NMR investigation of DNA primer-template models: structural insights into dislocation mutagenesis in DNA replication

Slipped frameshift intermediates can occur when DNA polymerase slows or stalls at sites of DNA lesions. However, this phenomenon is much less common when unmodified DNA is replicated. In order to study the effect of templating bases on the alignment of primer-templates, NMR structural investigation has been performed on primer-template oligonucleotide models which mimic the situation that dNTP has just been incorporated opposite template. NMR evidence reveals the occurrence of misalignment when dGTP is incorporated opposite template T with a downstream nucleotide C. Depending on the template sequence, further extension of the primer can lead to realignment.     

Assembly of an active chromatin structure during replication.

MSB cells were pulse labeled with 3H-thymidine and the isolated nuclei digested with either staphylococcal nuclease (to about 40% acid solubility) or DNase I (to 15% acid solubility). The purified, nuclease resistant single-copy DNA was then hybridized to nuclear RNA (nRNA). The results of these experiments show that actively transcribed genes are assembled into nucleosome-like structures within 5-10 nucleosomes of the replication fork and that they also acquire a conformation characteristic of actively transcribed nucleosomes (ie, a DNase I sensitive structure) within 20 nucleosomes of the fork. Assuming DNA sequence specific interactions are required for establishing a DNase I sensitive conformation on active genes during each round of replication, our results indicate that a specific recognition event can occur very rapidly and very specifically in eukaryotic cells. The results are discussed in terms of the possible mechanisms responsible for propagating active, chromosomal conformations from mother cells to daughter cells.     

Shaping time: chromatin structure and the DNA replication programme

DNA is replicated according to a precise and reproducible temporal pattern. The S-phase programme has previously been analyzed in metazoan and yeast cells using different methods: cytological chromosome banding in human cells and DNA isotopic-labeling techniques in yeast. Microarray-based approaches for the analysis of the replication programme and chromatin structure are bringing us closer to a molecular understanding of the factors that determine replication time. In this article, I assess the impact of recent investigations and compare our knowledge of DNA replication-timing controls in yeast with those of metazoans.     

The chromatin structure of the lysozyme GAS41 origin of DNA replication changes during the cell cycle

We used a rapid and simple protocol using lysolecithin for mapping HS sites in vivo. The protocol is based on partial digestion with DNase I of exponentially growing cells following permeabilization by short treatment with lysolecithin. Using this protocol, we analyzed the chromatin structure of the region surrounding two overlapping elements, an origin of bidirectional DNA replication and the GAS41 promoter, in chicken myelomonocytic HD11 cells arrested in G0, G1 and S phases as well as at the G1/S border. The results show that the chromatin of this region became more nuclease sensitive when cells were arrested in G1 phase and that this change in chromatin structure was reversible after the cells began to enter S phase.     

Metazoan origins of DNA replication: Regulation through dynamic chromatin structure

DNA replication in eukaryotes is initiated at multiple replication origins distributed over the entire genome, which are normally activated once per cell cycle. Due to the complexity of the metazoan genome, the study of metazoan replication origins and their activity profiles has been less advanced than in simpler genome systems. DNA replication in eukaryotes involves many protein–protein and protein–DNA interactions, occurring in multiple stages. As in prokaryotes, control over the timing and frequency of initiation is exerted at the initiation site. A prerequisite for understanding the regulatory mechanisms of eukaryotic DNA replication is the identification and characterization of the cis‐acting sequences that serve as replication origins and the trans‐acting factors (proteins) that interact with them. Furthermore, in order to understand how DNA replication may become deregulated in malignant cells, the distinguishing features between normal and malignant origins of DNA replication as well as the proteins that interact with them must be determined. Based on advances that were made using simple genome model systems, several proteins involved in DNA replication have been identified. This review summarizes the current findings about metazoan origins of DNA replication and their interacting proteins as well as the role of chromatin structure in their regulation. Furthermore, progress in origin identification and isolation procedures as well as potential mechanisms to inhibit their activation in cancer development and progression are discussed. J. Cell. Biochem. 106: 512–520, 2009. © 2009 Wiley‐Liss, Inc.     

Hemicatenanes form upon inhibition of DNA replication

Plasmid DNA incubated in interphase Xenopus egg extracts is normally assembled into chromatin and then into synthetic nuclei which undergo one round of regulated replication. During a study of restriction endonuclease cut plasmid replication intermediates (RIs) by the Brewer–Fangman 2D gel electrophoresis technique, we have observed the formation of a strong spike of X-shaped DNA molecules in extracts that otherwise yield very little or no RIs. Formation of these joint molecules is also efficiently induced in replication-competent extracts upon inhibition of replication fork progression by aphidicolin. Although their electrophoretic properties are quite similar to those of Holliday junctions, 2D gels of doubly cut plasmids show that these junctions can link two plasmid molecules at any pair of DNA sequences, with no regard for sequence homology at the branch points. Neutral–neutral–alkaline 3D gels show that the junctions only contain single strands of parental size and no recombinant strands. A hemicatenane, in which one strand of a duplex is wound around one strand of another duplex, is the most likely structure to account for these observations. The mechanism of formation of these novel joint DNA molecules and their biological implications are discussed.     

A role for chromatin remodellers in replication of damaged DNA

In eukaryotic cells, replication past damaged sites in DNA is regulated by the ubiquitination of proliferating cell nuclear antigen (PCNA). Little is known about how this process is affected by chromatin structure. There are two isoforms of the Remodels the Structure of Chromatin (RSC) remodelling complex in yeast. We show that deletion of RSC2 results in a dramatic reduction in the level of PCNA ubiquitination after DNA-damaging treatments, whereas no such effect was observed after deletion of RSC1. Similarly, depletion of the BAF180 component of the corresponding PBAF (Polybromo BRG1 (Brahma-Related Gene 1) Associated Factor) complex in human cells led to a similar reduction in PCNA ubiquitination. Remarkably, we found that depletion of BAF180 resulted after UV-irradiation, in a reduction not only of ubiquitinated PCNA but also of chromatin-associated unmodified PCNA and Rad18 (the E3 ligase that ubiquitinates PCNA). This was accompanied by a modest decrease in fork progression. We propose a model to account for these findings that postulates an involvement of PBAF in repriming of replication downstream from replication forks blocked at sites of DNA damage. In support of this model, chromatin immunoprecipitation data show that the RSC complex in yeast is present in the vicinity of the replication forks, and by extrapolation, this is also likely to be the case for the PBAF complex in human cells.     

The N-terminal noncatalytic region of Xenopus RecQ4 is required for chromatin binding of DNA polymerase alpha in the initiation of DNA replication

Recruitment of DNA polymerases onto replication origins is a crucial step in the assembly of eukaryotic replication machinery. A previous study in budding yeast suggests that Dpb11 controls the recruitment of DNA polymerases alpha and epsilon onto the origins. Sld2 is an essential replication protein that interacts with Dpb11, but no metazoan homolog has yet been identified. We isolated Xenopus RecQ4 as a candidate Sld2 homolog. RecQ4 is a member of the metazoan RecQ helicase family, and its N-terminal region shows sequence similarity with Sld2. In Xenopus egg extracts, RecQ4 is essential for the initiation of DNA replication, in particular for chromatin binding of DNA polymerase alpha. An N-terminal fragment of RecQ4 devoid of the helicase domain could rescue the replication activity of RecQ4-depleted extracts, and antibody against the fragment inhibited DNA replication and chromatin binding of the polymerase. Further, N-terminal fragments of RecQ4 physically interacted with Cut5, a Xenopus homolog of Dpb11, and their ability to bind to Cut5 closely correlated with their ability to rescue the replication activity of the depleted extracts. Our data suggest that RecQ4 performs an essential role in the assembly of replication machinery through interaction with Cut5 in vertebrates.     

Stepwise assembly of chromatin during DNA replication in vitro.

A cell free system that supports replication-dependent chromatin assembly has been used to determine the mechanism of histone deposition during DNA replication. CAF-I, a human cell nuclear factor, promotes chromatin assembly on replicating SV40 DNA in the presence of a crude cytosol replication extract. Biochemical fractionation of the cytosol extract has allowed separation of the chromatin assembly reaction into two steps. During the first step, CAF-I targets the deposition of newly synthesized histones H3 and H4 to the replicating DNA. This reaction is dependent upon and coupled with DNA replication, and utilizes the newly synthesized forms of histones H3 and H4, which unlike bulk histone found in chromatin, do not bind to DNA by themselves. The H3/H4-replicated DNA complex is a stable intermediate which exhibits a micrococcal nuclease resistant structure and can be isolated by sucrose gradient sedimentation. In the second step, this replicated precursor is converted to mature chromatin by the addition of histones H2A and H2B in a reaction that can occur after DNA replication. The requirement for CAF-I in at least the first step of the reaction suggests a level of cellular control for this fundamental process.     

Abiotic stress and induced DNA hypomethylation cause interphase chromatin structural changes in rice rDNA loci

Global climate change, i.e. higher and more variable temperatures, and a gain in soil salinity are increasing plant stress with direct consequences on crop yield and quality levels. Rice productivity is strongly affected by abiotic stress conditions. The regulation of chromatin structure in response to environmental stress is poorly understood. We investigated the interphase chromatin organization from rice plants in non-stress versus stress conditions. We have used a cytogenetic approach, based on fluorescence in situ hybridization (FISH) with 45S, 5S rDNA and centromeric probes on rice tissue sections. The abiotic stress conditions included cold, heat and mild salinity and were applied during seed germination. In contrast to cold, saline and heat stresses caused extensive decondensation of 45S rDNA chromatin and also an increase in the distance between the 2 homologous 5S rDNA loci. 5-Azacytidine (5-AC), a DNA hypomethylating drug, greatly increased 45S rDNA chromatin decondensation and interestingly was able to induce polarization of centromeres in rice interphase nuclei. The abiotic stresses tested did not perturb the spatial position of centromeres, typically with circular arrangement around the nucleolus. The results suggest a role for chromatin plasticity in a world of climate changes.     

Linker histones: conformational changes and the role in the structural organization of chromatin

Histones, linker histones of the H1 family, their postsyntetic modifications, DNA-histone H1 interaction are reviewed. A question of protein change in spermatogenesis at the formation of inactive nucleus with high degree of DNA density is considered. Special attention was paid to sperm-specific histones of the H1 family of sperm cells. Their role in organization of high-order chromatin structure of sperm cells is discussed. Also, results of different studies on the structural organization of chromatin (nucleosomes, 30-nm fibers, chromatin loops and metaphase chromosomes) are discussed.     

Structural changes in RepA,a plasmid replication initiator,upon binding to origin DNA

RepA protein is the DNA replication initiator of the Pseudomonas plasmid pPS10. RepA dimers bind to an inversely repeated operator sequence in repA promoter, thus repressing its own synthesis, whereas monomers bind to four directly repeated sequences (iterons) to initiate DNA replication. We had proposed previously that RepA is composed of two winged-helix (WH) domains, a structural unit also present in eukaryotic and archaeal initiators. To bind to the whole iteron sequence through both domains, RepA should couple monomerization to a conformational change in the N-terminal WH, which includes a leucine zipper-like sequence motif. We show for the first time that, by itself, binding to iteron DNA in vitro dissociates RepA dimers into monomers and alters RepA conformation, suggesting an allosteric effect. Furthermore, we also show that similar changes in RepA are promoted by mutations that substitute two Leu residues of the putative leucine zipper by Ala, destabilizing the hydrophobic core of the first WH. We propose that this mutant (RepA-2L2A) resembles a transient folding intermediate in the pathway leading to active monomers. These findings, together with the known activation of other Rep-type proteins by chaperones, are relevant to understand the molecular basis of plasmid DNA replication initiation.     

DNA replication in eukaryotes: a model for the specific involvement of chromatin subunits.

A model is proposed whereby eukaryotic DNA replication is specifically directed by the 200 base pair repeat structure of the DNA-histone complex. The model proposes a mechanism for the sequential, bidirectional replication of DNA from initial origin points on the chromatin fibre and is consistent with the known properties of eukaryotic DNA replication. Several predictions can be made from the model which are amenable to testing.     

A new structural framework for integrating replication protein A into DNA processing machinery

By coupling the protection and organization of single-stranded DNA (ssDNA) with recruitment and alignment of DNA processing factors, replication protein A (RPA) lies at the heart of dynamic multi-protein DNA processing machinery. Nevertheless, how RPA coordinates biochemical functions of its eight domains remains unknown. We examined the structural biochemistry of RPA’s DNA-binding activity, combining small-angle X-ray and neutron scattering with all-atom molecular dynamics simulations to investigate the architecture of RPA’s DNA-binding core. The scattering data reveal compaction promoted by DNA binding; DNA-free RPA exists in an ensemble of states with inter-domain mobility and becomes progressively more condensed and less dynamic on binding ssDNA. Our results contrast with previous models proposing RPA initially binds ssDNA in a condensed state and becomes more extended as it fully engages the substrate. Moreover, the consensus view that RPA engages ssDNA in initial, intermediate and final stages conflicts with our data revealing that RPA undergoes two (not three) transitions as it binds ssDNA with no evidence for a discrete intermediate state. These results form a framework for understanding how RPA integrates the ssDNA substrate into DNA processing machinery, provides substrate access to its binding partners and promotes the progression and selection of DNA processing pathways.     

Urea-induced structural changes in chromatin obtained by sedimentation.

Sedimentation coefficients have been determined for fractionated preparations of whole and stripped (depleted of very lysine-rich histones and non-histone proteins) chicken erythrocyte chromatin fragments in 0-10 M urea. Significant differences in urea effects are observed between these preparations; differences which can be interpreted structurally by use of Kirkwood's dynamical theory of the translational frictional coefficient. This type of analysis implies that urea-induced chain-swelling in stripped chromatin is due largely to the urea effect upon the constituent nu-bodies, whereas the much larger swelling observed in whole chromatin appears to involve also the effect of urea upon the region between adjacent nu-bodies.     

Direction of SPP1 DNA replication in transfected B. subtilis cells

Summary The origin and the direction of replication of the SPP1 chromosome, which has a unique, nonpermuted sequence of markers, was established by determination of the frequency distribution of various markers along the SPP1 map. For this purpose replicating DNA was isolated from transfected competent B. subtilis cells. Marker frequencies were measured by means of helper mediated transfection. In the range defined by the genetic map, replication is unidirectional, originating from a point in the left part of the map. Shearing the DNA into halves prior to transfection permits only one round of replication of that half molecule which carries the origin.