Origin Remodeling and Opening in Bacteria Rely on Distinct Assembly States of the DnaA Initiator

The initiation of DNA replication requires the melting of chromosomal origins to provide a template for replisomal polymerases. In bacteria, the DnaA initiator plays a key role in this process, forming a large nucleoprotein complex that opens DNA through a complex and poorly understood mechanism. Using structure-guided mutagenesis, biochemical, and genetic approaches, we establish an unexpected link between the duplex DNA-binding domain of DnaA and the ability of the protein to both self-assemble and engage single-stranded DNA in an ATP-dependent manner. Intersubunit cross-talk between this domain and the DnaA ATPase region regulates this link and is required for both origin unwinding in vitro and initiator function in vivo. These findings indicate that DnaA utilizes at least two different oligomeric conformations for engaging single- and double-stranded DNA, and that these states play distinct roles in controlling the progression of initiation.     

The N-terminal Domain of the Drosophila Mitochondrial Replicative DNA Helicase Contains an Iron-Sulfur Cluster and Binds DNA

The metazoan mitochondrial DNA helicase is an integral part of the minimal mitochondrial replisome. It exhibits strong sequence homology with the bacteriophage T7 gene 4 protein primase-helicase (T7 gp4). Both proteins contain distinct N- and C-terminal domains separated by a flexible linker. The C-terminal domain catalyzes its characteristic DNA-dependent NTPase activity, and can unwind duplex DNA substrates independently of the N-terminal domain. Whereas the N-terminal domain in T7 gp4 contains a DNA primase activity, this function is lost in metazoan mtDNA helicase. Thus, although the functions of the C-terminal domain and the linker are partially understood, the role of the N-terminal region in the metazoan replicative mtDNA helicase remains elusive. Here, we show that the N-terminal domain of Drosophila melanogaster mtDNA helicase coordinates iron in a 2Fe-2S cluster that enhances protein stability in vitro. The N-terminal domain binds the cluster through conserved cysteine residues (Cys⁶⁸, Cys⁷¹, Cys¹⁰², and Cys¹⁰⁵) that are responsible for coordinating zinc in T7 gp4. Moreover, we show that the N-terminal domain binds both single- and double-stranded DNA oligomers, with an apparent K_d of ∼120 nm. These findings suggest a possible role for the N-terminal domain of metazoan mtDNA helicase in recruiting and binding DNA at the replication fork.     

Replication Intermediates of the Linear Mitochondrial DNA of Candida parapsilosis Suggest a Common Recombination Based Mechanism for Yeast Mitochondria

Variation in the topology of mitochondrial DNA (mtDNA) in eukaryotes evokes the question if differently structured DNAs are replicated by a common mechanism. RNA-primed DNA synthesis has been established as a mechanism for replicating the circular animal/mammalian mtDNA. In yeasts, circular mtDNA molecules were assumed to be templates for rolling circle DNA-replication. We recently showed that in Candida albicans, which has circular mapping mtDNA, recombination driven replication is a major mechanism for replicating a complex branched mtDNA network. Careful analyses of C. albicans-mtDNA did not reveal detectable amounts of circular DNA molecules. In the present study we addressed the question of how the unit sized linear mtDNA of Candida parapsilosis terminating at both ends with arrays of tandem repeats (mitochondrial telomeres) is replicated. Originally, we expected to find replication intermediates diagnostic of canonical bi-directional replication initiation at the centrally located bi-directional promoter region. However, we found that the linear mtDNA of Candida parapsilosis also employs recombination for replication initiation. The most striking findings were that the mitochondrial telomeres appear to be hot spots for recombination driven replication, and that stable RNA:DNA hybrids, with a potential role in mtDNA replication, are also present in the mtDNA preparations.     

A257T linker region mutant of T7 helicase-primase protein is defective in DNA loading and rescued by T7 DNA polymerase

The helicase and primase activities of the hexameric ring-shaped T7 gp4 protein reside in two separate domains connected by a linker region. This linker region is part of the subunit interface between monomers, and point mutations in this region have deleterious effects on the helicase functions. One such linker region mutant, A257T, is analogous to the A359T mutant of the homologous human mitochondrial DNA helicase Twinkle, which is linked to diseases such as progressive external opthalmoplegia. Electron microscopy studies show that A257T gp4 is normal in forming rings with dTTP, but the rings do not assemble efficiently on the DNA. Therefore, A257T, unlike the WT gp4, does not preassemble on the unwinding DNA substrate with dTTP without Mg(II), and its DNA unwinding activity in ensemble assays is slow and limited by the DNA loading rate. Single molecule assays measured a 45 times slower rate of A257T loading on DNA compared with WT gp4. Interestingly, once loaded, A257T has almost WT-like translocation and DNA unwinding activities. Strikingly, A257T preassembles stably on the DNA in the presence of T7 DNA polymerase, which restores the ensemble unwinding activity of A257T to ～75% of WT, and the rescue does not require DNA synthesis. The DNA loading rate of A257T, however, remains slow even in the presence of the polymerase, which explains why A257T does not support T7 phage growth. Similar types of defects in the related human mitochondrial DNA helicase may be responsible for inefficient DNA replication leading to the disease states.     

An analysis of CAF-1-interacting proteins reveals dynamic and direct interactions with the KU complex and 14-3-3 proteins

CAF-1 is essential in human cells for the de novo deposition of histones H3 and H4 at the DNA replication fork. Depletion of CAF-1 from various cell lines causes replication fork arrest, activation of the intra-S phase checkpoint, and global defects in chromatin structure. CAF-1 is also involved in coordinating inheritance of states of gene expression and in chromatin assembly following DNA repair. In this study, we generated cell lines expressing RNAi-resistant versions of CAF-1 and showed that the N-terminal 296 amino acids are dispensable for essential CAF-1 function in vivo. N-terminally truncated CAF-1 p150 was deficient in proliferating cell nuclear antigen (PCNA) binding, reinforcing the existence of two PCNA binding sites in human CAF-1, but the defect in PCNA binding had no effect on the recruitment of CAF-1 to chromatin after DNA damage or to resistance to DNA-damaging agents. Tandem affinity purification of CAF-1-interacting proteins under mild conditions revealed that CAF-1 was directly associated with the KU70/80 complex, part of the DNA-dependent protein kinase, and the phosphoserine/threonine-binding protein 14-3-3 ζ. CAF-1 was a substrate for DNA-dependent protein kinase, and the 14-3-3 interaction in vitro is dependent on DNA-dependent protein kinase phosphorylation. These results highlight that CAF-1 has prominent interactions with the DNA repair machinery but that the N terminus is dispensable for the role of CAF-1 in DNA replication- and repair-coupled chromatin assembly.     

Uncoupling the roles of the SUV3 helicase in maintenance of mitochondrial genome stability and RNA degradation

Yeast SUV3 is a nuclear encoded mitochondrial RNA helicase that complexes with an exoribonuclease, DSS1, to function as an RNA degradosome. Inactivation of SUV3 leads to mitochondrial dysfunctions, such as respiratory deficiency; accumulation of aberrant RNA species, including excised group I introns; and loss of mitochondrial DNA (mtDNA). Although intron toxicity has long been speculated to be the major reason for the observed phenotypes, direct evidence to support or refute this theory is lacking. Moreover, it remains unknown whether SUV3 plays a direct role in mtDNA maintenance independently of its degradosome activity. In this paper, we address these questions by employing an inducible knockdown system in Saccharomyces cerevisiae with either normal or intronless mtDNA background. Expressing mutants defective in ATPase (K245A) or RNA binding activities (V272L or ΔCC, which carries an 8-amino acid deletion at the C-terminal conserved region) resulted in not only respiratory deficiencies but also loss of mtDNA under normal mtDNA background. Surprisingly, V272L, but not other mutants, can rescue the said deficiencies under intronless background. These results provide genetic evidence supporting the notion that the functional requirements of SUV3 for degradosome activity and maintenance of mtDNA stability are separable. Furthermore, V272L mutants and wild-type SUV3 associated with an active mtDNA replication origin and facilitated mtDNA replication, whereas K245A and ΔCC failed to support mtDNA replication. These results indicate a direct role of SUV3 in maintaining mitochondrial genome stability that is independent of intron turnover but requires the intact ATPase activity and the CC conserved region.     

Epstein-Barr nuclear antigen 1 (EBNA1)-dependent recruitment of origin recognition complex (Orc) on oriP of Epstein-Barr virus with purified proteins: stimulation by Cdc6 through its direct interaction with EBNA1

Origin recognition complex (Orc) plays an essential role in directing assembly of prereplicative complex at selective sites on chromosomes. However, Orc from vertebrates is reported to bind to DNA in a sequence-nonspecific manner, and it is still unclear how it selects specific genomic loci and how Cdc6, another conserved AAA(+) factor known to interact with Orc, participates in this process. Replication from oriP, the latent origin of Epstein-Barr virus, provides an excellent model system for the study of initiation on the host chromosomes because it is known to depend on prereplicative complex factors, including Orc and Mcm. Here, we show that Orc is recruited selectively at the essential dyad symmetry element in nuclear extracts in a manner dependent on EBNA1, which specifically binds to dyad symmetry. With purified proteins, EBNA1 can recruit both Cdc6 and Orc independently on a DNA containing EBNA1 binding sites, and Cdc6 facilitates the Orc recruitment by EBNA1. Purified Cdc6 directly binds to EBNA1, whereas association of Orc with EBNA1 requires the presence of the oriP DNA. Nuclease protection assays suggest that Orc associates with DNA segments on both sides adjacent to the EBNA1 binding sites and that this process is stimulated by the presence of Cdc6. Thus, EBNA1 can direct localized assembly of Orc in a process that is facilitated by Cdc6. The possibility of similar modes of recruitment of Orc/Cdc6 at the human chromosomal origins will be discussed.     

Double-stranded DNA binding domain of poly(ADP-ribose) polymerase-1 and molecular insight into the regulation of its activity

Poly(ADP-ribose) polymerase-1 (PARP-1) modifies various proteins, including itself, with ADP-ribose polymers (automodification). Polymer synthesis is triggered by binding of its zinc finger 1 (Zn1) and 2 (Zn2) to DNA breaks and is followed by inactivation through automodification. The multiple functional domains of PARP-1 appear to regulate activation and automodification-mediated inactivation of PARP-1. However, the roles of these domains in activation-inactivation processes are not well understood. Our results suggest that Zn1, Zn2, and a domain identified in this study, the double-stranded DNA binding (DsDB) domain, are involved in DNA break-dependent activation of PARP-1. We found that binding of the DsDB domain to double-stranded DNA and DNA break recognition by Zn1 and Zn2, whose actual binding targets are likely to be single-stranded DNA, lead to the activation of PARP-1. In turn, the displacement of single- and double-stranded DNA from Zn2 and the DsDB domain caused by ADP-ribose polymer synthesis results in the dissociation of PARP-1 from DNA breaks and thus its inactivation. We also found that the WGR domain is one of the domains involved in the RNA-dependent activation of PARP-1. Furthermore, because zinc finger 3 (Zn3) has the ability to bind to single-stranded RNA, it may have an indirect role in RNA-dependent activation. PARP-1 functional domains, which are involved in oligonucleic acid binding, therefore coordinately regulate PARP-1 activity depending on the status of the neighboring oligonucleic acids. Based on these results, we proposed a model for the regulation of PARP-1 activity.     

Mapping Topoisomerase Sites in Mitochondrial DNA with a Poisonous Mitochondrial Topoisomerase I (Top1mt)

Mitochondrial topoisomerase I (Top1mt) is a type IB topoisomerase present in vertebrates and exclusively targeted to mitochondria. Top1mt relaxes mitochondrial DNA (mtDNA) supercoiling by introducing transient cleavage complexes wherein the broken DNA strand swivels around the intact strand. Top1mt cleavage complexes (Top1mtcc) can be stabilized in vitro by camptothecin (CPT). However, CPT does not trap Top1mtcc efficiently in cells and is highly cytotoxic due to nuclear Top1 targeting. To map Top1mtcc on mtDNA in vivo and to overcome the limitations of CPT, we designed two substitutions (T546A and N550H) in Top1mt to stabilize Top1mtcc. We refer to the double-mutant enzyme as Top1mt*. Using retroviral transduction and ChIP-on-chip assays with Top1mt* in Top1mt knock-out murine embryonic fibroblasts, we demonstrate that Top1mt* forms high levels of cleavage complexes preferentially in the noncoding regulatory region of mtDNA, accumulating especially at the heavy strand replication origin O_H, in the ribosomal genes (12S and 16S) and at the light strand replication origin O_L. Expression of Top1mt* also caused rapid mtDNA depletion without affecting mitochondria mass, suggesting the existence of specific mitochondrial pathways for the removal of damaged mtDNA.     

Replacement of nucleosomal histones by histone H1-like proteins during spermiogenesis in Cnidaria: Evolutionary implications

We have analyzed the chromosomal protein composition of the sperm from several species belonging to three different classes (Hydrozoa, Scyphozoa, Anthozoa) of the phylum Cnidaria. In every instance, the sperm nuclear basic proteins (SNBPs) were found to consist of one to two major protein fractions that belong to the histone H1 family, as can be deduced from their amino acid composition and solubility in dilute perchloric acid, and the presence of a trypsin-resistant core. In those species where mature spawned sperm could be obtained, we were able to show that these proteins completely replace the somatic histones from the stem cells that are present at the onset of spermatogenesis. The presence of a highly specialized histone H1 molecule in the sperm of this phylum provides support for the idea that the protamine-like proteins (PL) from higher groups in the phylogenetic tree (and possibly protamines as well) may all have evolved from a primitive histone H1 ancestor.     

Mechanisms for defining supercoiling set point of DNA gyrase orthologs: II. The shape of the GyrA subunit C-terminal domain (CTD) is not a sole determinant for controlling supercoiling efficiency

DNA topoisomerases are essential enzymes that can overwind, underwind, and disentangle double-helical DNA segments to maintain the topological state of chromosomes. Nearly all bacteria utilize a unique type II topoisomerase, gyrase, which actively adds negative supercoils to chromosomes using an ATP-dependent DNA strand passage mechanism; however, the specific activities of these enzymes can vary markedly from species to species. Escherichia coli gyrase is known to favor supercoiling over decatenation (Zechiedrich, E. L., Khodursky, A. B., and Cozzarelli, N. R. (1997) Genes Dev. 11, 2580-2592), whereas the opposite has been reported for Mycobacterium tuberculosis gyrase (Aubry, A., Fisher, L. M., Jarlier, V., and Cambau, E. (2006) Biochem. Biophys. Res. Commun. 348, 158-165). Here, we set out to understand the molecular basis for these differences using structural and biochemical approaches. Contrary to expectations based on phylogenetic inferences, we find that the dedicated DNA wrapping domains (the C-terminal domains) of both gyrases are highly similar, both architecturally and in their ability to introduce writhe into DNA. However, the M. tuberculosis enzyme lacks a C-terminal control element recently uncovered in E. coli gyrase (see accompanying article (Tretter, E. M., and Berger, J. M. (2012) J. Biol. Chem. 287, 18636-18644)) and turns over ATP at a much slower rate. Together, these findings demonstrate that C-terminal domain shape is not the sole regulatory determinant of gyrase activity and instead indicate that an inability to tightly couple DNA wrapping to ATP turnover is why M. tuberculosis gyrase cannot supercoil DNA to the same extent as its γ-proteobacterial counterpart. Our observations demonstrate that gyrase has been modified in multiple ways throughout evolution to fine-tune its specific catalytic properties.     

Multiple DNA binding domains mediate the function of the ERCC1-XPF protein in nucleotide excision repair

ERCC1-XPF is a heterodimeric, structure-specific endonuclease that cleaves single-stranded/double-stranded DNA junctions and has roles in nucleotide excision repair (NER), interstrand crosslink (ICL) repair, homologous recombination, and possibly other pathways. In NER, ERCC1-XPF is recruited to DNA lesions by interaction with XPA and incises the DNA 5' to the lesion. We studied the role of the four C-terminal DNA binding domains in mediating NER activity and cleavage of model substrates. We found that mutations in the helix-hairpin-helix domain of ERCC1 and the nuclease domain of XPF abolished cleavage activity on model substrates. Interestingly, mutations in multiple DNA binding domains were needed to significantly diminish NER activity in vitro and in vivo, suggesting that interactions with proteins in the NER incision complex can compensate for some defects in DNA binding. Mutations in DNA binding domains of ERCC1-XPF render cells more sensitive to the crosslinking agent mitomycin C than to ultraviolet radiation, suggesting that the ICL repair function of ERCC1-XPF requires tighter substrate binding than NER. Our studies show that multiple domains of ERCC1-XPF contribute to substrate binding, and are consistent with models of NER suggesting that multiple weak protein-DNA and protein-protein interactions drive progression through the pathway. Our findings are discussed in the context of structural studies of individual domains of ERCC1-XPF and of its role in multiple DNA repair pathways.     

Interactions and subcellular distribution of DNA replication initiation proteins in eukaryotic cells

For initiation of eukaryotic DNA replication the origin recognition complex (ORC) associates with chromatin sites and constitutes a landing pad allowing Cdc6, Cdt1 and MCM proteins to accomplish the pre-replication complex (pre-RC). In S phase, the putative MCM helicase is assumed to move away from the ORC to trigger DNA unwinding. By using the fluorescence-based assays bioluminescence resonance energy transfer (BRET) and bimolecular fluorescence complementation (BiFC) we show in live mammalian cells that one key interaction in pre-RC assembly, the interaction between Orc2 and Orc3, is not restricted to the nucleus but also occurs in the cytoplasm. BRET assays also revealed a direct interaction between Orc2 and nuclear localization signal (NLS)-depleted Orc3. Further, we assessed the subcellular distribution of Orc2 and Orc3 in relation to MCM proteins Mcm3 and Mcm6 as well as to a key protein involved in elongation of DNA replication, proliferating nuclear cell antigen (PCNA). Our findings illustrate the spatial complexity of the elaborated process of DNA replication as well as that the BRET and BiFC techniques are novel tools that could contribute to our understanding of the processes at the very beginning of the duplication of the genome.     

The recognition specificity of the CHD1 chromodomain with modified histone H3 peptides

Histone tail peptides comprise the flexible portion of chromatin, the substance which serves as the packaging for the eukaryotic genome. According to the histone code hypothesis, reader protein domains (chromodomains) can recognize modifications of amino acid residues within these peptides, regulating the expression of genes. We have performed simulations on models of chromodomain helicase DNA-binding protein 1 complexed with a variety of histone H3 modifications. Binding free energies for both the overall complexes and the individual residues within the protein and peptides were computed with molecular mechanics-generalized Born surface area. The simulation results agree well with experimental data and identify several chromodomain helicase DNA-binding protein 1 residues that play key roles in the interaction with each of the H3 modifications. We identified one class of protein residues that bind to H3 in all of the complexes (generally interacting hydrophobically), and a second class of residues that bind only to particular H3 modifications (generally interacting electrostatically). Additionally, we found that modifications of H3R2 and H3T3 have a dominant effect on the binding affinity; methylation of H3K4 has little effect on the interaction strength when H3R2 or H3T3 is modified. Our findings with regard to the specificity shown by the latter class of protein residues in their binding affinity to certain modifications of H3 support the histone code hypothesis.     

Differential binding of nonhistone chromosomal proteins to the putative mouse origin of replication

Ehrlich ascites tumour (EAT) cells were treated with trioxsalen and ultraviolet light to crosslink DNA in vivo. After the treatment initiation of DNA replication can still occur but elongation is blocked by the crosslinks and this leads to the formation of short DNA fragments containing the origin of replication that can be isolated in double-stranded form after S₁ nuclease cutting of the crosslinked DNA (Russev, G. and Vassilev, L. (1982) J. Mol. Biol. 161, 77–87). To assess the affinity of these DNA fragments toward different chromosomal proteins, chromatin was fractionated by SDS-polyacrylamide gel electrophoresis, proteins were transferred to nitrocellulose filters and allowed to interact with in vivo labelled [³²P]DNA. The autoradiography of the filters showed that the DNA fraction synthesized between crosslinks and containing the putative mouse origin of replication bound preferentially to several nonhistone proteins, the most strongly binding ones having molecular weights of 64, 68, 72 and 150 kDa.     

Intraspecific heterogeneity of the Vicia faba mitochondrial genome: evidence for multiregional rearrangements in the mitochondrial chromosome associated with coxII-orf192 chimeric gene formation

Summary Previous RFLP-analysis of mtDNA isolated from different lines and cultivars of Vicia faba with respect to variability of the coxII gene revealed two types of mitochondrial genome: one with a normal coxII gene and the other with both normal coxII and chimeric coxII-orf192 genes. In this study we analyzed other regions of these two types of mitochondrial genome and found significant differences in the arrangement of regions around the coxII, coxIII, cob, rrn26 and atpA genes. More detailed analysis of the rrn26 and atpA gene regions showed that these genes are associated with recombinationally active repeats. Restriction maps of the rrn26 and atpA gene regions in different recombinative variants are presented.     

Novel Function of the Fanconi Anemia Group J or RECQ1 Helicase to Disrupt Protein-DNA Complexes in a Replication Protein A-stimulated Manner

Understanding how cellular machinery deals with chromosomal genome complexity is an important question because protein bound to DNA may affect various cellular processes of nucleic acid metabolism. DNA helicases are at the forefront of such processes, yet there is only limited knowledge how they remodel protein-DNA complexes and how these mechanisms are regulated. We have determined that representative human RecQ and Fe-S cluster DNA helicases are potently blocked by a protein-DNA interaction. The Fanconi anemia group J (FANCJ) helicase partners with the single-stranded DNA-binding protein replication protein A (RPA) to displace BamHI-E111A bound to duplex DNA in a specific manner. Protein displacement was dependent on the ATPase-driven function of the helicase and unique properties of RPA. Further biochemical studies demonstrated that the shelterin proteins TRF1 and TRF2, which preferentially bind the telomeric repeat found at chromosome ends, effectively block FANCJ from unwinding the forked duplex telomeric substrate. RPA, but not the Escherichia coli single-stranded DNA-binding protein or shelterin factor Pot1, stimulated FANCJ ejection of TRF1 from the telomeric DNA substrate. FANCJ was also able to displace TRF2 from the telomeric substrate in an RPA-dependent manner. The stimulation of helicase-catalyzed protein displacement is also observed with the DNA helicase RECQ1, suggesting a conserved functional interaction of RPA-interacting helicases. These findings suggest that partnerships between RPA and interacting human DNA helicases may greatly enhance their ability to dislodge proteins bound to duplex DNA, an activity that is likely to be highly relevant to their biological roles in DNA metabolism.     

In vivo conformation of mitochondrial DNA revealed by pulsed-field gel electrophoresis in the true slime mold, Physarum polycephalum.

Pulsed-field gel electrophoresis (PFGE) was used to examine the in vivo and in vitro conformations of Physarum polycephalum mitochondrial DNA (mtDNA). We used plugs containing isolated mitochondria, isolated mitochondrial nucleoids (mt-nuclei), and isolated mtDNA, in addition to whole cells. The mtDNA contained in the myxamoebae, plasmodia, isolated mitochondria, and isolated mt-nuclei was circular, but most of the isolated mtDNA had been site-specifically fragmented and linearized during DNA preparation and storage under low ionic strength conditions. Restriction mapping of Physarum mtDNA by the direct digestion of the isolated mt-nuclei from two different strains, DP89 x AI16 and KM88 x AI16, resulted in the circular form. A linear mitochondrial plasmid, mF, is known to promote mitochondrial fusion and integration of itself into the mtDNA in Physarum. Linearization of mtDNA by the integration of the mF plasmid was demonstrated when we used PFGE to analyze isolated mitochondria from the plasmodial strain DP89 x NG7 carrying the mF plasmid (mF+). The PFGE system can be used not only to determine whether the form of mtDNA is linear or circular but also to analyze the dynamic conformational changes of mtDNA.