Protein Identified That Regulates Cellular Trafficking, Potential for Anti-Cancer Therapy
Molecular microbiologists at the University of Southern California (USC) have uncovered intricate regulatory mechanisms within the cell that could lead to novel therapeutics for the treatment of cancer and other diseases. Their findings, which have long-standing significance in the basic understanding of cell biology, appear in the journal Nature Cell Biology.
“Our research reveals a new regulatory mechanism that coordinates two distinct intracellular processes that are critical to cellular homeostasis and disease development,” said Chengyu Liang, M.D., Ph.D., a member of the USC Norris Comprehensive Cancer Center and principal investigator of the study.
The endoplasmic reticulum (ER) and Golgi apparatus are cellular organelles in eurkaryotic organisms where proteins are synthesized and packaged for secretion through the body. The trafficking of proteins between the ER and Golgi must be tightly modulated to maintain the health of the cell and prevent diseases like cancer from taking hold.
“Interest in the role of ER-Golgi network during cancer cell death has been gaining momentum,” said Shanshan He, Ph.D., research associate at the Keck School of Medicine of USC and one of the study’s first authors. “In this study, we identified a novel regulatory factor for the Golgi-ER retrograde transport and a new mechanistic connection between the physiological trafficking and the autophagic transportation of cellular material.”
The researchers discovered that the UV irradiation resistance-associated gene protein (UVRAG), which has been implicated in the suppression of colon and breast cancer, coordinates trafficking of proteins between the ER and Golgi apparatus and also autophagy, the natural process of breaking down cellular components.
“Given that the ER-Golgi network is often dismantled in malignant conditions and that UVRAG is intensively involved in different types of human cancers, this study gives us a new avenue to investigate anti-cancer agents that target UVRAG and/or the ER-Golgi pathway in cancer and other relevant diseases,” Liang said.
source:http://www.sciencedaily.com/releases/2013/09/130922155124.htm
Novel gene discovery could lead to new HIV treatments
A team of researchers led by King’s College London has for the first time identified a new gene which may have the ability to prevent HIV, the virus that causes AIDS, from spreading after it enters the body.
Published in Nature today, the study is the first to identify a role for the human MX2 gene in inhibiting HIV. Researchers say this gene could be a new target for effective, less toxic treatments where the body’s own natural defence system is mobilised against the virus.
The work was funded by the Medical Research Council and the National Institute for Health Research (NIHR) Biomedical Research Centre at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London. The study was also supported by the Wellcome Trust and European Commission.
Scientists carried out experiments on human cells in the lab, introducing the virus to two different cell lines and observing the effects. In one cell line the MX2 gene was expressed or ‘switched on’, and in the other it was not, or ‘silenced’. They saw that in the cells where MX2 was silenced, the virus replicated and spread. In the cells where the MX2 gene was expressed, the virus was not able to replicate and new viruses were not produced.
The work was led by Dr Caroline Goujon and Professor Mike Malim at the Department of Infectious Diseases, King’s College London. Professor Malim said: “This is an extremely exciting finding which advances our understanding of how HIV virus interacts with the immune system and opens up opportunities to develop new therapies to treat the disease. Until now we knew very little about the MX2 gene, but now we recognise both its potent anti-viral function and a key point of vulnerability in the life cycle of HIV.
“Developing drugs to stimulate the body’s natural inhibitors is a very important approach because you are triggering a natural process and therefore won’t have the problem of drug resistance. There are two possible routes – it may be possible to develop either a molecule that mimics the role of MX2 or a drug which activates the gene’s natural capabilities.
“Although people with HIV are living longer, healthier lives with the virus thanks to current effective treatments, they can often be toxic for the body and drug resistance can become an issue with long-term use.
It is important to continue to find new ways of mobilising the body’s natural defence systems and this gene appears to be a key player in establishing viral control in people with HIV.”
Researchers identify a switch that controls growth of most aggressive brain tumor cells
DALLAS — Researchers at UT Southwestern Medical Center have identified a cellular switch that potentially can be turned off and on to slow down, and eventually inhibit the growth of the most commonly diagnosed and aggressive malignant brain tumor.
Findings of their investigation show that the protein RIP1 acts as a mediator of brain tumor cell survival, either protecting or destroying cells. Researchers believe that the protein, found in most glioblastomas, can be targeted to develop a drug treatment for these highly malignant brain tumors. The study was published online Aug. 22 in Cell Reports.
“Our study identifies a new mechanism involving RIP1that regulates cell division and death in glioblastomas,” said senior author Dr. Amyn Habib, associate professor of neurology and neurotherapeutics at UT Southwestern, and staff neurologist at VA North Texas Health Care System. “For individuals with glioblastomas, this finding identified a target for the development of a drug treatment option that currently does not exist.”
In the study, researchers used animal models to examine the interactions of the cell receptor EGFRvIII and RIP1. Both are used to activate NFκB, a family of proteins that is important to the growth of cancerous tumor cells. When RIP1 is switched off in the experimental model, NFκB and the signaling that promotes tumor growth is also inhibited. Furthermore, the findings show that RIP1 can be activated to divert cancer cells into a death mode so that they self-destruct.
According to the American Cancer Society, about 30 percent of brain tumors are gliomas, a fast-growing, treatment-resistant type of tumor that includes glioblastomas, astrocytomas, oligodendrogliomas, and ependymomas. In many cases, survival is tied to novel clinical trial treatments and research that will lead to drug development.
The Department of Neurology and Neurotherapeutics at UT Southwestern is ranked in the top 20 in the nation, according to U.S. News & World Report. UT Southwestern physicians routinely deal with the most difficult neurology cases referred from around the region, state, and nation.
The research was conducted with support from the National Institutes of Health, NASA, and the Cancer Prevention and Research Institute of Texas.
UT Southwestern investigators who participated in the study include former postdoctoral researcher Dr. Vineshkumar Puliyappadamba, senior research associate Dr. Sharmistha Chakraborty, former research assistant Sandili Chauncey, and senior research scientist Dr. Li Li, all from the Department of Neurology and Neurotherapeutics. Dr. Kimmo Hatanpaa, associate professor of pathology; Dr. Bruce Mickey, director of the Annette G. Strauss Center in Neuro-Oncology; Dr. David Boothman, professor of radiation oncology and pharmacology in the Harold C. Simmons Comprehensive Cancer Center; and Dr. Sandeep Burma, associate professor of radiation oncology, also contributed to the research.
source4:http://www.eurekalert.org/pub_releases/2013-09/usmc-ria092013.php
Bacteria can cause pain on their own
Microbes caused discomfort in mice by activating nervous system, not immune response
Mice with infected paws were most sensitive to being prodded when bacterial numbers were at their highest, not when the immune response was peaking
Bacteria can directly trigger the nerves that sense pain, suggesting that the body’s own immune reaction is not always to blame for the extra tenderness of an infected wound. In fact, mice with staph-infected paws showed signs of pain even before immune cells had time to arrive at the site, researchers report online August 21 in Nature.
“Most people think that when they get pain during infection it’s due to the immune system,” says coauthor Isaac Chiu of Boston Children’s Hospital and Harvard Medical School. Indeed, immune cells do release pain-causing molecules while fighting off invading microbes. But in recent years scientists have started uncovering evidence that bacteria can also cause pain.
Chiu and his colleagues stumbled on this idea when they grew immune cells and pain-sensing cells together in a dish. The researchers were trying to activate the immune cells by adding bacteria to the mix but were surprised to see an immediate response in the nerve cells instead. This made them suspect that nerve cells were sensing the bacteria directly.
To take a closer look at a real infection, the team injected the back paws of mice with Staphylococcus aureus, a bacterium that causes painful sores in humans. The researchers measured how tender the infected area was by poking it with flexible filaments of plastic. If the mouse didn’t like being prodded, it would lift its paw, giving a sensitive measure of each infection’s ouch factor.
The mice’s paws were most sensitive when bacterial cell numbers were at their peak, six hours after infection. By the time the immune response caught up, at 48 hours after infection, the pain had largely ebbed away. The researchers identified two protein factors released by S. aureus that could trigger nerve cells in dishes and that were also painful when injected into the mice.
These factors seemed to be more important than the immune system in making mouse paws achy, since mice with faulty immune responses were at least as tender as normal mice, the researchers report. However, pain from immune reactions might play a more significant role in other kinds of infections, Chiu cautions, since not all bacterial species are as good as S. aureus at evading the immune system.
The team guessed that the nerves were helping to alert the immune system to the presence of bacteria, but when they tested this idea, they got a surprise. “We saw the opposite of what we expected,” Chiu says. When the researchers infected mice that lacked pain-sensing nerve cells, even more immune cells rushed to the site of infection than in normal mice. This implies that the nerve cells normally suppress the immune system, Chiu says.
Chiu doesn’t know why pain should dampen the body’s defenses against pathogens but speculates that when tissue is damaged by injury, an overenthusiastic immune system may need to be held back. Bacteria like S. aureus might take advantage of pain’s anti-immune effects to avoid detection, he suggests, “but it’s an open question.”
Kevin Tracey, an immunologist and president of the Feinstein Institute for Medical Research in Manhasset, N.Y., says the results fit with his own studies that show nerve signals can put the brakes on immune responses. “It’s a beautiful study,” Tracey says. “It’s important because it shows that in order to understand the immune system, you really have to understand the nervous system.”
source:http://www.sciencenews.org/view/access/id/352641/description/Bacteria_Brings_Pain
RNA Structure and Function
DNA vs RNA
Diffen › Science › Biology › Microbiology
The main difference between DNA and RNA is the sugar present in the molecules. While the sugar present in an RNA molecule is ribose, the sugar present in a molecule of DNA is deoxyribose. Deoxyribose is the same as ribose, except that the former has one more OH.
DNA does not usually exist as a single molecule, but instead as a tightly-associated pair of molecules. These two long strands entwine like vines, in the shape of a double helix. This arrangement of DNA strands is called antiparallel. The asymmetric ends of DNA strands are referred to as the 5′ (five prime) and 3′ (three prime) ends. One of the major differences between DNA and RNA is the sugar, with 2-deoxyribose being replaced by the alternative pentose sugar ribose in RNA. The four bases found in DNA are adenine (abbreviated A), cytosine (C), guanine (G) and thymine (T). A fifth pyrimidine base, called uracil (U), usually takes the place of thymine in RNA and differs from thymine by lacking a methyl group on its ring.
Comparison chart EMBED THIS CHART
DNA RNA
Stands for: DeoxyriboNucleicAcid RiboNucleicAcid
Definition: A nucleic acid that contains the genetic instructions used in the development and functioning of all modern living organisms(scientists believe that RNA may have been the main genetic material in primitive life forms). A single-stranded chain of alternating phosphate and ribose units with the bases Adenine, Guanine, Cytosine, and Uracil bonded to the ribose. RNA molecules are involved in protein synthesis and sometimes in the transmission of genetic information.
Job/Role: Medium of long-term storage and transmission of genetic information Transfer the genetic code needed for the creation of proteins from the nucleus to the ribosome.
Unique Features: The helix geometry of DNA is of B-Form. DNA is completely protected by the body, i.e., the body destroys enzymes that cleave DNA. DNA can be damaged by exposure to Ultra-violet rays The helix geometry of RNA is of A-Form. RNA strands are continually made, broken down and reused. RNA is more resistant to damage by Ultra-violet rays.
Predominant Structure: Double- stranded molecule with a long chain of nucleotides A single-stranded molecule in most of its biological roles and has a shorter chain of nucleotides
Bases & Sugars: Deoxyribose sugar; phosphate backbone; Four bases: adenine, guanine, cytosine and thymine Ribose sugar; phosphate backbone. Four bases: adenine, guanine, cytosine, and uracil
Pairing of Bases: A-T(Adenine-Thymine), G-C(Guanine-Cytosine) A-U(Adenine-Uracil), G-C(Guanine-Cytosine)
Stability: Deoxyribose sugar in DNA is less reactive because of C-H bonds. Stable in alkaline conditions. DNA has smaller grooves, which makes it harder for enzymes to “attack” DNA. Ribose sugar is more reactive because of C-OH (hydroxyl) bonds. Not stable in alkaline conditions. RNA has larger grooves, which makes it easier to be attacked by enzymes.
Propagation: DNA is self-replicating. RNA is synthesized from DNA when needed.
DNA’s B Form, A Form and Z Form
In a DNA molecule, the two strands are not parallel, but intertwined with each other. Each strand looks like a helix. The two strands form a “double helix” structure, which was first discovered by James D. Watson and Francis Crick in 1953. In this structure, also known as the B form, the helix makes a turn every 3.4 nm, and the distance between two neighboring base pairs is 0.34 nm. Hence, there are about 10 pairs per turn. The intertwined strands make two grooves of different widths, referred to as the major groove and the minor groove, which may facilitate binding with specific proteins.
The normal right-handed “double helix” structure of DNA, also known as the B form.
In a solution with higher salt concentrations or with alcohol added, the DNA structure may change to an A form, which is still right-handed, but every 2.3 nm makes a turn and there are 11 base pairs per turn.
Another DNA structure is called the Z form, because its bases seem to zigzag. Z DNA is left-handed. One turn spans 4.6 nm, comprising 12 base pairs. The DNA molecule with alternating G-C sequences in alcohol or high salt solution tends to have such structure.
Comparison between B form and Z form.
Bacteriological Media
| Chapter 5-Bacteriological Media |
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The role of suitable quality culture media for cultivation of microorganisms cannot be over emphasised. On it depends the very success of isolation of aetiological agents. Only in exceptional cases, can an organism be identified on the basis of its morphological characteristics alone.
Types of media Bacteriological media can be broadly sub-divided into four categories. 1. Ordinary culture media These are routinely employed in a laboratory e.g. nutrient broth, nutrient agar, infusion broth and lysate media. 2. Enriched media Certain organisms do not grow on ordinary nutrient media. They require growth- promoting ingredients such as blood, glucose, serum, egg, etc. The media containing ingredients which enhance their growth-promoting qualities are enriched media e.g. blood agar, chocolate agar and Loeffler medium. 3. Enrichment media Enrichment media are liquid media containing chemical constituents which inhibit some normal flora and allow pathogens which may be present in very small number in the specimen, to grow unhampered and thus enriching them. Isolated colonies of these organisms may be obtained by subculturing onto solid media. An example of enrichment media is selenitebroth used for primary isolation of enteric bacteria. 4. Differential and selective media Differential media have got some chemical constituents which characterize different bacteria by their special colonial appearances in the culture e.g. MacConkey agar contains lactose as a substrate and neutral red as an indicator. Bacteria fermenting lactose produce acid and this will change the colour of the indicator and thus the colonies will turn red. The red lactose fermenting colonies can be differentiated from the pale non-lactose fermenting colonies. Selective media will selectively permit the growth of pathogens and inhibit the commensals. In addition, it may differentiate the pathogen from commensals that grow by the colour and opacity of the colonies e.g. blood tellurite medium for C.diphtheriae. In addition, transport media are also frequently used to sustain the viability of organisms when a clinical specimen is to be transported from the periphery to laboratory. The transport medium prevents the outgrowth of contaminants during transit and sustains the pathogen. Cary and Blair and Stuart media are two examples of this group of media. Preparation of media and checking of pH Presently, a wide range of culture media are available commercially in the form of dehydrated media. These media are simply reconstituted by weighing the required quantities and by adding distilled water, as per the manufacturer’s instructions. The pH determination can be conveniently done with the use of Lovibond comparator with phenol red indicator disc. Take two clean test tubes and add 5 ml of the medium to each of the tubes. One serves as a blank while phenol red indicator is added to the other tube. Compare the colour of the medium with the phenol red indicator at the appropriate pH marking. Add N/10 NaOH or N/10 HCl, drop by drop till the colour of the medium matches the colour of the disc at the required pH reading. Calculate the volume of the NaOH or HCL of 1/10 strength for 5 ml of the medium to get the required pH. Based on the calculation, the volume of 1N NaOH or IN HCl required for the total volume of medium can be calculated and added. Check the pH of the medium once again before use. The quantity of agar given in the formulae of media may have to be changed depending upon the quality of agar used. The concentration varies from batch to batch and should be such that will produce a sufficiently firm surface on solidification. This can be tested by streaking with inoculating wire. In some laboratories media are prepared by individual measurement of ingredients and then mixing the same. Hence the method of preparation is given likewise: Nutrient broth Meat extract 10.0 gm Mix the ingredients and dissolve them by heating in a steamer. When cool, adjust the pH to 7.5-7.6. Nutrient agar To the ingredients as in nutrient broth, add 15 gm agar per litre. Dissolve the agar in nutrient broth and sterilize by autoclaving at 121oC for 15 minutes. Prepare plates and slopes as required. Glucose broth Nutrient broth 900 ml Dissolve 9 gm glucose in distilled water and sterilize by tyndallisation. Add l00 ml of the glucose solution to 900 ml of sterile nutrient broth. Dispense 60 ml each in 100 ml pre-sterilized culture bottles. Sterilize by open steaming at l00oC for one hour.
Blood agar Nutrient agar 100 ml Melt the sterile nutrient agar by steaming, cool to 45oC. Add required amount of sheep blood aseptically with constant shaking. Mix the blood with molten nutrient agar thoroughly but gently, avoiding froth formation. Immediately pour into petri dishes or test tubes and allow to set.
Chocolate agar
The ingredients are essentially the same as in blood agar. Melt the sterile nutrient agar by steaming and cool to about 75oC. Add blood to the molten nutrient agar and allow to remain at 75oC after gently mixing till it is chocolate brown in colour. Pour in petri dishes or test tubes for slopes as desired.
XLD agar
Xylose 3.5 gm Weigh the ingredients into a flask and add distilled water. Mix the contents well and steam it for 15 minutes (do not autoclave). Cool to 56oC and pour in plates. Buffered glycerol saline Glycerol 300 ml Dissolve NaCl in water and add glycerol. Add disodium hydrogen phosphate to dissolve. Add phenol red and adjust pH to 8.4. Distribute 6 ml in universal containers (screw -capped bottles of 30 ml capacity). Autoclave at 115oC for 15 minutes.
Loeffler serum medium
Nutrient broth 100 ml Dissolve glucose in nutrient broth and sterilize at 121oC for 15 minutes. Add serum aseptically. Mix thoroughly but gently, avoiding froth formation. Distribute in sterile test tubes or quarter ounce screw-cap bottles. Inspissate the medium in a slanting position in a water inspissator at 82oC for two hours. In the absence of an inspissator, the medium may be coagulated by standing over the top of a steam sterilizer for 6-7 minutes.
Blood tellurite agar
Agar base Meat extract 5.0 gm Dissolve the ingredients and adjust the pH to 7.6. Distribute in 100 ml quantities in a bottle and autoclave at 121oC for 15 minutes. Glycerolated blood tellurite mixture Sterile defibrinated sheep blood 14 ml Sterilize the glycerol in hot air oven at 160oC for 60 minutes and the tellurite solution by autoclaving at 115oC for 20 minutes. Mix the ingredients in a sterile flask, incubate for 1-2 hrs.at 37oC, then refrigerate. Haemolysis is complete after 24 hrs. The mixture keeps well in a refrigerator. One per cent solution of good quality tellurite is sufficient but 2% of some batches may be required. Preparation of complete medium Glycerolated blood tellurite mixture 24 ml Melt the agar, cool to 45oC, add blood and tellurite and pour in sterile petri dishes. |
DNA Replication and Repair
Introduction
DNA replication is the process in which genetic material that is encoded in a DNA sequence is copied so that it can be passed to new cells and offspring through. For humans, DNA replication is the reason we bear similarities to our parents and relatives.
While the aim of replication is to yield an identical copy of the original DNA, errors in the process make this impossible and lead to mutations. Although these mutations can be fatal to cells, many are not and act as a means of diversifying cell offspring. The replication process is essential for cell growth and reproduction.
We will begin our discussion by looking globally at how DNA replication occurs and what types of mutations commonly occur. We will then look more specifically at the molecules responsible for facilitation of replication and at the chemical mechanisms behind the process. Finally, we will look at how the built-in repair systems that DNA uses to produce a faithful copies maintain low mutation rates.
Terms
2′ deoxyribonucleoside triphosphate – The building blocks of DNA replication. A five-membered, oxygen-containing ribose sugar ring that has three phosphate groups attached to its 5′ carbon and either an adenine, cytosine, guanine, or thymine base group attached to its 1′ carbon.
Base-pair excision – One class of DNA repair system. Recognizes and removes single nucleotide mutations that result from unnatural bases.
Daughter strand – Refers to the newly synthesized strand of DNA that is copied via the addition of complementary nucleotides from one strand of pre-existing DNA during DNA replication.
DNA Helicase – The enzyme responsible for separating the two strands of DNA in a helix so that they can be copied during DNA replication.
DNA Ligase – The enzyme responsible for sealing together breaks or nicks in a DNA strand. Responsible for patching together Okazaki fragments on the lagging strand during DNA replication.
DNA Polymerase – The enzyme responsible for catalyzing the addition of nucleotide substrates to DNA both during and after DNA replication.
Primase – The enzyme responsible for initiating synthesis of RNA primers on the lagging strand during DNA replication.
Holoenzyme – A term used to describe a collection of different enzymes that work together in a given process such as DNA replication.
Hydrolysis – The process in which water is chemically added to a molecule.
Lagging strand – In DNA replication, the strand of pre-existing DNA that is oriented in the 5′ to 3′ direction with respect to the direction of replication on which synthesis is discontinuous.
Leading strand – In DNA replication, the strand of pre-existing DNA that is oriented in the 3′ to 5′ direction with respect to the direction of replication on which replication is continuous.
Mismatch repair – One class of DNA repair system. Recognizes and removes mutations that result from base-pairing that is not complementary.
Okazaki fragment – Short stretches of newly synthesized DNA found on the lagging strand during DNA replication.
Origin of replication – Site of initiation of DNA replication. Short, usually internal stretch in a DNA helix that opens so that each strand is separate for DNA replication.
Parent strand – In DNA replication, refers to the pre-existing single strand of DNA that is copied into a new strand of DNA via complementary base pairing.
Pyrophosphate – A two phosphate-containing molecule. In DNA replication, it is released from a 2′ deoxyribonucleoside triphosphate during its addition to a growing, newly synthesized DNA strand. Its subsequent hydrolysis provides the energy for the addition reaction.
Replication fork – Term used to describe the junction at which nucleotide substrates are being added to a growing DNA chain during DNA replication. Its shape resembles a “Y” where the two branches represent single stranded daughter strands of DNA and the base represents helical DNA.
RNA Primer – Short stretches of ribonucleotides (RNA substrates) found on the lagging strand during DNA replication. Helps initiate lagging strand replication and are later removed.
Semi-conservative – Refers to the fact that after the replication of one DNA helix each of the two daughter helices that result contain one newly-synthesized and one pre- existing strand of DNA.
Short-patch excision – One class of DNA repair system. Recognizes and removes short stretches of DNA that surround mutations resulting from large adducts on a DNA strand that impede DNA replication.
Single-stranded binding protein – A protein involved in helping to keep strands of DNA that have been separated by DNA helicase from recoiling in a helix. It works by coating the single strands in such a way as not to cover the bases, allowing them to remain free for base pairing.
Thymine dimer – A form of DNA damage that results from radiation. Adjacent thymines on the same strand of DNA form a bond that results in a bulky adduct that can impede DNA replication.
Tautomerization – A process in which a molecule undergoes an electron rearrangement that results in a slightly different organization of the same molecule. The two forms of the same molecule are called “tautomers” of each other.
DNA Replication
DNA Replication is Semi-Conservative
DNA replication of one helix of DNA results in two identical helices. If the original DNA helix is called the “parental” DNA, the two resulting helices can be called “daughter” helices. Each of these two daughter helices is a nearly exact copy of the parental helix (it is not 100% the same due to mutations). DNA creates “daughters” by using the parental strands of DNA as a template or guide. Each newly synthesized strand of DNA (daughter strand) is made by the addition of a nucleotide that is complementary to the parent strand of DNA. In this way, DNA replication is semi-conservative, meaning that one parent strand is always passed on to the daughter helix of DNA.
Daughter helices
Replication Forks and Origins of Replication
The first step in DNA replication is the separation of the two DNA strands that make up the helix that is to be copied. DNA Helicase untwists the helix at locations called replication origins. The replication origin forms a Y shape, and is called a replication fork. The replication fork moves down the DNA strand, usually from an internal location to the strand’s end. The result is that every replication fork has a twin replication fork, moving in the opposite direction from that same internal location to the strand’s opposite end. Single-stranded binding proteins (SSB) work with helicase to keep the parental DNA helix unwound. It works by coating the unwound strands with rigid subunits of SSB that keep the strands from snapping back together in a helix. The SSB subunits coat the single-strands of DNA in a way as not to cover the bases, allowing the DNA to remain available for base-pairing with the newly synthesized daughter strands.
when the two parent strands of DNA are separated to begin replication, one strand is oriented in the 5′ to 3′ direction while the other strand is oriented in the 3′ to 5′ direction. DNA replication, however, is inflexible: the enzyme that carries out the replication, DNA polymerase, only functions in the 5′ to 3′ direction. This characteristic of DNA polymerase means that the daughter strands synthesize through different methods, one adding nucleotides one by one in the direction of the replication fork, the other able to add nucleotides only in chunks. The first strand, which replicates nucleotides one by one is called the leading strand; the other strand, which replicates in chunks, is called the lagging strand.
The Leading and Lagging Strands
The Leading Strand
Since DNA replication moves along the parent strand in the 5′ to 3′ direction, replication can occur very easily on the leading strand. As seen in , the nucleotides are added in the 5′ to 3′ direction. Triggered by RNA primase, which adds the first nucleotide to the nascent chain, the DNA polymerase simply sits near the replication fork, moving as the fork does, adding nucleotides one after the other, preserving the proper anti-parallel orientation. This sort of replication, since it involves one nucleotide being placed right after another in a series, is called continuous.
The Lagging Strand
Whereas the DNA polymerase on the leading strand can simply follow the replication fork, because DNA polymerase must move in the 5′ to 3′ direction, on the lagging strand the enzyme must move away from the fork. But if the enzyme moves away from the fork, and the fork is uncovering new DNA that needs to be replicated, then how can the lagging strand be replicated at all? The problem posed by this question is answered through an ingenious method. The lagging strand replicates in small segments, called Okazaki fragments. These fragments are stretches of 100 to 200 nucleotides in humans (1000 to 2000 in bacteria) that are synthesized in the 5′ to 3′ direction away from the replication fork. Yet while each individual segment is replicated away from the replication fork, each subsequent Okazaki fragment is replicated more closely to the receding replication fork than the fragment before. These fragments are then stitched together by DNA ligase, creating a continuous strand. This type of replication is called discontinuous.
As you can see in the figure above, the first synthesized Okazaki fragment on the lagging strand is the furthest away from the replication fork, which is itself receding to the right. Each subsequent Okazaki fragment starts at the replication fork and continues until it meets the previous fragment. The two fragments are then stitched together by DNA ligase.
In figure above, we can also see how replication on the lagging strand remains slightly behind that on the leading strand. Because synthesis on the lagging strand takes place in a “backstitching” mechanism, its replication is slightly delayed in relation to synthesis on the leading strand. The lagging strand must wait for a patch of the parent helix to open up a short distance in front of the newly synthesized strand before it can begin its synthesis back to the end of the daughter strand. This “lag” time does not occur in the leading strand because it synthesizes the new strand by following right behind as the helix unwinds at the replication fork.
Another complication to replication on the lagging strand is the initiation of replication. Whereas the RNA primer on the leading strand only has to trigger the initiation of the strand once, on the lagging strand each individual Okazaki fragment must be triggered. On the lagging strand, then, an enzyme called primase that moves with the replication fork synthesizes numerous RNA primers, each of which triggers the growth of an Okazaki fragment. The RNA primers are eventually removed leaving gaps that are filled by the replication machinery.
Problems
Problem : Why is DNA replication called “semi-conservative”?
DNA replication is semi-conservative because each helix that is created contains one strand from the helix from which it was copied. The replication of one helix results in two daughter helices each of which contains one of the original parental helical strands. It is semi- conservative because half of each parent helix is conserved in each daughter helix.
Problem : The number of replication forks in a DNA helix is often an even number. Explain this finding.
Since replication origins are usually not found at the end of a helix, but rather internally to a helix, one replication origin leads to the formation of two replication forks. Replication forks, thus, are usually found in pairs.
Problem : In what direction (5′ to 3′ or 3′ to 5′) does biological DNA replication take place?
Biological DNA replication ALWAYS takes place in the 5′ to 3′ direction.
Problem : The strand on which DNA replication is continuos is called the ___________. The strand on which DNA replication is discontinuous is called the ___________.
Leading strand. Lagging strand.
Problem : Replication on the lagging strand is mediated by small segments of nucleotides called what?
Okazaki fragments.
The Chemistry of the Addition of Substrates of DNA Replication
While the leading strand and lagging strand replicate differently, each individual nucleotide added to each strand is attached through the same mechanism. In this section, we will examine the mechanism of nucleotide attachment. Note: classes such as AP Biology do not require you to know the topics covered in this section.
The Building Blocks of DNA Replication are Deoxyribonucleotides
The building blocks added on to a growing daughter strand are individual nucleotides. Remember, in DNA the -OH group at the 2′ position of the ribose ring is missing. As a result, the substrates for DNA synthesis are called 2′ deoxyribonucleotides.
Figure %: 2′ Deoxyribonucleoside triphosphate
Attached to each deoxyribose ring is a base group (C, G, A, or T) and a triphosphate group. The three phosphates are designated alpha, beta, and gamma (alpha being the closest to the ribose ring). These phosphates play key roles in the addition of subsequent nucleotides to the daughter strand.
Addition Occurs Via a Nucleophilic Attack
Deoxyribonucleoside triphosphates, as we just stated, are the building blocks of DNA. Recall, furthermore, that a complete polynucleotide strand of DNA has only one phosphate group and that through this phosphate group each nucleotide is attached to the next. Why then is the substrate a triphosphate instead of just a monophosphate? The answer to this question lies in the chemistry underlying the addition of nucleotides to a growing daughter strand of DNA.
While each nucleotide added to a growing DNA chain lacks an -OH group at its 2′ position, it retains its 3′ -OH. This hydroxyl group is used to attack the alpha phosphate group of an incoming nucleoside triphosphate. In the attack, the 3′ -OH replaces the beta and gamma phosphates that are ejected from the complex as a pyrophosphate molecule. The result is the formation of the phosphodiester bond between the growing daughter strand and the next nucleotide. The 3′ -OH of the newly added nucleotide is now exposed on the end of the growing chain and can attack the next nucleotide in the same way.
Figure %: Addition of Nucleotides to a Growing Daughter Strand
The figure above presents a simplified schematic of a growing polynucleotide chain. The lines represent the ribose sugar with one 3′ -OH branching from it. Each p represents a phosphate group. This figure illustrates a number of key points of DNA replication. First, we see that the parent strand is oriented in the 3′ to 5′ direction. Second, each new nucleotide added to the growing daughter strand is complementary to the nucleotide on the parent strand that is across from it and a bond forms between them. Finally, we see how the 3′ -OH group displaces the two outermost phosphate groups of an incoming nucleotide in order to add it to the growing chain.
The Driving Force of the Addition Reaction
Each incoming nucleotide supplies the energy for its addition in the high-energy bond between the beta and gamma phosphates that are ejected upon addition. It is not the release of the pyrophosphate that drives the reaction, but rather the subsequent hydrolysis that takes place. A much larger amount of energy is released when the two phosphates are separated into individual phosphates through the hydrolysis reaction.
Problems →
Problem : What dictates which nucleoside triphosphate will be added next to a growing DNA chain?
The chemical addition of nucleotides to a growing DNA chain is dictated by the parent strand that is being copied. A nucleoside triphosphate with a complementary base to the one on the parent strand will be added to a growing chain.
Problem : What side group on a nucleoside triphosphate (building block of DNA) is responsible for mediating the addition of the next nucleotide?
The addition of nucleotides occurs through a nucleophilic attack by the 3′ –OH on the deoxyribose sugar group of a nucleotide located at the end of a growing DNA chain.
Problem : What provides the energy for the addition of nucleotides to a growing DNA chain during replication?
During the addition reaction, a pyrophosphate group is released from the nucleoside triphosphate being added. The pyrophosphate’s subsequent hydrolysis provides the energy that drives the addition reaction.
Problem : True or False. DNA replication occurs in the 5′ to 3′ direction because 3′ to 5′ replication is chemically impossible. Explain your answer.
False. While DNA replication does occur in the 5′ to 3′ direction, the reason is not because 3′ to 5′ replication is chemically impossible. 3′ to 5′ replication can in principle occur. The 3′ –OH of the incoming nucleotide instead of the nucleotide attached to the end of the growing chain would be the attacking group.
Problem : Why is lagging strand replication more complicated than leading strand replication?
Lagging strand synthesis is more complex because of the requirement for DNA replication to take place in the 5′ to 3′ direction. Since parent strands are oriented in an anti-parallel fashion, one strand is oriented in the 5′ to 3′ direction, while the other strand is oriented in the 3′ to 5′ direction. Synthesis on the strand that is oriented in the 3′ to 5′ (leading strand) direction can occur easily because replication simply begins at the 3′ end (synthesizing the 5′ end of the daughter strand) and continues along with the replication fork. Synthesis on the strand that is oriented in the 5′ to 3′ direction (lagging strand) can not follow the direction of the replication fork because that would lead to 3′ to 5′ synthesis of the daughter strand. Instead synthesis must occur in small segments to preserve the proper synthesis direction.
DNA Proof-Reading and Repair
Errors in DNA Replication
The low overall rate of mutation during DNA replication (1 base pair change in one billion base pairs per replication cycle) does not reflect the true number of errors that take place during the replication process. The number is kept so low by a proof-reading system that checks newly synthesized DNA for errors and corrects them when they are found. Errors in DNA replication can take different forms, but usually revolve around the addition of a nucleotide with the incorrect base, meaning the pairing between the parent and daughter strand bases is not complementary. The addition of an incorrect base can take place by a process called tautomerization. A tautomer of a base group is a slight rearrangement of its electrons that allows for different bonding patterns between bases. This can lead to the incorrect pairing of C with A instead of G, for example.
Figure %: Tautomerization of Cytosine
DNA retains its high level of accuracy is with its proof-reading function.
The 3′ to 5′ Proof-Reading Exonuclease
The 3′ to 5′ proof-reading exonuclease works by scanning along directly behind as the DNA polymerase adds new nucleotides to the growing strand. If the last nucleotide added is mismatched, then the entire replication holoenzyme backs up, removes the last incorrect base, and attempts to add the correct base again. The enzyme is “3′ to 5′” because it scans in the opposite direction of DNA replication, which we learned must always be 5′ to 3′. The mechanism of the proof-reading system offers an explanation as to why DNA replication must occur in this direction.
Keeping in mind the chemical mechanism we learned for the addition of nucleotides to the growing DNA strand, imagine what happens when the proof- reading system removes an incorrectly paired base. The exonuclease removes the base by cleaving the phosphodiester bond that had just been formed. In 5′ to 3′ synthesis, this leaves the 3′ -OH still attached to the terminal end of the growing strand ready to attack another nucleotide.
Figure %: 3′ to 5′ Exonuclease Action
If synthesis occurred in the opposite direction, the terminal end of a growing chain would contain a triphosphate group instead of an -OH group. This triphosphate would become the target of the proof-reading exonuclease and its removal would halt DNA replication.
Figure %: Incorrect 3′ to 5′ Synthesis
Types of DNA Damage
After DNA has been completely replicated, the daughter strand is often not a perfect copy of the parent strand it came from. Mutations during replication and damage after replication make it necessary for there to be a repair system to fix any errors in newly synthesized DNA. There are three main sources of damage to DNA.
Attack by water which can lead to the removal of an amine group from the base group of a nucleotide or the loss of the entire base group.
Chemical damage that permanently alters the structure of the DNA.
Radiation damage which can lead to nicks in the backbone of DNA or the formation of thymine dimers, which will be discussed later.
These different sources of damage lead to different categories of DNA damage. The damage that is caused by water attack can lead to unnatural bases. Chemical and radiation damage leads to the formation of bulky adducts to, or breaks in, the growing DNA strand. In the previous section we discussed the 3′ to 5′ proof-reading exonuclease that is responsible for fixing mismatches. Because it is not a perfect system, it can miss mismatched bases. As a result, a third category of DNA damage is mismatched bases.
Because these categories of DNA damage are different, there is a need for multiple repair systems.
Excision Repair System
The first type of repair system we will discuss is the excision repair system. To excise simply means to remove, so this repair systems works by removing the area of damage. Special enzymes recognize damaged DNA. This repair system comes in two forms: Base-excision repair and short-patch nucleotide excision.
Base-pair Excision Repair
In base-pair excision, single base-pairs are identified and removed. The resultant gap is then filled with a DNA polymerase and the nick is sealed by a DNA ligase.
Short-patch Excision Repair
Short-patch excision varies from base-pair excision in that its enzymes will recognize and remove “short patches” of DNA that are damaged. These short patches of damage arise from bulky lesions such as thymine dimers. This form of damage is radiation-induced and leads to the formation of a bond between adjacent thymine bases on the same strand of DNA. This bond leads to a distortion in the DNA that makes a short stretch around the thymine dimer unable to base pair correctly. The short-patch excision repair system recognizes such distortions and cuts the damaged strand on both sides of the damaged region leaving a 12 base pair gap in the strand. A helicase then unwinds the stretch of the helix with the damage that can then be filled and sealed with DNA polymerase and ligase. The short-patch excision repair can also be used to correct damage resulting from unnatural bases.
Repair of Mismatched Bases
The other major type of repair systems corrects damage resulting from mismatched bases, called the mismatch repair system. The mismatch repair system is able to identify mismatch errors because such damage leads to a small distortion in the DNA backbone. Once it has identified a mismatched base pair, it marks the spot with a cut and then uses an exonuclease to digest or “eat up” the DNA at the marker. A DNA polymerase can then fill in the gap with the appropriate base.
There is one major question that remains: How does the mismatch repair system know the difference between strand contains the correct base and the one on which it should make the incision? The way that it tells the two strands apart is by a marker that is added to the parent strand during replication. An additional methyl (-CH3) added to the adenine base groups of the parent strand and acts as a flag for the mismatch repair system so that it knows to make its cuts on the opposite strand.
Methylated Adenines Found on the Parent Strand Only
Problems
Problem : How does the 3′ to 5′ proof-reading exonuclease work?
The proof-reading exonuclease scans the newly synthesized DNA strand in the opposite direction of DNA replication for errors in base pairing. When it finds an error, it cuts the incorrect base pair from the newly synthesized strand out and the entire replication holoenzyme backs up and attempts to introduce the correct base all over again.
Problem : Where does the exonuclease cut the daughter strand to remove an improperly paired base?
The exonuclease cleaves the phosphodiester bond that is located between the phosphate group of the incorrect base and the 3′ –OH of the previous base on the daughter strand.
Problem : What are the three main sources of DNA damage?
The three main sources are hydrolysis, chemical damage, and radiation damage.
Problem : Thymine dimer mutations are corrected by what DNA repair system?
Short-patch excision repair system.
Problem : In the mismatch repair system, how does the exonuclease distinguish which base is the correct one?
Adenine bases that are located on the original or parent DNA strand are methylated during replication. When the mismatch repair exonuclease finds a mismatched base pair, it removes the base that is on the strand that lacks methylated adenines.
source:http://www.sparknotes.com/biology/molecular/dnareplicationandrepair/summary.html
Central Dogma
Central Dogma
The following diagram displays the flow of genetic information from DNA to the protein. This can be interpreted in genetic terms by saying that information contained in genes (DNA) is eventually expressed as the phenotype (protein).
Compartmentalization of the Central Dogma of Molecular Genetics
The organelles of plants are both of a prokaryotic (chloroplast and mitochondria) and eukaryotic (nucleaus) origin. The organelles contain DNA as well as the nucleus. Also, found within each organelle are all of the steps of the central dogma of molecular genetics. Reverse transcription has not been demonstrated in the organelles, but plant mitochondria undergo mRNA editing, a feature not known for nuclear expressed genes.
http://www.ndsu.edu/pubweb/~mcclean/plsc731/dna/dna1.htm
source:
Double helix DNA
DNA Structure
Watson and Crick Model of DNA
The following are the features of the DNA molecule as described by Watson and Crick in 1953.
2 chains
purine opposite a pyrimidine
chains held together by H-bonds
Guanine is paired with cytosine by three H-bonds
Adenine is paired with thymine by two H-bonds
anti-parallel orientation of the two chains
5′—————>3′
3′<—————5'
the molecule is stabilized by:
large # of H-bonds
hydrophobic bonding between the stacked bases
Components of DNA
DNA is composed of two chains of repeating nucleotides. Each nucleotide consists of three components. These components are:
Phosphate Group
2-deoxyribose sugar
A nitrogen containing base
cytosine
adenine
guanine
thymine
Types of DNA
The DNA molecule that Watson and Crick described was in the B form. It is now known that DNA can exist in several other forms. The primary difference between the forms is the direction that the helix spirals.
A, B, C = right-handed helix
Z = left-handed helix (found in vitro under high salt)
B is the major form that is found in the cell. Z-DNA was initially found only under high salt conditions, but the cellular environment is actually a low-salt environment. The question then is whether type Z exist under cellular conditions. Several features have been discovered that can stablize Z-DNA under in a low salt environment.
Alternating purine/pyrimidine tracts
poly GC or poly AT stretchesvvv
5-methyl-cytosine
Because both of these conditions can exists in the cell, it is suggested that stretches of Z-DNA may actually exists in the cell along with other stretches of B-DNA.
In addition to the direction the molecule turns, several other differences exists between the various forms of DNA. The following table summarizes the features of the different forms of DNA.
Form Direction Bases/
360o Turn Helix
Diameter
A Right 11.0 23A
B Right 10.0 19A
C Right 9.3 19A
Z Left 12.0 18A
source: http://www.ndsu.edu/pubweb/~mcclean/plsc731/dna/dna4.htm
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