Establishing a biochemical understanding of the initiation of chromosome replication in bacteria.

In the mid-1950s, Arthur Kornberg elucidated the enzymatic synthesis of DNA by DNA polymerase, for which he was recognized with the 1959 Nobel Prize in Physiology or Medicine. He then identified many of the proteins that cooperate with DNA polymerase to replicate duplex DNA of small bacteriophages. However, one major unanswered problem was understanding the mechanism and control of the initiation of chromosome replication in bacteria.

Origins of DNA replication in eukaryotes.

Errors occurring during DNA replication can result in inaccurate replication, incomplete replication, or re-replication, resulting in genome instability that can lead to diseases such as cancer or disorders such as autism. A great deal of progress has been made toward understanding the entire process of DNA replication in eukaryotes, including the mechanism of initiation and its control. This review focuses on the current understanding of how the origin recognition complex (ORC) contributes to determining the location of replication initiation in the multiple chromosomes within eukaryotic cells, as well as methods for mapping the location and temporal patterning of DNA replication.

Prevalent and dynamic binding of the cell cycle checkpoint kinase Rad53 to gene promoters.

Replication of the genome must be coordinated with gene transcription and cellular metabolism, especially following replication stress in the presence of limiting deoxyribonucleotides. The Rad53 (CHEK2 in mammals) checkpoint kinase plays a major role in cellular responses to DNA replication stress. Cell cycle regulated, genome-wide binding of Rad53 to chromatin was examined.

Pelvic floor behavioral treatment for fecal incontinence and constipation in quiescent inflammatory bowel disease.

Background And Aim: Refractory bowel symptoms in quiescent inflammatory bowel disease (IBD) are common but evidence for effective management is limited. We aimed to determine whether behavioral treatment, including pelvic floor muscle training, decreases the severity of functional bowel symptoms in patients with quiescent IBD. Secondary aims were to evaluate the treatment effect on quality of life, psychological well-being and pelvic floor muscle function.

The remarkable gymnastics of ORC.

As a cell prepares to divide, a molecular actor known as the Origin Recognition Complex makes intricate ATP-driven movements to recruit proteins required to duplicate DNA.

The structure of ORC-Cdc6 on an origin DNA reveals the mechanism of ORC activation by the replication initiator Cdc6.

The Origin Recognition Complex (ORC) binds to sites in chromosomes to specify the location of origins of DNA replication. The S. cerevisiae ORC binds to specific DNA sequences throughout the cell cycle but becomes active only when it binds to the replication initiator Cdc6.

Multiple, short protein binding motifs in ORC1 and CDC6 control the initiation of DNA replication.

The initiation of DNA replication involves cell cycle-dependent assembly and disassembly of protein complexes, including the origin recognition complex (ORC) and CDC6 AAA ATPases. We report that multiple short linear protein motifs (SLiMs) within intrinsically disordered regions (IDRs) in ORC1 and CDC6 mediate cyclin-CDK-dependent and independent protein-protein interactions, conditional on the cell cycle phase. A domain within the ORC1 IDR is required for interaction between the ORC1 and CDC6 AAA domains in G1, whereas the same domain prevents CDC6-ORC1 interaction during mitosis.

The human origin recognition complex is essential for pre-RC assembly, mitosis, and maintenance of nuclear structure.

The origin recognition complex (ORC) cooperates with CDC6, MCM2-7, and CDT1 to form pre-RC complexes at origins of DNA replication. Here, using tiling-sgRNA CRISPR screens, we report that each subunit of ORC and CDC6 is essential in human cells. Using an auxin-inducible degradation system, we created stable cell lines capable of ablating ORC2 rapidly, revealing multiple cell division cycle phenotypes.

Evolution of DNA replication origin specification and gene silencing mechanisms.

DNA replication in eukaryotic cells initiates from replication origins that bind the Origin Recognition Complex (ORC). Origin establishment requires well-defined DNA sequence motifs in Saccharomyces cerevisiae and some other budding yeasts, but most eukaryotes lack sequence-specific origins. A 3.

The dynamic nature of the human origin recognition complex revealed through five cryoEM structures.

Genome replication is initiated from specific origin sites established by dynamic events. The Origin Recognition Complex (ORC) is necessary for orchestrating the initiation process by binding to origin DNA, recruiting CDC6, and assembling the MCM replicative helicase on DNA. Here we report five cryoEM structures of the human ORC (HsORC) that illustrate the native flexibility of the complex.

Structural mechanism of helicase loading onto replication origin DNA by ORC-Cdc6.

DNA replication origins serve as sites of replicative helicase loading. In all eukaryotes, the six-subunit origin recognition complex (Orc1-6; ORC) recognizes the replication origin. During late M-phase of the cell-cycle, Cdc6 binds to ORC and the ORC-Cdc6 complex loads in a multistep reaction and, with the help of Cdt1, the core Mcm2-7 helicase onto DNA.

Borrelia burgdorferi Antibody Test Results in Dogs Administered 4 Different Vaccines.

Vaccines against Borrelia burgdorferi are administered frequently to dogs in areas endemic for the infection. These vaccines produce an antibody response to spirochetal proteins that cross-react in many antibody tests, including immunofluorescence assay, Western blot, and whole cell ELISA used to document exposure to B. burgdorferi.

Histone Modifications: Insights into Their Influence on Gene Expression.

This year's Albert Lasker Basic Medical Research Award honors David Allis and Michael Grunstein for their pioneering research that highlighted the importance of histones and their post-translational modifications in the direct control of gene expression.

The dNTP triphosphohydrolase activity of SAMHD1 persists during S-phase when the enzyme is phosphorylated at T592.

SAMHD1 is the major catabolic enzyme regulating the intracellular concentrations of DNA precursors (dNTPs). The S-phase kinase CDK2-cyclinA phosphorylates SAMHD1 at Thr-592. How this modification affects SAMHD1 function is highly debated.

Cryo-EM structure of Mcm2-7 double hexamer on DNA suggests a lagging-strand DNA extrusion model.

During replication initiation, the core component of the helicase-the Mcm2-7 hexamer-is loaded on origin DNA as a double hexamer (DH). The two ring-shaped hexamers are staggered, leading to a kinked axial channel. How the origin DNA interacts with the axial channel is not understood, but the interaction could provide key insights into Mcm2-7 function and regulation.

Structure of the active form of human origin recognition complex and its ATPase motor module.

Binding of the Origin Recognition Complex (ORC) to origins of replication marks the first step in the initiation of replication of the genome in all eukaryotic cells. Here, we report the structure of the active form of human ORC determined by X-ray crystallography and cryo-electron microscopy. The complex is composed of an ORC1/4/5 motor module lobe in an organization reminiscent of the DNA polymerase clamp loader complexes.

Opposing roles for DNA replication initiator proteins ORC1 and CDC6 in control of Cyclin E gene transcription.

Newly born cells either continue to proliferate or exit the cell division cycle. This decision involves delaying expression of Cyclin E that promotes DNA replication. ORC1, the Origin Recognition Complex (ORC) large subunit, is inherited into newly born cells after it binds to condensing chromosomes during the preceding mitosis.

Targeted Doxorubicin Delivery to Brain Tumors via Minicells: Proof of Principle Using Dogs with Spontaneously Occurring Tumors as a Model.

Background: Cytotoxic chemotherapy can be very effective for the treatment of cancer but toxicity on normal tissues often limits patient tolerance and often causes long-term adverse effects. The objective of this study was to assist in the preclinical development of using modified, non-living bacterially-derived minicells to deliver the potent chemotherapeutic doxorubicin via epidermal growth factor receptor (EGFR) targeting. Specifically, this study sought to evaluate the safety and efficacy of EGFR targeted, doxorubicin loaded minicells (designated EGFRminicellsDox) to deliver doxorubicin to spontaneous brain tumors in 17 companion dogs; a comparative oncology model of human brain cancers.

Concerted activities of Mcm4, Sld3, and Dbf4 in control of origin activation and DNA replication fork progression.

Eukaryotic chromosomes initiate DNA synthesis from multiple replication origins in a temporally specific manner during S phase. The replicative helicase Mcm2-7 functions in both initiation and fork progression and thus is an important target of regulation. Mcm4, a helicase subunit, possesses an unstructured regulatory domain that mediates control from multiple kinase signaling pathways, including the Dbf4-dependent Cdc7 kinase (DDK).

Reconsidering DNA Polymerases at the Replication Fork in Eukaryotes.

The distribution of DNA polymerase activities at the eukaryotic DNA replication fork was "established," but recent genetic studies in this issue of Molecular Cell raise questions about which polymerases are copying the leading and lagging strand templates (Johnson et al, 2015).