Role In DNA Replication Research Articles

Generation of an induced pluripotent stem cell line from an individual with a heterozygous RECQL4 mutation

The DNA helicase RECQL4 is known for its roles in DNA replication and repair. RECQL4 mutations cause several genetic disorders including Rothmund-Thomson syndrome (RTS), characterized by developmental defects and predisposition to osteosarcoma. Here we reprogrammed fibroblasts with a heterozygous RECQL4 mutation (c.1878 + 32_1878 + 55del24) to induced pluripotent stem cells (iPSCs). These iPSCs are pluripotent and are able to be differentiated into all three germ layers, providing a novel tool to further interrogate the role of RECQL4 DNA helicase in vitro.

SUMO-1 modification of FEN1 facilitates its interaction with Rad9-Rad1-Hus1 to counteract DNA replication stress.

Human flap endonuclease 1 (FEN1) is a structure-specific, multi-functional endonuclease essential for DNA replication and repair. We and others have shown that during DNA replication, FEN1 processes Okazaki fragments via its interaction with the proliferating cell nuclear antigen (PCNA). Alternatively, in response to DNA damage, FEN1 interacts with the PCNA-like Rad9–Rad1–Hus1 complex instead of PCNA to engage in DNA repair activities, such as homology-directed repair of stalled DNA replication forks. However, it is unclear how FEN1 is able to switch between these interactions and its roles in DNA replication and DNA repair. Here, we report that FEN1 undergoes SUMOylation by SUMO-1 in response to DNA replication fork-stalling agents, such as UV irradiation, hydroxyurea, and mitomycin C. This DNA damage-induced SUMO-1 modification promotes the interaction of FEN1 with the Rad9–Rad1–Hus1 complex. Furthermore, we found that FEN1 mutations that prevent its SUMO-1 modification also impair its ability to interact with HUS1 and to rescue stalled replication forks. These impairments lead to the accumulation of DNA damage and heightened sensitivity to fork-stalling agents. Altogether, our findings suggest an important role of the SUMO-1 modification of FEN1 in regulating its roles in DNA replication and repair.

Computational Determination of the Effects of Bacteriophage Bacteriophage Interactions in Human Body.

Chronic diseases are becoming more serious and widely spreading and this carries a heavy burden on doctors to deal with such patients. Although many of these diseases can be treated by bacteriophages, the situation is significantly dangerous in patients having concomitant more than one chronic disease, where conflicts between phages used in treating these diseases are very closer to happen. This research paper presents a method to detecting the Bacteriophage-Bacteriophage Interaction. This method is implemented based on Domain-Domain Interactions model and it was used to infer Domain-Domain Interactions between the bacteriophages injected in the human body at the same time. By testing the method over bacteriophages that are used to treat tuberculosis, salmonella and virulent E.coli, many interactions have been inferred and detected between these bacteriophages. Several effects were detected for the resulted interactions such as: playing a role in DNA repair such as nonhomologous end joining, playing a role in DNA replication, playing a role in the interaction between the immune system and the tumor cells and playing a role in the stiff man syndrome. We revised all patents relating to bacteriophage bacteriophage interactions and phage therapy. The proposed method is developed to help doctors to realize the effect of simultaneously injecting different bacteriophages into the human body to treat different diseases.

T4 DNA ligase structure reveals a prototypical ATP-dependent ligase with a unique mode of sliding clamp interaction.

DNA ligases play essential roles in DNA replication and repair. Bacteriophage T4 DNA ligase is the first ATP-dependent ligase enzyme to be discovered and is widely used in molecular biology, but its structure remained unknown. Our crystal structure of T4 DNA ligase bound to DNA shows a compact α-helical DNA-binding domain (DBD), nucleotidyl-transferase (NTase) domain, and OB-fold domain, which together fully encircle DNA. The DBD of T4 DNA ligase exhibits remarkable structural homology to the core DNA-binding helices of the larger DBDs from eukaryotic and archaeal DNA ligases, but it lacks additional structural components required for protein interactions. T4 DNA ligase instead has a flexible loop insertion within the NTase domain, which binds tightly to the T4 sliding clamp gp45 in a novel α-helical PIP-box conformation. Thus, T4 DNA ligase represents a prototype of the larger eukaryotic and archaeal DNA ligases, with a uniquely evolved mode of protein interaction that may be important for efficient DNA replication.

Analyte-driven self-assembly of graphene oxide sheets onto hydroxycamptothecin-functionalized upconversion nanoparticles for the determination of type I topoisomerases in cell extracts.

Type I topoisomerases (TOPOI), a potential diagnostic biomarker and a target for chemotherapeutic agents, play essential roles in DNA replication, transcription, chromosome segregation, and recombination. It is essential to develop analytical methods for accurate detection of TOPOI in biological fluids for early diagnosis of diseases. Here we show an assay for TOPOI on the basis of the target-induced self-assembly of graphene oxide (GO) sheets onto hydroxycamptothecin-functionalized upconversion nanoparticles (HCPT-UCNPs). The dipole-dipole coupling of HCPT-UCNPs (donor) and GO (acceptor) regulated by TOPOI enables Förster resonance energy transfer between the donor and the acceptor. Integration of minimal autofluorescence and highly specific affinity into the developed nanosensor allows reliable detection of TOPOI in the nanomolar range with the detection limit of 0.29nM. The detection of TOPOI in breast cancer cells with recoveries from 96.3 to 103.7% shows the availability of the proposed assay in complicated samples. Graphical abstract ᅟ.

Perturbing cohesin dynamics drives MRE11 nuclease-dependent replication fork slowing.

Pds5 is required for sister chromatid cohesion, and somewhat paradoxically, to remove cohesin from chromosomes. We found that Pds5 plays a critical role during DNA replication that is distinct from its previously known functions. Loss of Pds5 hinders replication fork progression in unperturbed human and mouse cells. Inhibition of MRE11 nuclease activity restores fork progression, suggesting that Pds5 protects forks from MRE11-activity. Loss of Pds5 also leads to double-strand breaks, which are again reduced by MRE11 inhibition. The replication function of Pds5 is independent of its previously reported interaction with BRCA2. Unlike Pds5, BRCA2 protects forks from nucleolytic degradation only in the presence of genotoxic stress. Moreover, our iPOND analysis shows that the loading of Pds5 and other cohesion factors on replication forks is not affected by the BRCA2 status. Pds5 role in DNA replication is shared by the other cohesin-removal factor Wapl, but not by the cohesin complex component Rad21. Interestingly, depletion of Rad21 in a Pds5-deficient background rescues the phenotype observed upon Pds5 depletion alone. These findings support a model where loss of either component of the cohesin releasin complex perturbs cohesin dynamics on replication forks, hindering fork progression and promoting MRE11-dependent fork slowing.

First-in-human phase 1 study of TAK-931, an oral cell division cycle 7 (CDC7) inhibitor, in patients (pts) with advanced solid tumors.

2506Background: TAK-931 is an oral selective inhibitor of CDC7, a protein kinase with key roles in DNA replication and DNA damage response. TAK-931 has demonstrated potent antitumor activity in var...

Overexpression of MCM6 predicts poor survival in patients with glioma

Minichromosome maintenance proteins (MCMs) play an essential role in DNA replication and other cellular activities. However, their expression levels and clinical value in glioma are unclear. In the present study, we analyzed the relationship between MCM mRNA expression and clinical parameters in 325 gliomas and found that MCM6 presented high expression and was associated with poor survival. Immunohistochemistry analysis of an independent data set of 423 glioma tissues confirmed the overexpression of MCM6 protein, especially in glioblastomas with shorter overall survival. Importantly, a combination of MCM6 overexpression with IDH1 mutation further improved the prediction of the prognosis of glioblastomas. Patients with IDH1 mutation and low MCM6 expression exhibited the longest survival, whereas those with high MCM6 expression and wild-type IDH1 showed the shortest. Collectively, our observation indicates that MCM6 is a novel potential biomarker for predicting poor prognosis of the patients with glioma.

Characterization of the DNA binding activity of structural protein VP1 from chicken anaemia virus

BackgroundChicken anaemia virus (CAV) is commonly found in poultry. VP1 is the sole structural protein of CAV, which is the major component responsible for capsid assembly. The CAV virion consists of the VP1 protein and a viral genome. However, there is currently no information on the protein-nucleic acid interactions between VP1 and DNA molecules.ResultsIn this study, the recombinant VP1 protein of CAV was expressed and purified to characterize its DNA binding activity. When VP1 protein was incubated with a DNA molecule, the DNA molecule exhibited retarded migration on an agarose gel. Regardless of whether the sequence of the viral genome was involved in the DNA molecule, DNA retardation was not significantly influenced. This outcome indicated VP1 is a DNA binding protein with no sequence specificity. Various DNA molecules with different conformations, such as circular dsDNA, linear dsDNA, linear ssDNA and circular ssDNA, interacted with VP1 proteins according to the results of a DNA retardation assay. Further quantification of the amount of VP1 protein required for DNA binding, the circular ssDNA demonstrated a high affinity for the VP1 protein. The preferences arranged in the order of affinity for the VP1 protein with DNA are circular ssDNA, linear ssDNA, supercoiled circular dsDNA, open circular DNA and linear dsDNA.ConclusionsThe results of this study demonstrated that the interaction between VP1 and DNA molecules exhibited various binding preferences that were dependent on the structural conformation of DNA. Taken together, the results of this report are the first to demonstrate that VP1 has no sequence-specific DNA binding activity. The particular binding preferences of VP1 might play multiple roles in DNA replication or encapsidation during the viral life cycle.

The role of the C-domain of bacteriophage T4 gene 32 protein in ssDNA binding and dsDNA helix-destabilization: Kinetic, single-molecule, and cross-linking studies.

The model single-stranded DNA binding protein of bacteriophage T4, gene 32 protein (gp32) has well-established roles in DNA replication, recombination, and repair. gp32 is a single-chain polypeptide consisting of three domains. Based on thermodynamics and kinetics measurements, we have proposed that gp32 can undergo a conformational change where the acidic C-terminal domain binds internally to or near the single-stranded (ss) DNA binding surface in the core (central) domain, blocking ssDNA interaction. To test this model, we have employed a variety of experimental approaches and gp32 variants to characterize this conformational change. Utilizing stopped-flow methods, the association kinetics of wild type and truncated forms of gp32 with ssDNA were measured. When the C-domain is present, the log-log plot of k vs. [NaCl] shows a positive slope, whereas when it is absent (*I protein), there is little rate change with salt concentration, as expected for this model.A gp32 variant lacking residues 292–296 within the C-domain, ΔPR201, displays kinetic properties intermediate between gp32 and *I. The single molecule force-induced DNA helix-destabilizing activitiesas well as the single- and double-stranded DNA affinities of ΔPR201 and gp32 truncated at residue 295 also fall between full-length protein and *I. Finally, chemical cross-linking of recombinant C-domain and gp32 lacking both N- and C-terminal domains is inhibited by increasing concentrations of a short single-stranded oligonucleotide, and the salt dependence of cross-linking mirrors that expected for the model. Taken together, these results provide the first evidence in support of this model that have been obtained through structural probes.

Arabidopsis replication factor C4 is critical for DNA replication during the mitotic cell cycle.

Replication factor C (RFC) is a conserved eukaryotic complex consisting of RFC1/2/3/4/5. It plays important roles in DNA replication and the cell cycle in yeast and fruit fly. However, it is not very clear how RFC subunits function in higher plants, except for the Arabidopsis (At) subunits AtRFC1 and AtRFC3. In this study, we investigated the functions of AtRFC4 and found that loss of function of AtRFC4 led to an early sporophyte lethality that initiated as early as the elongated zygote stage, all defective embryos arrested at the two- to four-cell embryo proper stage, and the endosperm possessed six to eight free nuclei. Complementation of rfc4-1/+ with AtRFC4 expression driven through the embryo-specific DD45pro and ABI3pro or the endosperm-specific FIS2pro could not completely restore the defective embryo or endosperm, whereas a combination of these three promoters in rfc4-1/+ enabled the aborted ovules to develop into viable seeds. This suggests that AtRFC4 functions simultaneously in endosperm and embryo and that the proliferation of endosperm is critical for embryo maturation. Assays of DNA content in rfc4-1/+ verified that DNA replication was disrupted in endosperm and embryo, resulting in blocked mitosis. Moreover, we observed a decreased proportion of late S-phase and M-phase cells in the rfc4-1/-FIS2;DD45;ABI3pro::AtRFC4 seedlings, suggesting that incomplete DNA replication triggered cell cycle arrest in cells of the root apical meristem. Therefore, we conclude that AtRFC4 is a crucial gene for DNA replication.

Identification of Poxvirus Genome Uncoating and DNA Replication Factors with Mutually Redundant Roles.

Genome uncoating is essential for replication of most viruses. For poxviruses, the process is divided into two stages: removal of the envelope, allowing early gene expression, and breaching of the core wall, allowing DNA release, replication, and late gene expression. Subsequent studies showed that the host proteasome and the viral D5 protein, which has an essential role in DNA replication, are required for vaccinia virus (VACV) genome uncoating. In a search for additional VACV uncoating proteins, we noted a report that described a defect in DNA replication and late expression when the gene encoding a 68-kDa ankyrin repeat/F-box protein (68k-ank), associated with the cellular SCF (Skp1, cullin1, F-box-containing complex) ubiquitin ligase complex, was deleted from the attenuated modified vaccinia virus Ankara (MVA). Here we showed that the 68k-ank deletion mutant exhibited diminished genome uncoating, formation of DNA prereplication sites, and degradation of viral cores as well as an additional, independent defect in DNA synthesis. Deletion of the 68k-ank homolog of VACV strain WR, however, was without effect, suggesting the existence of compensating genes. By inserting VACV genes into an MVA 68k-ank deletion mutant, we discovered that M2, a member of the poxvirus immune evasion (PIE) domain superfamily and a regulator of NF-κB, and C5, a member of the BTB/Kelch superfamily associated with cullin-3-based ligase complexes, independently rescued the 68k-ank deletion phenotype. Thus, poxvirus uncoating and DNA replication are intertwined processes involving at least three viral proteins with mutually redundant functions in addition to D5.IMPORTANCE Poxviruses comprise a family of large DNA viruses that infect vertebrates and invertebrates and cause diseases of medical and zoological importance. Poxviruses, unlike most other DNA viruses, replicate in the cytoplasm, and their large genomes usually encode 200 or more proteins with diverse functions. About 90 genes may be essential for chordopoxvirus replication based either on their conservation or individual gene deletion studies. However, this number may underestimate the true number of essential functions because of redundancy. Here we show that any one of three seemingly unrelated and individually nonessential proteins is required for the incompletely understood processes of genome uncoating and DNA replication, an example of synthetic lethality. Thus, poxviruses appear to have a complex genetic interaction network that has not been fully appreciated and which will require multifactor deletion screens to assess.

Chromatin-Bound Cullin-Ring Ligases: Regulatory Roles in DNA Replication and Potential Targeting for Cancer Therapy.

Cullin-RING (Really Interesting New Gene) E3 ubiquitin ligases (CRLs), the largest family of E3 ubiquitin ligases, are functional multi-subunit complexes including substrate receptors, adaptors, cullin scaffolds, and RING-box proteins. CRLs are responsible for ubiquitination of ~20% of cellular proteins and are involved in diverse biological processes including cell cycle progression, genome stability, and oncogenesis. Not surprisingly, cullins are deregulated in many diseases and instances of cancer. Recent studies have highlighted the importance of CRL-mediated ubiquitination in the regulation of DNA replication/repair, including specific roles in chromatin assembly and disassembly of the replication machinery. The development of novel therapeutics targeting the CRLs that regulate the replication machinery and chromatin in cancer is now an attractive therapeutic strategy. In this review, we summarize the structure and assembly of CRLs and outline their cellular functions and their diverse roles in cancer, emphasizing the regulatory functions of nuclear CRLs in modulating the DNA replication machinery. Finally, we discuss the current strategies for targeting CRLs against cancer in the clinic.

RNA-DNA hybrids promote the expansion of Friedreich's ataxia(GAA)n repeats via break-induced replication.

Expansion of simple DNA repeats is responsible for numerous hereditary diseases in humans. The role of DNA replication, repair and transcription in the expansion process has been well documented. Here we analyzed, in a yeast experimental system, the role of RNA–DNA hybrids in genetic instability of long (GAA)n repeats, which cause Friedreich’s ataxia. Knocking out both yeast RNase H enzymes, which counteract the formation of RNA–DNA hybrids, increased (GAA)n repeat expansion and contraction rates when the repetitive sequence was transcribed. Unexpectedly, we observed a similar increase in repeat instability in RNase H-deficient cells when we either changed the direction of transcription-replication collisions, or flipped the repeat sequence such that the (UUC)n run occurred in the transcript. The increase in repeat expansions in RNase H-deficient strains was dependent on Rad52 and Pol32 proteins, suggesting that break-induced replication (BIR) is responsible for this effect. We conclude that expansions of (GAA)n repeats are induced by the formation of RNA–DNA hybrids that trigger BIR. Since this stimulation is independent of which strand of the repeat (homopurine or homopyrimidine) is in the RNA transcript, we hypothesize that triplex H-DNA structures stabilized by an RNA–DNA hybrid (H-loops), rather than conventional R-loops, could be responsible.

SAAV2152 is a single-stranded DNA binding protein: the third SSB in Staphylococcus aureus.

Single-stranded DNA-binding proteins (SSBs) play crucial roles in DNA replication, repair, and recombination. Unlike E. coli, which contains only one type of SSB (EcSSB), some bacteria have two paralogous SSBs, namely, SsbA and SsbB. In this study, we found the third SSB-like protein in Staphylococcus aureus, SAAV2152, which was designated as SaSsbC. SaSsbC is a protein of 131 amino acids and shares 38%, 36%, and 33% sequence identity to SaSsbB, SaSsbA, and EcSSB, respectively. Gene map analysis showed that unlike the E. coli ssb gene, which is adjacent to uvrA gene, the S. aureus ssb gene SAAV2152 is flanked by the putative SceD, the putative YwpF, and fabZ genes. A homology model showed that SaSsbC consists of the classic oligonucleotide/oligosaccharide-binding fold at the N-terminus. At the C-terminus, SaSsbC did not exhibit sequence similarity to that of EcSSB. Electrophoretic mobility shift analysis showed that SaSsbC formed a single complex with ssDNA of different lengths. Mutational analysis revealed that Tyr36, Tyr47, Phe53, and Tyr81 in SaSsbC are at positions that structurally correspond to the important residues of EcSSB for binding to ssDNA and are also critical for SaSsbC to bind ssDNA. Unlike EcSSB, which can stimulate EcPriA, SaSsbC did not affect the activity of SaPriA. In addition, SaSsbA inhibitor 9-methyl-2,3,7-trihydroxy-6-fluorone (NSC5426) could inhibit the ssDNA-binding activity of SaSsbC with IC50 of 78 μM. In conclusion, this study has identified and characterized SAAV2152 as a kind of SSB, and further research can directly focus on determining its actual physiological role in S. aureus.

Genetic Evidence for Roles of Yeast Mitotic Cyclins at Single-Stranded Gaps Created by DNA Replication.

Paused or stalled replication forks are major threats to genome integrity; unraveling the complex pathways that contribute to fork stability and restart is crucial. Experimentally, fork stalling is induced by growing the cells in presence of hydroxyurea (HU), which depletes the pool of deoxynucleotide triphosphates (dNTPs) and slows down replication progression in yeast. Here, I report an epistasis analysis, based on sensitivity to HU, between CLB2, the principal mitotic cyclin gene in Saccharomyces cerevisiae, and genes involved in fork stability and recombination. clb2Δ cells are not sensitive to HU, but the strong synergistic effect of clb2Δ with most genes tested indicates, unexpectedly, that CLB2 has an important role in DNA replication, in the stability and restart of stalled forks, and in pathways dependent on and independent of homologous recombination. Results indicate that CLB2 functions in parallel with the SGS1 helicase and EXO1 exonuclease to allow proper Rad51 recombination, but also regulates a combined Sgs1–Exo1 activity in a pathway dependent on Mec1 and Rad53 checkpoint protein kinases. The data argue that Mec1 regulates Clb2 to prevent a deleterious Sgs1–Exo1 activity at paused or stalled forks, whereas Rad53 checkpoint activation regulates Clb2 to allow a necessary Sgs1–Exo1 activity at stalled or collapsed forks. Altogether, this study indicates that Clb2 regulates the activity of numerous nucleases at single-stranded gaps created by DNA replication. A model is proposed for the function and regulation of Clb2 at stalled forks. These data provide new perspectives on the role of mitotic cyclins at the end of S phase.

Efficacy of the MEK Inhibitor Cobimetinib and its Potential Application to Colorectal Cancer Cells

Background/Aims: Mutations in the Ras/Raf/MEK/ERK pathway are detected in 50% of colorectal cancer cases and play a crucial role in cancer development and progression. Cobimetinib is a MEK inhibitor approved for the treatment of advanced melanoma and inhibits the cell viability of other types of cancer cells. Methods: HCT116 colorectal cancer cells were treated with cobimetinib, and MTT assay, colony formation assay, and flow cytometry were used to evaluate cell viability, cell cycle, and apoptosis, respectively. The expression of genes associated with the cell cycle and apoptosis were evaluated by quantitative real-time PCR and western blotting. To explore use of cobimetinib in colorectal cancer treatment and further understand its mechanisms, RNA-seq technology was used to identify differentially expressed genes (DEGs) between cobimetinib-treated and untreated HCT116 cells. Furthermore, we compared these DEGs with Gene Expression Omnibus data from colorectal cancer tissues and normal colonic epithelial tissues. Results: We found that cobimetinib not only inhibited cell proliferation but also induced G1 phase arrest and apoptosis in HCT116 colorectal cancer cells, suggesting that cobimetinib may useful in colorectal cancer therapy. After cobimetinib treatment, 3,495 DEGs were obtained, including 2,089 upregulated genes and 1,406 downregulated genes, and most of these DEGs were enriched in the cell cycle, DNA replication, and DNA damage repair pathways. Our results revealed that some genes with high expression in colorectal cancer tissues were downregulated by cobimetinib in HCT116 cells, including CCND1, E2F1, CDC25C, CCNE2, MYC, and PCNA. These genes have vital roles in DNA replication and the cell cycle. Furthermore, genes with low expression in colorectal cancer tissues were upregulated by cobimetinib, including PRKCA, PI3K, RTK, and PKC. Based on our results, the PKC and PI3K pathways were activated after cobimetinib treatment, and inhibition of these two pathways can increase the cytotoxicity of cobimetinib in HCT116 cells. Notably, cobimetinib appeared to enhance the efficacy of 5-fluorouracil (5-FU) by decreasing TYMS expression, high expression of which is responsible for 5-FU resistance in colorectal cancer. Conclusions: Our results suggest the potential use of cobimetinib in colorectal cancer therapy.

4β-amidotriazole linked podophyllotoxin congeners: DNA topoisomerase-IIα inhibition and potential anticancer agents for prostate cancer

Topoisomerases (topo-I and topo-II) have occupied a significant role in DNA replication, transcription, and are a promising set of antitumor targets. In the present approach, a series of new 4β-amidotriazole linked podophyllotoxin derivatives (10a-i and 11a-k) were designed, synthesized by employing the click chemistry and their biological activities were evaluated. The majority of derivatives showed promising antiproliferative activity with IC50 values ranging from 1 to 10 μM on the six human cancer cell lines; cervical (HeLa), breast (MCF-7), prostate (DU-145), lung (A549), liver (HepG2) and colon (HT-29). Among them, some of the congeners 10b, 10g and 10i have shown remarkable cytotoxicity with IC50 values of, < 1 μM against the tested cancer cell lines and found to be more active than etoposide. Topoisomerase-mediated DNA relaxation assay results showed that the derivatives could efficiently inhibit the activity of topoisomerase-II. In addition, flow cytometry analysis on DU-145 cells revealed that these compounds arrest G2/M phase of cell cycle. Further apoptotic studies were also performed on these DU-145 cells, which showed that this class of compounds could induce apoptosis effectively.

Rv0004 is a new essential member of the mycobacterial DNA replication machinery.

DNA replication is fundamental for life, yet a detailed understanding of bacterial DNA replication is limited outside the organisms Escherichia coli and Bacillus subtilis. Many bacteria, including mycobacteria, encode no identified homologs of helicase loaders or regulators of the initiator protein DnaA, despite these factors being essential for DNA replication in E. coli and B. subtilis. In this study we discover that a previously uncharacterized protein, Rv0004, from the human pathogen Mycobacterium tuberculosis is essential for bacterial viability and that depletion of Rv0004 leads to a block in cell cycle progression. Using a combination of genetic and biochemical approaches, we found that Rv0004 has a role in DNA replication, interacts with DNA and the replicative helicase DnaB, and affects DnaB-DnaA complex formation. We also identify a conserved domain in Rv0004 that is predicted to structurally resemble the N-terminal protein-protein interaction domain of DnaA. Mutation of a single conserved tryptophan within Rv0004’s DnaA N-terminal-like domain leads to phenotypes similar to those observed upon Rv0004 depletion and can affect the association of Rv0004 with DnaB. In addition, using live cell imaging during depletion of Rv0004, we have uncovered a previously unappreciated role for DNA replication in coordinating mycobacterial cell division and cell size. Together, our data support that Rv0004 encodes a homolog of the recently identified DciA family of proteins found in most bacteria that lack the DnaC-DnaI helicase loaders in E. coli and B. subtilis. Therefore, the mechanisms of Rv0004 elucidated here likely apply to other DciA homologs and reveal insight into the diversity of bacterial strategies in even the most conserved biological processes.

DNA damage and neurodegenerative phenotypes in aged Ciz1 null mice

Cell-cycle dysfunction and faulty DNA repair are closely intertwined pathobiological processes that may contribute to several neurodegenerative disorders. CDKN1A interacting zinc finger protein 1 (CIZ1) plays a critical role in DNA replication and cell-cycle progression at the G1/S checkpoint. Germline or somatic variants in CIZ1 have been linked to several neural and extra-neural diseases. Recently, we showed that germline knockout of Ciz1 is associated with motor and hematological abnormalities in young adult mice. However, the effects of CIZ1 deficiency in much older mice may be more relevant to understanding age-related declines in cognitive and motor functioning and age-related neurologic disorders such as isolated dystonia and Alzheimer disease. Mouse embryonic fibroblasts from Ciz1−/− mice showed abnormal sensitivity to the effects of γ-irradiation with persistent DNA breaks, aberrant cell-cycle progression, and apoptosis. Aged (18-month-old) Ciz1−/− mice exhibited marked deficits in motor and cognitive functioning, and, in brain tissues, overt DNA damage, NF-κB upregulation, oxidative stress, vascular dysfunction, inflammation, and cell death. These findings indicate that the deleterious effects of CIZ1 deficiency become more pronounced with aging and suggest that defects of cell-cycle control and associated DNA repair pathways in postmitotic neurons could contribute to global neurologic decline in elderly human populations. Accordingly, the G1/S cell-cycle checkpoint and associated DNA repair pathways may be targets for the prevention and treatment of age-related neurodegenerative processes.