Fission yeast Ku protein is required for recovery from DNA replication stress

The fundamental function of the conserved Ku70–Ku80 heterodimer is to promote the non-homologous end-joining (NHEJ) pathway in double-strand break repair. Although it is thought that Ku plays several roles other than NHEJ in maintaining chromosomal integrity including telomere protection, these precise functions remain unclear. In this study, we describe a novel role of fission yeast Ku proteins encoded by pku70 ⁺ and pku80 ⁺ genes in dealing with DNA replication stress. In the absence of Rqh1, the fission yeast RecQ helicase, the cells are sensitive to reagents inducing replication stress. pku Δ rqh1 Δ double mutant showed synergistic sensitivities to these reagents. However, this synthetic phenotype was not observed when rqh1 Δ mutant was coupled with the deletion of lig4 ⁺ that encodes a ligase essential for NHEJ, indicating that the role of Ku in replication stress is NHEJ independent. pku Δ rqh1 Δ double mutant also showed highly variable copy numbers of rDNA repeats even under unstressed condition. Furthermore, the double mutant exhibited inefficient replication resumption after transient replication stalling. These results suggest the possibility that Ku proteins play an important role in genome integrity recovering replication stress.     

Kinetics of DNA replication and telomerase reaction at a single-seeded telomere in human cells

In most cancer cells, telomerase is activated to elongate telomere DNA, thereby ensuring numerous rounds of cell divisions. It is thus important to understand how telomerase and the replication fork react with telomeres in human cells. However, the highly polymorphic and repetitive nature of the nucleotide sequences in human subtelomeric regions hampers the precise analysis of sequential events taking place at telomeres in S phase. Here, we have established HeLa cells harboring a single-seeded telomere abutted by a unique subtelomere DNA sequence, which has enabled us to specifically focus on the seeded telomere. We have also developed a modified chromatin immunoprecipitation (ChIP) method that uses restriction digestion instead of sonication to fragment chromatin DNA (RES-ChIP), and a method for immunoprecipitating 5-bromo-2'-deoxyuridine (BrdU)-labeled single-stranded DNA by incubating DNA with anti-BrdU antibody in the nondenaturing condition. We have shown that DNA replication of the seeded telomere takes place during a relatively narrow time window in S phase, and telomerase synthesizes telomere DNA after the replication. Moreover, we have demonstrated that the telomerase catalytic subunit TERT associates with telomeres before telomere DNA replication. These results provide a temporal and spatial framework for understanding DNA replication and telomerase reaction at human telomeres.     

Rif1 controls DNA replication by directing Protein Phosphatase 1 to reverse Cdc7-mediated phosphorylation of the MCM complex

Initiation of eukaryotic DNA replication requires phosphorylation of the MCM complex by Dbf4-dependent kinase (DDK), composed of Cdc7 kinase and its activator, Dbf4. We report here that budding yeast Rif1 (Rap1-interacting factor 1) controls DNA replication genome-wide and describe how Rif1 opposes DDK function by directing Protein Phosphatase 1 (PP1)-mediated dephosphorylation of the MCM complex. Deleting RIF1 partially compensates for the limited DDK activity in a cdc7-1 mutant strain by allowing increased, premature phosphorylation of Mcm4. PP1 interaction motifs within the Rif1 N-terminal domain are critical for its repressive effect on replication. We confirm that Rif1 interacts with PP1 and that PP1 prevents premature Mcm4 phosphorylation. Remarkably, our results suggest that replication repression by Rif1 is itself also DDK-regulated through phosphorylation near the PP1-interacting motifs. Based on our findings, we propose that Rif1 is a novel PP1 substrate targeting subunit that counteracts DDK-mediated phosphorylation during replication. Fission yeast and mammalian Rif1 proteins have also been implicated in regulating DNA replication. Since PP1 interaction sites are evolutionarily conserved within the Rif1 sequence, it is likely that replication control by Rif1 through PP1 is a conserved mechanism.     

The viral DNA replication complex of adenovirus 12

After infection of human embryo kidney cells in a resting state with adenovirus 12, viral DNA synthesis began at 24 hr post infection (p.i.) and reached its peak at 32 hr p.i. Both parental and nascent DNAs were present in the M-band fraction of nuclei of infected cells at 32 hr p.i. The DNA synthesizing activity in vitro with isolated nuclei and the M-band fractions began to increase at 24 hr p.i. and reached its maximum at 32 hr p.i. The DNA synthesized in vitro was viral as revealed by DNA-DNA hybridization. Electron microscopic autoradiograms of infected cells revealed that silver grains were found in association with bandlike inclusion bodies in the interior of the nucleus and did not associate with the nuclear membrane. After chase for 1 hr, most of the grains were found in the same state. These observations indicate that the viral DNA replication complex is formed in the interior of the nucleus and contained in the M-band fraction after fractionation.     

An in vitro DNA replication complex from Brassica mitochondria

Summary An in vitro complex capable of supporting DNA replication was isolated from Brassica L. mitochondria. Template preferences and inhibitor studies confirmed DNA polymerase characteristics. Comparison of complexes isolated from B. napus, B. campestris and B. napus/campestris cybrid, indicated that both nuclear and cytoplasmic background influenced the template specificity and response to inhibitors.     

The plastidic DNA replication enzyme complex of Plasmodium falciparum

The replication and repair of organellar genomes in the malaria parasite Plasmodium falciparum is poorly understood. We have assessed the properties of an open reading frame Pfprex (formerly known as pom1) and confirm that it specifies a multi-domain polypeptide with DNA primase, DNA helicase, DNA polymerase and 3'-5' exonuclease activities. The sequence of the primase/helicase domain is phylogenetically related to the T7-bacteriophage gene 4 product and mammalian mitochondrial helicase, Twinkle. Despite that, the N-terminal sequence of this multi-domain polypeptide directs a green fluorescent protein reporter specifically to the P. falciparum apicoplast and not to the mitochondrion. Phylogenetic analysis placed the DNA polymerase sequence with the family A bacterial polymerases, most closely to those of the thermophilic Aquifex species. Notably, the malarial enzyme was optimally active at 75 degrees C. Pfprex is the first example of a gene encoding contiguous DNA polymerase, DNA primase and DNA helicase components. We propose it has a key role in replication of the malarial plastid genome, a validated drug target.     

Ku acts in a unique way at the mammalian telomere to prevent end joining

Telomeres are specialized DNA/protein structures that act as protective caps to prevent end fusion events and to distinguish the chromosome ends from double-strand breaks. We report that TRF1 and Ku form a complex at the telomere. The Ku and TRF1 complex is a specific high-affinity interaction, as demonstrated by several in vitro methods, and exists in human cells as determined by coimmunoprecipitation experiments. Ku does not bind telomeric DNA directly but localizes to telomeric repeats via its interaction with TRF1. Primary mouse embryonic fibroblasts that are deficient for Ku80 accumulated a large percentage of telomere fusions, establishing that Ku plays a critical role in telomere capping in mammalian cells. We propose that Ku localizes to internal regions of the telomere via a high-affinity interaction with TRF1. Therefore, Ku acts in a unique way at the telomere to prevent end joining.     

Candidate DNA replication initiation regions at human trinucleotide repeat disease loci

The positions of DNA replication initiation regions (IRs) at three human trinucleotide repeat (TNR) disease loci were examined in order to characterize the role played by IRs in explaining the known locus-specific variation in TNR instability levels. Using three different normal cell lines, candidate IRs were identified at the HD, SCA-7 and SBMA loci. At each locus the IR is less than 3.6 kb from the CAG/CTG repeat tract. Preliminary studies with a cell line homozygous for an HD disease mutation indicated no change in the position of the candidate IR in spite of the mutation. Comparison with experimental results from model systems suggests that a complex relationship may exist between instability and the proximity and/or orientation of the repeats with respect to an IR.     

DNA replication is not restricted to specific regions in young vegetative Streptomyces mycelia

In order to determine the localization of DNA-synthesis in Streptomyces granaticolor and Streptomyces hygroscopicus, mycelia (growing either on agar or in liquid medium) were pulse-labelled with 3H-thymidine and prepared for autoradiography. The distribution of silver grains showed no regions of preferential incorporation of 3H-thymidine in mycelia up 300 micron in length. Since mycelia grow by apical elongation of hyphae, the frequency of silver grains was quantitatively analysed along individual main hyphase. No significant difference of labelling was found within zones of different age up to a distance of 80 micron from the hyphal tip. Also, the very youngest part of the hyphae enclosing only the most apically situated nucleoid did not show any deviation from the average frequency of silver grains.     

Sequence-independent DNA binding and replication initiation by the human origin recognition complex

We report that a highly purified human origin recognition complex (HsORC) has intrinsic DNA-binding activity, and that this activity is modestly stimulated by ATP. HsORC binds preferentially to synthetic AT-rich polydeoxynucleotides, but does not effectively discriminate between natural DNA fragments that contain known human origins and control fragments. The complex fully restores DNA replication to ORC-depleted Xenopus egg extracts, providing strong evidence for its initiator function. Strikingly, HsORC stimulates initiation from any DNA sequence, and it does not preferentially replicate DNA containing human origin sequences. These data provide a biochemical explanation for the observation that in metazoans, initiation of DNA replication often occurs in a seemingly random pattern, and they have important implications for the nature of human origins of DNA replication.     

DNA replication error in endometrial carcinoma and complex atypical endometrial hyperplasia.

We attempted to define the relation between DNA replication errors (RERs) in endometrial carcinomas and the precancerous lesion complex atypical endometrial hyperplasia (ATH) and clinicopathological characteristics. Tissue samples from 93 patients with endometrial carcinoma diagnosed as endometrioid adenocarcinoma and 26 patients with ATH (including 21 in whom endometrial carcinoma also was found) were prepared as formalin-fixed, paraffin-embedded sections. The samples were examined for the presence of RERs by the polymerase chain reaction with the use of five microsatellite markers. RERs were observed at > or = 1 loci in 32 endometrial carcinoma patients (34%); all 26 ATH patients were RER negative. RERs were observed in 25% of stage I and stage II cancer patients (16/64) and in 55% of stage III and stage IV cancer patients (16/29) (P = 0.009), as well as in 63% (10/16) of cancer patients with and in 27% (20/75) of patients without lymph-node metastases (P = 0.013). The incidence of RERs was not related to patient age, histological tumor grade, or prognosis. These results suggest that RER may be involved in the advanced rather than the early stages of endometrioid adenocarcinoma. There appears to be little association between RER and ATH.     

Ku complex interacts with and stimulates the Werner protein

Werner syndrome (WS) is the hallmark premature aging disorder in which affected humans appear older than their chronological age. The protein WRNp, defective in WS, has helicase function, DNA-dependent ATPase, and exonuclease activity. Although WRNp functions in nucleic acid metabolism, there is little or no information about the pathways or protein interactions in which it participates. Here we identify Ku70 and Ku86 as proteins that interact with WRNp. Although Ku proteins had no effect on ATPase or helicase activity, they strongly stimulated specific exonuclease activity. These results suggest that WRNp and the Ku complex participate in a common DNA metabolic pathway.     

A novel ring-like complex of Xenopus proteins essential for the initiation of DNA replication

We have identified Xenopus homologs of the budding yeast Sld5 and its three interacting proteins. These form a novel complex essential for the initiation of DNA replication in Xenopus egg extracts. The complex binds to chromatin in a manner dependent on replication licensing and S-phase CDK. The chromatin binding of the complex and that of Cdc45 are mutually dependent and both bindings require Xenopus Cut5, the yeast homolog of which interacts with Sld5. On replicating chromatin the complex interacts with Cdc45 and MCM, putative components of replication machinery. Electron microscopy further reveals that the complex has a ring-like structure. These results suggest that the complex plays an essential role in the elongation stage of DNA replication as well as the initiation stage.     

起始识别复合物1基因在血管平滑肌细胞DNA复制中的作用

目的探讨起始识别复合物1（ORC1）在血管平滑肌细胞（VSMC）DNA复制中的表达及其意义。方法采用组织块贴片法原代培养的大鼠胸主动脉VSMC,利用双胸苷阻断、秋水仙素阻抑法和血清饥饿法使VSMC达到细胞周期同步化,用逆转录聚合酶链反应（RT-PCR）技术和Western blot检测不同细胞周期的VSMC ORC1 mRNA和蛋白表达。结果同步化的VSMC DNA含量百分比,血清饥饿法以G0G1期为主（P〈0．01）,双胸苷阻断14h以G0G0期为主（P〈0．01）,双胸苷阻断14h后10％胎牛血清刺激6h以S期为主（P〈0．01）,秋水仙素培养12h以G2／M期为主（P〈0．05）。静止状态VSMC的ORC1 mRNA和蛋白质无表达,处于G1／S期VSMC的ORC1 mRNA表达量显著高于S期和G2／M期。VSMC的ORC1蛋白质表达量与ORC1 mRNA变化规律相似。结论ORC1随细胞的分裂周期而表达,ORC1可能是启动VSMC DNA复制的关键因子。     

Differential expression of DNA nonhomologous end-joining proteins Ku70 and Ku80 in melanoma progression.

Ku70 and Ku80 heterodimers function as regulatory subunits of the DNA-dependent protein kinase and play a very important role in the repairing of DNA double-strand breaks. Although Ku70 is proposed as a candidate for a tumor suppressor gene, not many data are available on Ku70 and Ku80 expression in human tumors. The main aim of this study was to investigate the expression of Ku70 and Ku80 in the ultraviolet-induced lesions-nevus cell nevi, lentigos maligna, and malignant melanomas. Nineteen nevus cell nevi, 23 lentigos maligna, 76 primary melanomas, and 31 melanoma metastases were stained immunohistochemically for the presence of Ku70 and Ku80 proteins. Ku70 and Ku80 expression was preserved in about 80% of nevi, 26% of lentigo maligna, 45% of primary melanomas, and 67% of melanoma metastases. Highly significant differences in Ku70 and Ku80 expression were found between nevi, lentigo maligna, and melanomas. In Cox regression, Ku70 and Ku80 were shown to be highly significant influences on patients' prognosis. Significant correlations between Ku70 and Ku80 expressions were found in nevi, lentigo maligna, and primary melanomas. These correlations were not more present in melanoma metastases. To summarize, earlier phases of melanoma progression seem to be connected with the loss of expression of Ku proteins. Metastatic spread is related to dysregulation of the Ku70 and Ku80 axis.     

Essential roles of Xenopus TRF2 in telomere end protection and replication

TRF1 and TRF2 are double-stranded (ds) telomere DNA-binding proteins and the core members of shelterin, a complex that provides the structural and functional basis of telomere functions. We have reported that unlike mammalian TRF1 that constitutively binds to chromatin, Xenopus TRF1 (xTRF1) associates with mitotic chromatin but dissociates from interphase chromatin reconstituted in Xenopus egg extracts. This finding raised the possibility that xTRF1 and Xenopus TRF2 (xTRF2) contribute to telomere functions in a manner different from mammalian TRF1 and TRF2. Here, we focused on the role of xTRF2. We prepared chromatin reconstituted in egg extracts immunodepleted for xTRF2. Compared to mock-depleted nuclei, DNA damage response at telomeres was activated, and bulk DNAs were poorly replicated in xTRF2-depleted nuclei. The replication defect was rescued by inactivating ATR through the addition of anti-ATR neutralizing antibody, suggesting that ATR plays a role in the defect. Interestingly, the bulk DNA replication defect, but not the DNA damage response at telomeres, was rescued by supplementing the xTRF2-depleted extracts with recombinant xTRF2 (rTRF2). We propose that xTRF2 is required for both efficient replication of bulk DNA and protection from the activation of the DNA damage checkpoints pathway, and that those two functions are mechanistically separable.