion photoelectron spectroscopy and first principles electronic structure calculations. It is shown that Al(3)X and Al(5)X exhibit enhanced energetic stability, as evidenced from calculated removal and embedding energies as well as chemical stability manifested through a large gap between the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO). However, the stabilities of these species are derived from different mechanisms. Al(3)As and Al(3)Sb, with HOMO-LUMO gaps of 1.86 and 1.73 eV, respectively, are shown to have planar geometries where the p orbitals combine to form one pi and two sigma aromatic orbitals reminiscent of conventional all-metal aromatic species. Al(5)As and Al(5)Sb, with 20 valence electrons, possess a closed electronic shell (1s(2), 1p(6), 1d(10), 2s(2)) within a jellium framework and have HOMO-LUMO gaps of 1.12 and 1.17 eV, respectively.

Structural evolution of triniobium carbide clusters: evidence of large Cn chains (n = 3-4) in Nb3Cn- (n = 5-10) clusters.

First-principle density functional calculations and photoelectron spectroscopy experiments show that triniobium carbide clusters exist in multiple motifs. The Nb(3)C(n)(-) (n = 5-10) series have isomers surrounding a triangular Nb(3) base while incorporating Nb-C bonding. We provide evidence of not only C(2) carbon chains but also stable isomers with previously unidentified C(3) and C(4) carbon chains in triniobium carbide clusters.

Al(n)Bi clusters: transitions between aromatic and jellium stability.

An experimental and theoretical study of bismuth-doped aluminum clusters in the gas phase has revealed two particularly stable clusters, namely, Al(3)Bi and Al(5)Bi. We show that their electronic structure can be understood in terms of the aromatic and "Jellium" models, respectively. Negative ion photodetachment spectra provide a fingerprint of the electronic states in Al(n)Bi(-) (n = 1-5) anions, while theoretical investigations reveal the nature of the electronic orbitals involved. Together, the findings reveal that the all-metal Al(3)Bi cluster with 14 valence electrons is a cyclic, planar structure with a calculated large ionization potential of 7.08 eV, a low electron affinity of 1.41 eV, and a large gap of 1.69 eV between the highest occupied and lowest unoccupied molecular orbitals (HOMO-LUMO gap). The Al(3)Bi cluster has molecular orbitals reminiscent of aromatic systems and is a neutral cluster with no need for counterion or ligand support. A slightly larger cluster, Al(5)Bi, has 20 valence electrons and is another highly stable compact structure with a calculated large ionization potential of 6.51 eV and a large HOMO-LUMO gap of 1.15 eV. This cluster's stability is rooted in a Jellium electronic shell closing. The formation of stable species using aromatic bonding allows us to extend the idea of cluster-assembled materials built out of stable clusters with Jellium shell closings (superatoms) to include ones involving aromatic building blocks.

Crystal structure of bacteriophage T4 5' nuclease in complex with a branched DNA reveals how flap endonuclease-1 family nucleases bind their substrates.

Bacteriophage T4 RNase H, a flap endonuclease-1 family nuclease, removes RNA primers from lagging strand fragments. It has both 5' nuclease and flap endonuclease activities. Our previous structure of native T4 RNase H (PDB code 1TFR) revealed an active site composed of highly conserved Asp residues and two bound hydrated magnesium ions. Here, we report the crystal structure of T4 RNase H in complex with a fork DNA substrate bound in its active site. This is the first structure of a flap endonuclease-1 family protein with its complete branched substrate. The fork duplex interacts with an extended loop of the helix-hairpin-helix motif class 2. The 5' arm crosses over the active site, extending below the bridge (helical arch) region. Cleavage assays of this DNA substrate identify a primary cut site 7-bases in from the 5' arm. The scissile phosphate, the first bond in the duplex DNA adjacent to the 5' arm, lies above a magnesium binding site. The less ordered 3' arm reaches toward the C and N termini of the enzyme, which are binding sites for T4 32 protein and T4 45 clamp, respectively. In the crystal structure, the scissile bond is located within the double-stranded DNA, between the first two duplex nucleotides next to the 5' arm, and lies above a magnesium binding site. This complex provides important insight into substrate recognition and specificity of the flap endonuclease-1 enzymes.

Architecture of the bacteriophage T4 replication complex revealed with nanoscale biopointers.

Our previous electron microscopy of DNA replicated by the bacteriophage T4 proteins showed a single complex at the fork, thought to contain the leading and lagging strand proteins, as well as the protein-covered single-stranded DNA on the lagging strand folded into a compact structure. "Trombone" loops formed from nascent lagging strand fragments were present on a majority of the replicating molecules (Chastain, P., Makhov, A. M., Nossal, N. G., and Griffith, J. D. (2003) J. Biol. Chem. 278, 21276-21285). Here we probe the composition of this replication complex using nanoscale DNA biopointers to show the location of biotin-tagged replication proteins. We find that a large fraction of the molecules with a trombone loop had two pointers to polymerase, providing strong evidence that the leading and lagging strand polymerases are together in the replication complex. 6% of the molecules had two loops, and 31% of these had three pointers to biotin-tagged polymerase, suggesting that the two loops result from two fragments that are being extended simultaneously. Under fixation conditions that extend the lagging strand, occasional molecules show two nascent lagging strand fragments, each being elongated by a biotin-tagged polymerase. T4 41 helicase is present in the complex on a large fraction of actively replicating molecules but on a smaller fraction of molecules with a stalled polymerase. Unexpectedly, we found that 59 helicase-loading protein remains on the fork after loading the helicase and is present on molecules with extensive replication.

Maturation of bacteriophage T4 lagging strand fragments depends on interaction of T4 RNase H with T4 32 protein rather than the T4 gene 45 clamp.

In the bacteriophage T4 DNA replication system, T4 RNase H removes the RNA primers and some adjacent DNA before the lagging strand fragments are ligated. This 5'-nuclease has strong structural and functional similarity to the FEN1 nuclease family. We have shown previously that T4 32 protein binds DNA behind the nuclease and increases its processivity. Here we show that T4 RNase H with a C-terminal deletion (residues 278-305) retains its exonuclease activity but is no longer affected by 32 protein. T4 gene 45 replication clamp stimulates T4 RNase H on nicked or gapped substrates, where it can be loaded behind the nuclease, but does not increase its processivity. An N-terminal deletion (residues 2-10) of a conserved clamp interaction motif eliminates stimulation by the clamp. In the crystal structure of T4 RNase H, the binding sites for the clamp at the N terminus and for 32 protein at the C terminus are located close together, away from the catalytic site of the enzyme. By using mutant T4 RNase H with deletions in the binding site for either the clamp or 32 protein, we show that it is the interaction of T4 RNase H with 32 protein, rather than the clamp, that most affects the maturation of lagging strand fragments in the T4 replication system in vitro and T4 phage production in vivo.

Mutations of bacteriophage T4 59 helicase loader defective in binding fork DNA and in interactions with T4 32 single-stranded DNA-binding protein.

Bacteriophage T4 gene 59 protein greatly stimulates the loading of the T4 gene 41 helicase in vitro and is required for recombination and recombination-dependent DNA replication in vivo. 59 protein binds preferentially to forked DNA and interacts directly with the T4 41 helicase and gene 32 single-stranded DNA-binding protein. The helicase loader is an almost completely alpha-helical, two-domain protein, whose N-terminal domain has strong structural similarity to the DNA-binding domains of high mobility group proteins. We have previously speculated that this high mobility group-like region may bind the duplex ahead of the fork, with the C-terminal domain providing separate binding sites for the fork arms and at least part of the docking area for the helicase and 32 protein. Here, we characterize several mutants of 59 protein in an initial effort to test this model. We find that the I87A mutation, at the position where the fork arms would separate in the model, is defective in binding fork DNA. As a consequence, it is defective in stimulating both unwinding by the helicase and replication by the T4 system. 59 protein with a deletion of the two C-terminal residues, Lys(216) and Tyr(217), binds fork DNA normally. In contrast to the wild type, the deletion protein fails to promote binding of 32 protein on short fork DNA. However, it binds 32 protein in the absence of DNA. The deletion is also somewhat defective in stimulating unwinding of fork DNA by the helicase and replication by the T4 system. We suggest that the absence of the two terminal residues may alter the configuration of the lagging strand fork arm on the surface of the C-terminal domain, so that it is a poorer docking site for the helicase and 32 protein.

Bacteriophage T4 32 protein is required for helicase-dependent leading strand synthesis when the helicase is loaded by the T4 59 helicase-loading protein.

In the bacteriophage T4 DNA replication system, T4 gene 59 protein binds preferentially to fork DNA and accelerates the loading of the T4 41 helicase. 59 protein also binds the T4 32 single-stranded DNA-binding protein that coats the lagging strand template. Here we explore the function of the strong affinity between the 32 and 59 proteins at the replication fork. We show that, in contrast to the 59 helicase loader, 32 protein does not bind forked DNA more tightly than linear DNA. 32 protein displays a strong binding polarity on fork DNA, binding with much higher affinity to the 5' single-stranded lagging strand template arm of a model fork, than to the 3' single-stranded leading strand arm. 59 protein promotes the binding of 32 protein on forks too short for cooperative binding by 32 protein. We show that 32 protein is required for helicase-dependent leading strand DNA synthesis when the helicase is loaded by 59 protein. However, 32 protein is not required for leading strand synthesis when helicase is loaded, less efficiently, without 59 protein. Leading strand synthesis by wild type T4 polymerase is strongly inhibited when 59 protein is present without 32 protein. Because 59 protein can load the helicase on forks without 32 protein, our results are best explained by a model in which 59 helicase loader at the fork prevents the coupling of the leading strand polymerase and the helicase, unless the position of 59 protein is shifted by its association with 32 protein.