Requirement for the replication protein SSB in human DNA excision repair

Replication and repair are essential processes that maintain the continuity of the genetic material. Dissection of simian virus 40 (SV40) DNA replication has resulted in the identification of many eukaryotic replication proteins, but the biochemistry of the multienzyme process of DNA excision repair is less well defined. One protein that is absolutely required for semiconservative replication of SV40 DNA in vitro is human single-stranded DNA-binding protein (SSB, also called RF-A and RP-A). SSB consists of three polypeptides of relative molecular mass 70,000, 34,000 and 13,000, and acts with T antigen and topoisomerases to unwind DNA, allowing the access of other replication proteins. Human SSB can also stimulate the activity of polymerases alpha and delta, suggesting a further role in elongation during DNA replication. We have now found a role for human SSB in DNA excision repair using a cell-free system that can carry out nucleotide excision repair in vitro. Monoclonal antibodies against human SSB caused extensive inhibition of DNA repair in plasmid molecules damaged by ultraviolet light or acetylaminofluorene. Addition of purified SSB reversed this inhibition and further stimulated repair synthesis by increasing the number of repair events. These results show that a mammalian DNA replication protein is also essential for repair.     

ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex.

A multiprotein complex that specifically recognizes cellular origins of DNA replication has been identified and purified from the yeast Saccharomyces cerevisiae. We observe a strong correlation between origin function and origin recognition by this activity. Interestingly, specific DNA binding by the origin recognition complex is dependent upon the addition of ATP. We propose that the origin recognition complex acts as the initiator protein for S. cerevisiae origins of DNA replication.     

Role for Slimb in the degradation of Drosophila Period protein phosphorylated by Doubletime

Protein phosphorylation has a key role in modulating the stabilities of circadian clock proteins in a manner specific to the time of day. A conserved feature of animal clocks is that Period (Per) proteins undergo daily rhythms in phosphorylation and levels, events that are crucial for normal clock progression. Casein kinase Iepsilon (CKIepsilon) has a prominent role in regulating the phosphorylation and abundance of Per proteins in animals. This was first shown in Drosophila with the characterization of Doubletime (Dbt), a homologue of vertebrate casein kinase Iepsilon. However, it is not clear how Dbt regulates the levels of Per. Here we show, using a cell culture system, that Dbt promotes the progressive phosphorylation of Per, leading to the rapid degradation of hyperphosphorylated isoforms by the ubiquitin-proteasome pathway. Slimb, an F-box/WD40-repeat protein functioning in the ubiquitin-proteasome pathway interacts preferentially with phosphorylated Per and stimulates its degradation. Overexpression of slimb or expression in clock cells of a dominant-negative version of slimb disrupts normal rhythmic activity in flies. Our findings suggest that hyperphosphorylated Per is targeted to the proteasome by interactions with Slimb.     

Fidelity of DNA replication catalysed in vitro on a natural DNA template by the T4 bacteriophage multi-enzyme complex

More than 50 copies of a phi X174 DNA template can be made in 60 min in an in vitro DNA replication system consisting of seven purfied replication proteins isolated from T4 bacteriophage-infected cells. By transfecting with the DNA products and assaying for the reversion of specific amber mutants, the high degree of base-pairing fidelity in this system is revealed; the in vitro system is also shown to respond to the mutagenic effect of Mn2+ and to display strong base-pair context effects on fidelity, as expected from in vivo studies.     

Role for cyclin A in the dependence of mitosis on completion of DNA replication.

THE cyclins were first identified by their cell-cycle-dependent synthesis and destruction and have a key role in the control of mitosis in Xenopus embryonic cell cycles. All higher eukaryotes have at least two types of cyclins, the A- and B-type, which can be distinguished by sequence motifs and the timing of their destruction in the cell cycle. The degradation of both cyclins is required for exit from mitosis, but the activation and destruction of cyclin A occur earlier in the cell cycle than with the B-type cyclins. This suggests that cyclin A has a distinct role in cell-cycle progression. We have used an antisense oligodeoxy-nucleotide directed against cyclin A to investigate this role. Ablation of cyclin A messenger RNA in cytostatic factor/metaphase-arrested extracts of Xenopus eggs, followed by in vitro progression into interphase, resulted in the premature appearance of cyclin B-cdc2-associated H1 kinase activity and premature entry into mitosis. Although cyclin A-ablated extracts could initiate DNA synthesis during interphase, S phase was not completed before entry into mitosis. The effects of cyclin A ablation were reversed by the addition of cyclin A mRNA or cyclin A protein to the extracts.     

The Cdt1 protein is required to license DNA for replication in fission yeast

To maintain genome stability in eukaryotic cells, DNA is licensed for replication only after the cell has completed mitosis, ensuring that DNA synthesis (S phase) occurs once every cell cycle. This licensing control is thought to require the protein Cdc6 (Cdc18 in fission yeast) as a mediator for association of minichromosome maintenance (MCM) proteins with chromatin. The control is overridden in fission yeast by overexpressing Cdc18 (ref. 11) which leads to continued DNA synthesis in the absence of mitosis. Other factors acting in this control have been postulated and we have used a re-replication assay to identify Cdt1 (ref. 14) as one such factor. Cdt1 cooperates with Cdc18 to promote DNA replication, interacts with Cdc18, is located in the nucleus, and its concentration peaks as cells finish mitosis and proceed to S phase. Both Cdc18 and Cdt1 are required to load the MCM protein Cdc21 onto chromatin at the end of mitosis and this is necessary to initiate DNA replication. Genes related to Cdt1 have been found in Metazoa and plants (A. Whitaker, I. Roysman and T. Orr-Weaver, personal communication), suggesting that the cooperation of Cdc6/Cdc18 with Cdt1 to load MCM proteins onto chromatin may be a generally conserved feature of DNA licensing in eukaryotes.     

DNA primase acts as a molecular brake in DNA replication

A hallmark feature of DNA replication is the coordination between the continuous polymerization of nucleotides on the leading strand and the discontinuous synthesis of DNA on the lagging strand. This synchronization requires a precisely timed series of enzymatic steps that control the synthesis of an RNA primer, the recycling of the lagging-strand DNA polymerase, and the production of an Okazaki fragment. Primases synthesize RNA primers at a rate that is orders of magnitude lower than the rate of DNA synthesis by the DNA polymerases at the fork. Furthermore, the recycling of the lagging-strand DNA polymerase from a finished Okazaki fragment to a new primer is inherently slower than the rate of nucleotide polymerization. Different models have been put forward to explain how these slow enzymatic steps can take place at the lagging strand without losing coordination with the continuous and fast leading-strand synthesis. Nonetheless, a clear picture remains elusive. Here we use single-molecule techniques to study the kinetics of a multiprotein replication complex from bacteriophage T7 and to characterize the effect of primase activity on fork progression. We observe the synthesis of primers on the lagging strand to cause transient pausing of the highly processive leading-strand synthesis. In the presence of both leading- and lagging-strand synthesis, we observe the formation and release of a replication loop on the lagging strand. Before loop formation, the primase acts as a molecular brake and transiently halts progression of the replication fork. This observation suggests a mechanism that prevents leading-strand synthesis from outpacing lagging-strand synthesis during the slow enzymatic steps on the lagging strand.     

The RCAF complex mediates chromatin assembly during DNA replication and repair

Chromatin assembly is a fundamental biological process that is essential for the replication and maintenance of the eukaryotic genome. In dividing cells, newly synthesized DNA is rapidly assembled into chromatin by the deposition of a tetramer of the histone proteins H3 and H4, followed by the deposition of two dimers of histones H2A and H2B to complete the nucleosome-the fundamental repeating unit of chromatin. Here we describe the identification, purification, cloning, and characterization of replication-coupling assembly factor (RCAF), a novel protein complex that facilitates the assembly of nucleosomes onto newly replicated DNA in vitro. RCAF comprises the Drosophila homologue of anti-silencing function 1 protein ASF1 and histones H3 and H4. The specific acetylation pattern of H3 and H4 in RCAF is identical to that of newly synthesized histones. Genetic analyses in Saccharomyces cerevisiae demonstrate that ASF1 is essential for normal cell cycle progression, and suggest that RCAF mediates chromatin assembly after DNA replication and the repair of double-strand DNA damage in vivo.     

Reagents for the site-specific cleavage of megabase DNA.

The physical mapping of chromosomes will be facilitated by methods of breaking large DNA into manageable fragments, or cutting uniquely at genetic markers of interest. Key issues in the design of sequence-specific DNA cleaving reagents are the specificity of binding, the number of different sequences that can be targeted and the cleavage yield.     

Need for DNA topoisomerase activity as a swivel for DNA replication for transcription of ribosomal RNA

Yeast strains with mutations in the genes for DNA topoisomerases I and II have been identified previously in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. The topoisomerase II mutants (top2) are conditional-lethal temperature-sensitive (ts) mutants. They are defective in the termination of DNA replication and the segregation of daughter chromosomes, but otherwise appear to replicate and transcribe DNA normally. Topoisomerase I mutants (top1), including strains with null mutations are viable and exhibit no obvious growth defects, demonstrating that DNA topoisomerase I is not essential for viability in yeast. In contrast to the single mutants, top1 top2 ts double mutants from both Schizosaccharomyces pombe and Saccharomyces cerevisiae grow poorly at the permissive temperature and stop growth rapidly at the non-permissive temperature. Here we report that DNA and ribosomal RNA synthesis are drastically inhibited in an S. cerevisiae top1 top2 ts double mutant at the restrictive temperature, but that the rate of poly(A)+ RNA synthesis is reduced only about threefold and transfer DNA synthesis remains relatively normal. The results suggest that DNA replication and at least ribosomal RNA synthesis require an active topoisomerase, presumably to act as a swivel to relieve torsional stress, and that either topoisomerase can perform the required function (except in termination of DNA replication where topoisomerase II is required).     

Distal-less encodes a homoeodomain protein required for limb development in Drosophila

The spatial organization of the Drosophila embryo depends on the activity of three axial pattern-forming systems. In addition to the anterior-posterior and dorsal-ventral systems that organize the segmented body plan, a proximal-distal pattern-forming system is required to provide positional information for the developing limbs. The development of both the larval and adult limbs depends directly on the activity of the Distal-less gene. Genetic analysis has shown that Distal-less functions as a developmental switch that is required to promote the development of limb structures above the evolutionary ground-state of body wall. Here we provide genetic evidence that indicates a graded requirement for Distal-less activity during limb development. Reduction of this activity has a global effect on pattern formation in the limb. The molecular structure of the Distal-less locus indicates that the gene encodes a homoeodomain-containing protein which is therefore likely to specify limb development through differential regulation of subordinate genes.     

Nuclear factor III, a novel sequence-specific DNA-binding protein from HeLa cells stimulating adenovirus DNA replication

Dissection and reconstitution of the adenovirus DNA replication machinery has led to the discovery of two HeLa nuclear proteins which are required in conjunction with three viral proteins. One of these, nuclear factor I (NF-I), recognizes an internal region of the origin between nucleotides 25 and 40 and by binding to one side of the helix stimulates the initiation reaction up to 30-fold. NFI-binding sites have been observed upstream of several cellular genes, such as chicken lysozyme, human IgM and human c-myc, and coincide in most cases with DNase I hypersensitive regions. Here we report the identification of a novel DNA-binding protein from HeLa nuclei, designated NF-III, that recognizes a sequence in the adenovirus origin very close to the NFI-binding site, between nucleotides 36 and 54. This sequence includes the partially conserved nucleotides TATGATAATGAG. NF-III stimulates DNA replication four- to sixfold by increasing the initiation efficiency. Potential cellular binding sites include promoter elements of the histone H2B gene, the human interferon beta gene, the human and mouse immunoglobulin VK and VH genes and the mammal/chicken/Xenopus laevis U1 and U2 small nuclear RNA genes. Furthermore, a subset of the herpes simplex virus immediate early promoter specific TAATGARAT elements is homologous with the adenovirus 2 (Ad-2) NFIII-binding site.     

Error-prone replication of oxidatively damaged DNA by a high-fidelity DNA polymerase

Aerobic respiration generates reactive oxygen species that can damage guanine residues and lead to the production of 8-oxoguanine (8oxoG), the major mutagenic oxidative lesion in the genome. Oxidative damage is implicated in ageing and cancer, and its prevalence presents a constant challenge to DNA polymerases that ensure accurate transmission of genomic information. When these polymerases encounter 8oxoG, they frequently catalyse misincorporation of adenine in preference to accurate incorporation of cytosine. This results in the propagation of G to T transversions, which are commonly observed somatic mutations associated with human cancers. Here, we present sequential snapshots of a high-fidelity DNA polymerase during both accurate and mutagenic replication of 8oxoG. Comparison of these crystal structures reveals that 8oxoG induces an inversion of the mismatch recognition mechanisms that normally proofread DNA, such that the 8oxoG.adenine mismatch mimics a cognate base pair whereas the 8oxoG.cytosine base pair behaves as a mismatch. These studies reveal a fundamental mechanism of error-prone replication and show how 8oxoG, and DNA lesions in general, can form mismatches that evade polymerase error-detection mechanisms, potentially leading to the stable incorporation of lethal mutations.