oxoG repair

MOLECULAR BIOLOGY: ON DNA-REPAIR ENZYMES: "A variety of agents cause oxidative damage to DNA, including oxygen radicals and ionizing radiation. Oxidation of G to form oxoG produces a subtle structural transformation that results in deleterious mutations because DNA polymerases misread oxoG as a thymine (T) base when the genome is being duplicated during cell division. The human oxoG repair enzyme (hOGG1) catalyses excision of oxoG in the first step of BER. Structural studies of glycosylases involved in the repair process reveal common features of damaged-base recognition that include enzyme-initiated DNA distortion and bending to flip the damaged base out from the DNA double helix for recognition within a base-specific cavity of the enzyme."

strathmin

CELL BIOLOGY: ON THE ORCHESTRATION OF THE MITOTIC SPINDLE: "Intracellular gradients can also be generated by enzyme activity. In this case, spatial information is provided by the concentration gradient of a diffusible substrate generated by a fixed enzyme. For instance, if phosphorylation of a diffusible protein is catalyzed by a localized protein kinase, and if the opposing protein phosphatase is dispersed, then a gradient in the phosphorylation status (and therefore in the activity of the substrate protein) can be generated[5]. Such a reaction-diffusion process creates a steadily changing gradient that is distinct from sharp concentration differences due to compartmentalization, such as the difference in ion concentration across an impermeable membrane. Previous work has shown, by mathematical simulation combined with molecular experiments using fluorescence reporters, that such a chemical gradient can be generated by phosphorylation of the microtubule-binding protein stathmin."

myosins

EVOLUTION: MYOSINS AND THE EARLIEST EUKARYOTES: "The myosins are a diverse group of motor proteins that move along the actin filaments that form a major component of the cell's internal scaffolding. Myosins are best known for powering muscle contraction, cell migration and cytokinesis (the separation of two daughter cells during cell division)[2,3]. These proteins typically consist of an amino-terminal motor domain that binds to the actin track and catalyses nucleotide hydrolysis -- the reaction used to harvest energy from ATP -- and a carboxy-terminal tail region. This directs the motor domain to targets within the cell, either binding to a cargo that is to be transported around the cell or anchoring the myosin to a particular site[2,3]."

Chk1 Rad53

Understanding How Cells Respond to DNA Damage May Lead to Better Cancer Treatments: "To prevent mutations that lead to uncontrolled growth, cells have special 'quality controlling' proteins that include dedicated scouts that look for damage to the DNA. When damaged DNA is detected, the scouts signal 'messenger' proteins, which place the cell on alert. When the alert is given, cell division is stopped, and the 'repair crew' is called. The 'scout' and 'messenger' proteins and the responses they regulate are part of what is called the checkpoint pathways. Once the damage is repaired, the cell is permitted to resume making a copy of the genetic information and proceed with cell division.
The researchers previously identified these 'scout' proteins that signal the presence of DNA damage and delay cell division, in order to allow time to repair the damage to the DNA. In their current paper, the same team of researchers describe the mechanism used by the signaling proteins which prevent the separation of chromosomes during mitosis to daughter cells. This inhibiting action occurs when damaged DNA has been detected. The task of preventing mitosis is shared between two proteins Chk1 and Rad53, which prevent chromosome separation (anaphase) and entry into the next cell cycle, respectively."

type II topoisomerases

BioMed Central | Full text | Characterisation of cytotoxicity and DNA damage induced by the topoisomerase II-directed bisdioxopiperazine anti-cancer agent ICRF-187 (dexrazoxane) in yeast and mammalian cells: "Type II topoisomerases are essential nuclear enzymes found in all living organisms [1]. Their basic role in cells is to catalyse the transport of one DNA double helix through a transient double strand break in another DNA molecule [2]. This activity helps relieve tensions built up in DNA during various DNA metabolic processes such as DNA replication, chromosome condensation and de-condensation, chromosome segregation and transcription [3]."

cyclin-D1

The Agami Group: "DNA-damage response to ionizing radiation is composed of two processes: initiation and maintenance. DNA damage causes a fast G1-arrest by rapidly degrading cyclin-D1. Later, this initial G1-response to DNA damage is maintained and further strengthened by the stabilization of p53, leading to the induction of p21cip1. "

Cdc6

The Agami Group: "activation of p53 by DNA damage results in enhanced Cdc6 destruction by the anaphase-promoting complex (APC). This destruction is triggered by inhibition of CDK2 mediated CDC6 phosphorylation at serine 54. Conversely, suppression of p53 expression results in stabilization of Cdc6."

ATR ATM

Natural killer cells: DNA damage link to innate immunity - Cell Signaling Update - Signaling Gateway: "DNA damage alerts cells of the immune system to attack damaged self cells, and it is possible that this mechanism also operates to trigger immune responses to virally infected cells. It remains to be established whether this mechanism can account for at least some of the effects of chemotherapy and radiation therapy, but the signalling pathway that involves ATR and/or ATM could become a target for the development of new therapeutics for cancer."

XRCC2 and XRCC3

MRC | Radiation and Genome Stability Unit: "Loss of XRCC2 or XRCC3 causes high levels of chromosomal mis-segregation and centrosome defects, leading to aneuploidy (a common feature of cancer cells)."

nuclear protein XRCC2 required for RAD51 focus formation

MRC | Radiation and Genome Stability Unit: "XRCC2 as a nuclear protein required for RAD51 focus formation in response to X-ray damage, implicating it in RAD51-dependent repair processes."

XRCC2 and RAD51L2 dimer

MRC | Radiation and Genome Stability Unit: "XRCC2 and RAD51L3 proteins form a heterodimer; while both have ATP-binding domains, XRCC2 does not require these domains for function."

RAD51 homologous recombination repair

MRC | Radiation and Genome Stability Unit: "RAD51 gene family, involved in DNA break repair through a process known as homologous recombination."

DNA mutation and alkylation

CROET Faculty: Mitchell Turker, PhD: "Abnormal gene inactivation results from two distinct types of events. The first is DNA mutation, which represents a change in the structure of DNA that alters expression of a given gene. The second type of event is DNA methylation, which causes silencing of a gene without affecting the gene sequence. "

cytochrome p450

CROET Faculty: Dennis R. Koop, PhD: "The cytochrome P450 system is essential in most organisms for the biosynthesis of endogenous compounds such as steroids, fatty acids, prostaglandins and pheromones. However, the largest number of P450 substrates are foreign chemicals that include drugs, plant metabolites, environmental pollutants and food additives. In general, the role of P450 is to detoxify chemicals and increase elimination from the body. However, in some cases the oxidized products can initiate cell toxicity, including chemical carcinogenesis, mutagenesis and teratogenesis. "

O radicals and guanine

Harvard Gazette: Repairing DNA damage: "During these searches, OGG1 finds millions of normal guanines for every aberrant one. It must be able to detect very subtle differences between good and bad, and the molecular snapshots taken by Verdine's group reveal how it does that.
OGG1 boasts a tiny pocket in its structure that acts as a trap. Normal guanine won't fit into the pocket. Mutated guanine, carrying an extra oxygen atom, fits smoothly.
That oxygen atom can cause problems. It may come from radiation received for treatment of cancer, breathing polluted air, or normal breakdown of food we eat. In the last case, the oxygen is usually converted to water, releasing life-giving energy in the process. But occasionally, a highly reactive form of oxygen escapes into the body, as can also happen when someone breathes polluted air or receives treatment with radiation.
Called 'free radicals,' these oxygen atoms react ferociously with anything nearby. Most of the molecules they attack are constantly being replenished, so no harm is done. But if they react with a base like guanine, problems occur. "

OGG1

Harvard Gazette: Repairing DNA damage: "As described in the April 1 issue of the scientific journal Nature, OGG1 moves up and down long segments of DNA looking for aberrations in a base known as guanine. If everything is normal, the enzyme moves on. But if it detects an abnormal mutation, or change, it removes the damaged part. Other enzymes then follow up and install a replacement. "

ATM Signaling and DNA Damage

ATM Signaling and DNA Damage: "The ATM protein kinase, the caretaker of the genome, plays an important role in this process of maintaining genomic integrity. In response to DNA damage, ATM and other checkpoint proteins initiate a complex signal transduction cascade to halt the cell cycle and facilitate repair.
This can involve different types of cellular machinery, depending on the nature of the lesion. Many of the phosphorylation events that are catalyzed by ATM are critical to the cell cycle arrest and the subsequent repair process."

p53 as anti-rheumatoid arthritis factor

Genome Biology | Full text | DNA-damage signaling and apoptosis: " Role for p53 as an anti-rheumatoid-arthritis factor, which suggests that it might perform a similar role in other hyper-proliferative diseases. Rheumatoid arthritis occurs when hyperplastic tissue invades and destroys joints. Reactive oxygen species, released at joints as a result of inflammatory damage, should normally result in p53-dependent apoptosis of synoviocytes (joint cells). Cells from the joints of rheumatoid arthritis patients have mutations in p53, however, suggesting a requirement for p53 in the efficient removal of hyperplastic tissue. Crucially, in a mouse model of rheumatoid arthritis, absence of p53 results in worse arthritis."

oncogene aa tumor suppressor

Genome Biology | Full text | DNA-damage signaling and apoptosis: "Unlike c-Abl which continuously shuttles between the nucleus and the cytoplasm, oncogenic forms of Abl, such as Bcr-Abl, are exclusively cytoplasmic. If Bcr-Abl is trapped in the nucleus, it activates apoptosis. Essentially, an oncogene has thereby been converted into a tumor suppressor. Furthermore, this observation establishes the principle that the decision to undergo apoptosis can be regulated by the subcellular localization of c-Abl."

Apoptosis and Caspase

Genome Biology Full text DNA-damage signaling and apoptosis: "Cytochrome c binds the apoptosis-activating factor 1 (Apaf1) protein, leading to oligomerization of Apaf1 and caspase 9 into a large 'apoptasome', which then initiates a cascade of caspase activation. Although some non-caspase targets of caspase activation are known, the consequences of proteolysis of these targets are not well understood. Similarly, the events upstream of activation of the caspase cascade in response to DNA damage are not well known; in particular, it is not clear what regulates the decision to undergo apoptosis or to arrest cell proliferation and repair the damage."

Three Peaks in SOS

Three New Phases of Repairing DNA Damage in E. coli: "Friedman et al. monitored the SOS response by attaching a green fluorescent protein (GFP) to the promoters (the section of DNA responsible for activating a gene) of three SOS genes (lexA, recA, and umuDC). Bacteria expressing these promoter-GFP fusions became fluorescent within minutes of being exposed to UV radiation, visualized using time-lapse fluorescence microscopy. Since GFP fluorescence is directly correlated with the expression of each of the chosen genes (i.e., their promoter activity), the authors could gauge the SOS response rate upon DNA damage.

To induce the SOS response, the authors exposed E. coli cells to UV radiation. By monitoring individual cells at two-minute intervals after this dose, Friedman et al. found up to three peaks of promoter activity at roughly 30, 60, and 100 minutes. Although the amount of this activity and the average number of peaks varied between cells, the timing was always similar in different cells, suggesting a highly structured, timed response. When the authors averaged this response over the population, it �washed out� into a single peak�which explains why the three peaks of expression were not previously detected.

A deeper look into the dynamics of the SOS response in single E. coli cells showed that it did not correlate with cell size, suggesting the SOS response is not synchronized with the cell cycle. In addition, Friedman et al. repeated their experiments in a bacterial strain lacking the SOS response gene umuDC. The peak pattern was altered in this mutant strain, and the precision in the appearance of the peaks was reduced. By re-examining the SOS response in single cells, Friedman et al. have visualized an accurately timed and synchronized DNA repair process. Modulations in response to DNA damage have also been observed recently in individua"

SOS

Three New Phases of Repairing DNA Damage in E. coli: "The E. coli SOS response has been used to study DNA repair for decades, and a great deal is known about how the more than 30 genes involved in the response function. Two proteins figure prominently in this response. The LexA protein acts as a repressor and inhibits the expression of SOS genes under normal conditions; in the event of DNA damage, the protein RecA inactivates the LexA repressor by enhancing its autocleavage into two fragments, which initiates the SOS response. While these initial stages are well understood, how all the SOS genes are coordinated, and ultimately turned off, is only beginning to be explored."

ATM

Newly discovered cellular process helps cells respond to DNA damage caused by radiation and environmental toxins: "A report on this discovery, published in the current issue of the journal Nature, describes this critical early step in a cell's response to DNA damage. This step, a chemical modification of an enzyme called ATM, allows the enzyme to initiate a series of events that ultimately halt the growth of a damaged cell and help the cell survive.

ATM is activated by a signal from damaged DNA only seconds after the damage occurs. The activated ATM, in turn, activates other proteins by attaching a molecule called “phosphate” to them in a process called phosphorylation. This sets off a cascade of biochemical reactions that amplifies the initial ATM response.

Among the proteins phosphorylated by ATM are Brca1 and p53. It was already known that these proteins play important roles in preventing cancer, and that mutated forms of Brca1 and p53 are responsible for inherited cancers, such as familial breast cancer."

T4 polynucleotide kinase and RNA repair

Structure and mechanism of T4 polynucleotide kinase: an RNA repair enzyme -- Wang et al. 21 (14): 3873 -- The EMBO Journal: "The use of bacteriophage T4 Pnk to label 5' DNA or RNA ends with 32P was instrumental in the development of methods for the analysis of nucleic acid structure, molecular cloning and nucleic acid sequencing. Although the historical importance of T4 Pnk in the recombinant DNA revolution is well known, it is less widely appreciated that T4 Pnk spearheads a pathway of �RNA repair� in vivo. During T4 infection, Pnk participates in an elaborate pathogen�host dynamic whereby the bacterium attempts to thwart T4 protein synthesis by inducing site-specific breakage of host-cell tRNAs, to which the phage responds by repairing the broken tRNAs using Pnk and a phage-encoded RNA ligase "

DNA Ligase and DNA Repair

Sloan-Kettering - Molecular Biology: Stewart Shuman: "ATP-dependent DNA ligases catalyze the joining of a 5'-phosphate-terminated strand to a 3'-hydroxyl-terminated strand via 3 sequential nucleotidyl transfer reactions. In the first step, ligase attacks the alpha-phosphorus of ATP to release PPi and form a covalent intermediate (ligase-adenylate) in which AMP is linked via a phosphoamide (P-N) bond to a lysine side chain on the enzyme. In the second step, the AMP is transferred to the 5'-end of the 5'-phosphate-terminated DNA strand to form a DNA-adenylate intermediate, A(5')pp(5')N. In the third step, ligase catalyzes attack by the 3'-OH of the nick on DNA-adenylate to join the 2 polynucleotides and release AMP.


ATP-dependent ligases are ubiquitous in eukaryotes; they are also encoded by certain eubacteria, bacteriophages, and eukaryotic DNA viruses. Sequence comparisons suggest that a catalytic domain common to all ATP-dependent ligases is embellished by additional isozyme-specific protein segments at the amino or carboxyl termini. The catalytic domain includes a set of 6 collinear motifs (I, III, IIIa, IV, V, and VI) that define a superfamily of covalent nucleotidyl transferases, encompassing the ATP-dependent polynucleotide ligases and GTP-dependent mRNA capping enzymes.


The nucleotide binding pocket of DNA ligase is composed of motifs I, III, IIIa, IV, and V. The lysine in motif I (KxDGxR) is the site of covalent attachment of AMP to the enzyme. Crystallography and mutagenesis have illuminated several of the enzymic functional groups that are involved in forming the ligase-adenylate intermediate."

Topoisomerase I Structure and Mechanism

Sloan-Kettering - Molecular Biology: Stewart Shuman: "The eukaryotic type IB topoisomerase family includes nuclear topo I and the topoisomerases encoded by vaccinia and other cytoplasmic poxviruses. These enzymes relax DNA supercoils by transiently breaking and rejoining 1 strand of the DNA duplex. They act via a common mechanism, involving a covalent DNA-(3'-phosphotyrosyl)-topo intermediate. The participation of type IB topoisomerases in DNA replication, genetic recombination, and transcription, plus the fact that nuclear topo I is the target of the camptothecin anti-tumor drugs, mandates a thorough understanding of their mechanism of action."

RNA triphosphatase

Sloan-Kettering - Molecular Biology: Stewart Shuman: "RNA triphosphatase hydrolyzes the gamma phosphate of mRNA. The triphosphatase component of the capping apparatus has diverged in structure and mechanism during the transition from fungal to metazoan species. The metazoan triphosphatases belong to a superfamily of phosphatases (which includes protein tyrosine phosphatases and dual-specificity protein phosphatases) that act via formation and hydrolysis of a cysteinyl phosphate intermediate; the reaction requires no metal cofactor. The yeast and poxvirus RNA triphosphatases comprise a novel family of metal-requiring phosphohydrolases that share several sequence motifs implicated in catalysis. "

T4 Pnk

Jay Bischoff presentation: "During T4 infection of a bacterial cell Pnk participates in an essential role to resist the bacterial attempts to stop T4 protein synthesis by inducing site-specific breakage of host cell tRNA�s. In turn the phage attempts to repair the broken tRNA�s using the Pnk and phage encoded RNA ligase."

RNA Ligase and Repair

UB Department of Biological Sciences: Kiong Ho: "RNA ligase participates in the repair, splicing and editing pathway of RNAs or in altering their primary structure. "

trans-splicing ribozymes : repair

Entrez PubMed: "Recent reports have demonstrated that trans-splicing ribozymes can be employed to repair mutant RNAs. One key factor that influences RNA repair efficiency is the accessibility of the substrate RNA for ribozyme binding, which is complicated by the fact that RNAs may assume multiple conformations and have proteins bound to them in vivo.

These results demonstrate that this novel RNA mapping strategy represents an effective means to determine the accessible regions of target RNAs and that combinations of trans-splicing ribozymes can be employed to enhance RNA repair efficiency of clinically relevant transcripts such as beta(s)-globin RNA."

RNA repair using spliceosome-mediated RNA trans-splicing.

Entrez PubMed: "RNA repair using spliceosome-mediated RNA trans-splicing."

Sparky RNA repair

RNA repair: "The researchers, led by Al George, used a dog model of myotonia congenita, a non-lethal condition that is characterized by muscle stiffness and caused by mutations in the chloride channels of skeletal muscle cells. Targeting a mutation discovered in an affected dog, they engineered a ribozyme that splices off the mutation-containing 3? section of the mRNA and replaces it with the wild-type sequence. This technique is called ribozyme-mediated trans-splicing. The ribozyme is produced as a DNA template, which is transfected into cells using a vector, in the same way as in standard gene therapy. The template is then transcribed within the cell to create the RNA ribozyme. In theory, the ribozyme is continuously transcribed and is over expressed, which should mean that enough is present to repair all mutant mRNA in the cell.

George's group used a plasmid vector to transfect their ribozyme into a cell line with impaired chloride transportation caused by the mutation, which was named Sparky after the dog in which it was identified.

These results offer the first direct proof that ribozymes can repair mutant mRNA to produce functional wild-type proteins."

FRET

Fluorescence Resonance Energy Transfer: "Fluorescence resonance energy transfer (FRET), a relatively new technique, that relies on transferring energy between donor and acceptor fluorescent tags on different parts of a molecule to reveal stunning details about the conformational changes molecules undergo during biological processes. FRET studies have revealed new information about how helicase moves along a DNA molecule as it unwinds the strands." The Ha Lab