HROB Is Implicated in DNA Replication.

DNA replication represents a series of precisely regulated events performed by a complex protein machinery that guarantees accurate duplication of the genetic information. Since DNA replication is permanently faced by a variety of exogenous and endogenous stressors, DNA damage response, repair and replication must be closely coordinated to maintain genomic integrity. HROB has been identified recently as a binding partner and activator of the Mcm8/9 helicase involved in DNA interstrand crosslink (ICL) repair.

Compromised CDK12 activity causes dependency on the high activity of O-GlcNAc transferase.

O-GlcNAc transferase (OGT) coordinates with regulators of transcription, including cyclin-dependent kinase 12 (CDK12), the major transcription elongation kinase. Here, we use inhibitor- and knockdown-based strategies to show that co-targeting of OGT and CDK12 is toxic to prostate cancer cells. OGT catalyzes all nucleocytoplasmic O-GlcNAcylation and due to its essentiality in higher eukaryotes, it is not an ideal drug target.

ABRAXAS1 orchestrates BRCA1 activities to counter genome destabilizing repair pathways-lessons from breast cancer patients.

It has been well-established that mutations in BRCA1 and BRCA2, compromising functions in DNA double-strand break repair (DSBR), confer hereditary breast and ovarian cancer risk. Importantly, mutations in these genes explain only a minor fraction of the hereditary risk and of the subset of DSBR deficient tumors. Our screening efforts identified two truncating germline mutations in the gene encoding the BRCA1 complex partner ABRAXAS1 in German early-onset breast cancer patients.

Partial Reduction in Gene Dose Modulates DNA Replication Stress Level and Thereby Contributes to Sensitivity or Resistance.

is a well-known breast cancer risk gene, involved in DNA damage repair via homologous recombination (HR) and replication fork protection. Therapy resistance was linked to loss and amplification of the gene causing inferior survival of breast cancer patients. Most studies have focused on the analysis of complete loss or mutations in functional domains of .

Multiple biochemical properties of the p53 molecule contribute to activation of polymerase iota-dependent DNA damage tolerance.

We have previously reported that p53 decelerates nascent DNA elongation in complex with the translesion synthesis (TLS) polymerase ι (POLι) which triggers a homology-directed DNA damage tolerance (DDT) pathway to bypass obstacles during DNA replication. Here, we demonstrate that this DDT pathway relies on multiple p53 activities, which can be disrupted by TP53 mutations including those frequently found in cancer tissues. We show that the p53-mediated DDT pathway depends on its oligomerization domain (OD), while its regulatory C-terminus is not involved.

BRCA1 mislocalization leads to aberrant DNA damage response in heterozygous ABRAXAS1 mutation carrier cells.

Whilst heterozygous germline mutations in the ABRAXAS1 gene have been associated with a hereditary predisposition to breast cancer, their effect on promoting tumourigenesis at the cellular level has not been explored. Here, we demonstrate in patient-derived cells that the Finnish ABRAXAS1 founder mutation (c.1082G > A, Arg361Gln), even in the heterozygous state leads to decreased BRCA1 protein levels as well as reduced nuclear localization and foci formation of BRCA1 and CtIP.

Local inhibition of rRNA transcription without nucleolar segregation after targeted ion irradiation of the nucleolus.

Nucleoli have attracted interest for their role as cellular stress sensors and as potential targets for cancer treatment. The effect of DNA double-strand breaks (DSBs) in nucleoli on rRNA transcription and nucleolar organisation appears to depend on the agent used to introduce DSBs, DSB frequency and the presence (or not) of DSBs outside the nucleoli. To address the controversy, we targeted nucleoli with carbon ions at the ion microbeam SNAKE.

The ATR-Activation Domain of TopBP1 Is Required for the Suppression of Origin Firing during the S Phase.

The mammalian DNA replication program is controlled at two phases, the licensing of potential origins of DNA replication in early gap 1 (G1), and the selective firing of a subset of licenced origins in the synthesis (S) phase. Upon entry into the S phase, serine/threonine-protein kinase ATR (ATR) is required for successful completion of the DNA replication program by limiting unnecessary dormant origin activation. Equally important is its activator, DNA topoisomerase 2-binding protein 1 (TopBP1), which is also required for the initiation of DNA replication after a rise in S-phase kinase levels.

Cdc45-induced loading of human RPA onto single-stranded DNA.

Cell division cycle protein 45 (Cdc45) is an essential component of the eukaryotic replicative DNA helicase. We found that human Cdc45 forms a complex with the single-stranded DNA (ssDNA) binding protein RPA. Moreover, it actively loads RPA onto nascent ssDNA.

TSC loss distorts DNA replication programme and sensitises cells to genotoxic stress.

Tuberous Sclerosis (TSC) is characterized by exorbitant mTORC1 signalling and manifests as non-malignant, apoptosis-prone neoplasia. Previous reports have shown that TSC-/- cells are highly susceptible to mild, innocuous doses of genotoxic stress, which drive TSC-/- cells into apoptotic death. It has been argued that this hypersensitivity to stress derives from a metabolic/energetic shortfall in TSC-/- cells, but how metabolic dysregulation affects the DNA damage response and cell cycle alterations in TSC-/- cells exposed to genotoxic stress is not understood.

Human DNA polymerase α interacts with mismatch repair proteins MSH2 and MSH6.

High fidelity of genome duplication is ensured by cooperation of polymerase proofreading and mismatch repair (MMR) activities. Here, we show that human mismatch recognizing proteins MutS homolog 2 (MSH2) and MSH6 copurify and interact with replicative Pol α. This enzyme also is the replicative primase and replicates DNA with poor fidelity.

S100A11 plays a role in homologous recombination and genome maintenance by influencing the persistence of RAD51 in DNA repair foci.

The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is an essential process in maintenance of chromosomal stability. A key player of HR is the strand exchange factor RAD51 whose assembly at sites of DNA damage is tightly regulated. We detected an endogenous complex of RAD51 with the calcium-binding protein S100A11, which is localized at sites of DNA repair in HaCaT cells as well as in normal human epidermal keratinocytes (NHEK) synchronized in S phase.

DNA damage tolerance pathway involving DNA polymerase ι and the tumor suppressor p53 regulates DNA replication fork progression.

DNA damage tolerance facilitates the progression of replication forks that have encountered obstacles on the template strands. It involves either translesion DNA synthesis initiated by proliferating cell nuclear antigen monoubiquitination or less well-characterized fork reversal and template switch mechanisms. Herein, we characterize a novel tolerance pathway requiring the tumor suppressor p53, the translesion polymerase ι (POLι), the ubiquitin ligase Rad5-related helicase-like transcription factor (HLTF), and the SWI/SNF catalytic subunit (SNF2) translocase zinc finger ran-binding domain containing 3 (ZRANB3).

Cdc45 is limiting for replication initiation in humans.

Cdc45 is an essential protein that together with Mcm2-7 and GINS forms the eukaryotic replicative helicase CMG. Cdc45 seems to be rate limiting for the initial unwinding or firing of replication origins. In line with this view, Cdc45-overexpressing cells fired at least twice as many origins as control cells.

Heterozygous PALB2 c.1592delT mutation channels DNA double-strand break repair into error-prone pathways in breast cancer patients.

Hereditary heterozygous mutations in a variety of DNA double-strand break (DSB) repair genes have been associated with increased breast cancer risk. In the Finnish population, PALB2 (partner and localizer of BRCA2) represents a major susceptibility gene for female breast cancer, and so far, only one mutation has been described, c.1592delT, which leads to a sixfold increased disease risk.

High levels of RAD51 perturb DNA replication elongation and cause unscheduled origin firing due to impaired CHK1 activation.

In response to replication stress ATR signaling through CHK1 controls the intra-S checkpoint and is required for the maintenance of genomic integrity. Homologous recombination (HR) comprises a series of interrelated pathways that function in the repair of DNA double strand breaks and interstrand crosslinks. In addition, HR, with its key player RAD51, provides critical support for the recovery of stalled forks during replication.

High levels of TopBP1 induce ATR-dependent shut-down of rRNA transcription and nucleolar segregation.

Nucleoli are not only organelles that produce ribosomal subunits. They are also overarching sensors of different stress conditions and they control specific nucleolar stress pathways leading to stabilization of p53. During DNA replication, ATR and its activator TopBP1 initiate DNA damage response upon DNA damage and replication stress.

The intrinsically disordered amino-terminal region of human RecQL4: multiple DNA-binding domains confer annealing, strand exchange and G4 DNA binding.

Human RecQL4 belongs to the ubiquitous RecQ helicase family. Its N-terminal region represents the only homologue of the essential DNA replication initiation factor Sld2 of Saccharomyces cerevisiae, and also participates in the vertebrate initiation of DNA replication. Here, we utilized a random screen to identify N-terminal fragments of human RecQL4 that could be stably expressed in and purified from Escherichia coli.

Gap-directed translesion DNA synthesis of an abasic site on circular DNA templates by a human replication complex.

DNA polymerase ε (pol ε) is believed to be the leading strand replicase in eukaryotes whereas pols λ and β are thought to be mainly involved in re-synthesis steps of DNA repair. DNA elongation by the human pol ε is halted by an abasic site (apurinic/apyrimidinic (AP) site). We have previously reported that human pols λ, β and η can perform translesion synthesis (TLS) of an AP site in the presence of pol ε.

DNA binding properties of human Cdc45 suggest a function as molecular wedge for DNA unwinding.

The cell division cycle protein 45 (Cdc45) represents an essential replication factor that, together with the Mcm2-7 complex and the four subunits of GINS, forms the replicative DNA helicase in eukaryotes. Recombinant human Cdc45 (hCdc45) was structurally characterized and its DNA-binding properties were determined. Synchrotron radiation circular dichroism spectroscopy, dynamic light scattering, small-angle X-ray scattering and atomic force microscopy revealed that hCdc45 exists as an alpha-helical monomer and possesses a structure similar to its bacterial homolog RecJ.

The activities of lysyl hydroxylase 3 (LH3) regulate the amount and oligomerization status of adiponectin.

Lysyl hydroxylase 3 (LH3) has lysyl hydroxylase, galactosyltransferase, and glucosyltransferase activities, which are sequentially required for the formation of glucosylgalactosyl hydroxylysines in collagens. Here we demonstrate for the first time that LH3 also modifies the lysine residues in the collagenous domain of adiponectin, which has important roles in glucose and lipid metabolism and inflammation. Hydroxylation and, especially, glycosylation of the lysine residues of adiponectin have been shown to be essential for the formation of the more active high molecular weight adiponectin oligomers and thus for its function.

Segregation of replicative DNA polymerases during S phase: DNA polymerase ε, but not DNA polymerases α/δ, are associated with lamins throughout S phase in human cells.

DNA polymerases (Pol) α, δ, and ε replicate the bulk of chromosomal DNA in eukaryotic cells, Pol ε being the main leading strand and Pol δ the lagging strand DNA polymerase. By applying chromatin immunoprecipitation (ChIP) and quantitative PCR we found that at G(1)/S arrest, all three DNA polymerases were enriched with DNA containing the early firing lamin B2 origin of replication and, 2 h after release from the block, with DNA containing the origin at the upstream promoter region of the MCM4 gene. Pol α, δ, and ε were released from these origins upon firing.

The N-terminus of the human RecQL4 helicase is a homeodomain-like DNA interaction motif.

The RecQL4 helicase is involved in the maintenance of genome integrity and DNA replication. Mutations in the human RecQL4 gene cause the Rothmund-Thomson, RAPADILINO and Baller-Gerold syndromes. Mouse models and experiments in human and Xenopus have proven the N-terminal part of RecQL4 to be vital for cell growth.

In vitro gap-directed translesion DNA synthesis of an abasic site involving human DNA polymerases epsilon, lambda, and beta.

DNA polymerase (pol) ε is thought to be the leading strand replicase in eukaryotes, whereas pols λ and β are believed to be mainly involved in re-synthesis steps of DNA repair. DNA elongation by the human pol ε is halted by an abasic site (apurinic/apyrimidinic (AP) site). In this study, we present in vitro evidence that human pols λ, β, and η can perform translesion synthesis (TLS) of an AP site in the presence of pol ε, likely by initiating the 3'OHs created at the lesion by the arrested pol ε.