Cellular and Molecular Biology Topics
DNA Synthesis
DNA replication requires a template (provides sequence information) and a primer with a free 3’ hydroxyl. It is a semi-conservative process: parental strands separate and each acts as a template for synthesis of a new strand. Newly synthesized DNA has one old parental strand and one new daughter strand. Synthesis goes from 5' to 3’. Deoxynucleotides (dNTPs) are added to the 3’ end of the growing chain.
Large macromolecular complexes are involved in unwinding and separating the strands, correcting errors, and other steps to insure accuracy. DNA replication must be accurate and regulated. Even a single base pair change can have serious consequences like disease or death. Cells need to replicate all their DNA only once per cell division. Failure to replicate all DNA or replicating portions more than once can lead to cell death or problems with gene expression.
A primer is added before synthesis can start. In eukaryotes, the primer is added by an enzyme that is part of the polymerase complex, while in prokaryotes the primer is added by primase.
3’-Exonuclease proofreads the newly added nucleotide. When the correct nucleotide is added, the daughter strand is a good substrate for DNA polymerase, but a poor substrate for 3’-endonuclease. When a mismatched nucleoside is added, the daughter strand becomes a poor substrate for polymerase and 3’exonuclease removes the newly added nucleoside before the next one is added.
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Back to Basics: Biochemistry
The Replication Fork
The section of the DNA molecule to be replicated is unwound by topoisomerase and the parental strands are separated by helicase, forming a replication fork. Single-strand binding proteins (SSBs) attach to the separated strands to keep them from rejoining. A clamp protein binds to the parental strand and binds to DNA polymerase allowing it to add nucleotides.
The new strands are antiparallel, yet the fork moves with both sides being replicated almost simultaneously. While one strand is continuously replicated, the other undergoes semi continuous replication. The continuous strand is known as the leading strand and the semi continuous as the lagging or retrograde strand. The initial pieces of the retrograde strand are known as okazaki fragments. After each fragment is completed, the primer is hydrolyzed by polymerase I in E. coli or RNase H in eukaryotes. The gap between two fragments is filled with one nucleotide by polymerase, leaving a nick, i.e. a missing phosphodiester bond that is later closed by DNA ligase.
DNA synthesis is initiated at specific regions called ori (origins). Prokaryotic chromosomes have only one ori, while eukaryotic chromosomes have many ori in tandem. Synthesis proceeds bi-directional away from the ori via two growing forks moving in opposite directions. This movement produces a replication bubble. When replication bubbles meet, a termination event occurs and the duplicate chromosomes are ready to separate.
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Back to Basics: Biochemistry
Cell Cycle
The eukaryotic cell cycle consists of a mitotic (M) phase, first gap (G1) phase,
DNA synthesis (S), and a second gap (G2) phase. Cells
do not initiate replication until conditions are favorable, for example having
all the materials needed for replication. Cofactors regulate replication so
that one piece of DNA is replicated only once, and that all the DNA is replicated
in a particular cell cycle.
The first protein needed for regulation is the ori recognition complex (ORC) which binds to the ori sequence. Next the mini chromosome maintenance (MCM) protein binds to the ori associated with ORC. After an appropriate signal is received (involving cell cycle regulation kinases) the initiation complex becomes active. The complex is assembled during G1 but does not become active until the cell cycle signal is received. Then topoisomerases and helicase unwind and separate the strands to make a small bubble and start replication.
Tumors are characterized by excessive cell division, therefore a larger fraction of cells are in S phase than in the normal tissue from which it arose. Pathological examination can detect the larger number of cycling cells in cancer tissues. Many chemotherapy agents attack cycling cells , particularly the process of DNA replication. These drugs include antimetabolites that interfere with the synthesis of 5’dNTP precursors. Other chemotherapy drugs have metabolites that are incorporated into DNA and lead to strand breaks. Topoisomerase poisons inhibit the resealing of the phosphodiester bond. Alkylating agents damage DNA but are both cytotoxic and Mutagenic, sometimes resulting in secondary cancers.
Cells actively dividing are more sensitive to these drugs than cells dividing slowly. In addition to cancer other tissues normally having rapid cell division are the intestinal track, bone marrow and hair follicles, explaining the limiting side effects of chemotherapy.
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Advance Topics: Medical Pharmacology
Mutation
Mutation is a permanent, transmissible change in the nucleotide sequence of a chromosome, usually in a single gene, which leads to a change of its normal function. Mutation may occur by point mutations (substitutions, missense, nonsense), frameshift in the ORF or triplet expansion. Mutation can alter mRNA expression or processing, not just the coding sequences. This includes insertions or deletions in promoters/enhancers, splicing sites and polyadenylation signals.
Point mutations are changes of a single nucleotide in DNA, especially in a region coding for a protein, and may be classified as either transitions or transversions. Transition is the substitution of one purine by another purine, or one pyrimidine for another pyrimidine. Transversion is the substitution of one purine for a pyrimidine or vice versa. Such mutation may result in either missense or nonsense mutation. A missense mutation leads to a different amino acid during translation. A nonsense mutation creates a stop codon, resulting in a truncated polypeptide which is nonfunctional or have altered function.
Deletions and insertions of one or more nucleotides lead to frameshift mutation, with possible introduction of incorrect amino acid and stop codons. Unless the deletions occur in exact multiples of three nucleotides, the whole reading fame will be affected, causing a frameshift mutation. A frameshift mutation will change all the amino acids transcribed beyond the mutation, and usually also creates a premature stop codon.
A triplet expansion is a mutation tat creates a large increase in the number of triplets sequence in a microsatellite. This can change either the coding or regulatory properties of the gene and can cause many kinds of diseases, for example neurological diseases.
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Advance Topics: Molecular Biology Lab
Recombination
Recombination is the exchange of genetic information between two DNA strands. During recombination, two chromosomes break and rejoin, transferring part of one chromosome to the other, reciprocally. The probability that a recombination will occur between any two points on a chromosome is roughly proportional to the physical distance between them. A 1% frequency of recombination between two genes or markers is defined as a genetic distance of 1 centimorgan (nm).
Recombination may be homologous or non-homologous. Homologous recombination (general recombination) occurs between identical or nearly identical sequences (ex. chromosome pairs in meiosis). Non-homologous recombination does not require extensive homology.
Recombination may also be site-specific, when specific enzymes allow the integration of a sequence into a particular site of DNA. This requires the recognition of unique nucleotide sequences. Transposition is the movement of specific pieces of DNA in a genome, catalyzed by specific enzymes or transposases. Transposons are DNA segments that encode transposases and have insertion sequences that are recognized by transposase. Together this allows DNA to move within the genome. Other pieces of DNA contain insertion sequences but do not encode their own transposases.
Transposons can move into new places within the genome or create a duplicate of themselves in a new place. Some repetitive elements in the human genome are transposable or have risen from transposable elements. Retrotransposons are transcribed into RNA and then reverse transcribed and integrated back into a random place in the genome.
Insertion of a transposon into a gene can lead to mutation and disease. Retroviruses make DNA copies of their RNA genome using reverse transcriptase (an enzyme that makes a DNA copy from RNA) and insert them into the host chromosome by a transposition catalyzed by viral-encoded integrase.
Gene therapy is the introduction of new or altered genes into cells to correct a genetic defect or treat disease. One limitation is the difficulty of introducing genes into relevant cells. This is being addressed by work on vectors for gene induction and steam cell biology. Another problem is the tendency of introduced genes to incorporate randomly into the genome, probably as a result of double-strand break repair. Random insertion can create new mutations by inserting a transgene into an inappropriate location. It can also create difficulties in sustaining gene expression over time, when the gene is wrongly in a region affecting regulation.
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Advance Topics: Molecular Biology Lab
Homologous Recombination
The main model of homologous recombination is the Holliday model. The key features of the Holliday model are homology, symmetry, the formation of the Holliday junction and reciprocal exchange.
Recombination starts when two homologous DNA molecules come together properly aligned and single strand breaks are made at homologous positions in each DNA molecule. The strands partly unwind and base pair with the opposite molecule, forming a structure in which two intact strands are joined by two crossed strands, i.e. a reciprocal event.
Then branch migration occurs: simultaneous winding and unwinding so there is no net change in the amount of base pairing but the position of the cross-over point moves, resulting in more heterogeneous duplex. After DNA ligase seals the nick, further branch migration occurs, still four strands are held together at the crossover point.
Rotation of the molecule forms a Holliday intermediate. The molecules are separated by a pair of symmetrical cuts, and the ends are resealed by DNA ligase. The direction of the cut determines whether flanking regions are exchanged. If the crossed strands are cut, the flanking markers would remain as they were and recombination could not be detected. If the intact strands are cut, the flanking markers are exchanged and the recombination event could be detected.
The Meselson-Radding model of homologous recombination is overall similar to the Holliday model, but initiation is by a single base pair rather than two. Therefore, this model is asymmetric. One strand out of two aligned DNA molecules is nicked and displaced by synthesis of new DNA in the same strand. The displaced strand invades the opposing duplex and base pairs with one of the opposing strands, forming a D-loop in the invaded strand.
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The D-loop is degraded and the invaded strand is repaired and resealed. As the strands swivel, a Holliday junction is formed and fixed with cuts and closing of nicks. If the crossed strand is cut, the flanking makers remain as they were. If the intact strands are cut, the flanking markers are exchanged.
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The double-strand break model of homologous recombination occurs in yeast and is initiated by a double strand break. One of the double strands is dissected (?) in the 5’ to 3’ direction, removing part of each strand after the nick. One of the broken strands invades the unbroken strands, forcing a loop in one of the unbroken strands, that pairs with the non-invading broken strand.
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There is mismatch repair (addition of nucleotides) and fixing of nicks resulting in two Holliday junctions. Each junction is resolved in one of four possible cutting combinations. Two ways of resolving the junction will leave the markers as they were, the other two ways will exchange flanking markers. Topoisomerases can also carry out the resolution.
After recombination there may be a small amount of heteroduplex DNA in the region were branch migration occurred. This may be resolved by the cell’s mismatch repair mechanisms resulting in a gene conversion event in which one sequence (allele) is changed into another. Asymmetric recombination leads to more frequent gene conversions (one strand donates more sequence than another).
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Advance Topics: Molecular Biology Lab
DNA Damage
Mechanisms of DNA damage include spontaneous or chemically-induced deamination, depurination/depyrimidation, alkylation, and radiation damage.
In spontaneous deamination, an hydroxyl attacks the amine group of a nucleotide, usually cysteine. This type of damage may convert a cytosine into uracil, which is recognized as an error and fixed by DNA glycosylase (which removes uracil from DNA).
+ -OH 
+ NH3
Spontaneous depurination or depyrimidation leads to apurinic or apyridimic (AP) sites and is usually due to acid conditions, heat or the repairing action of DNA glycosylase.
Alkylation damage is the addition of a methyl group to a base. O6-methylguanosine will pair up with thymine instead of cytosine, leading to a lot of mispairing and a very high rate of mutation. N3-methyladenosine is also common. Oxidative damage is due to cell metabolism and stimulated by oxidizing agents, and is an important cause of alkylation damage.
Radiation damage may occur by UV light or ionizing radiation. UV light creates
cyclobutane pyrimidine dimmers (the most common damage introduced by exposure
to UV ligth), 6-4-pyrimidine dimmers and other radiation products which distort
the double helix. Ionizing radiation creates strand breaks and base and sugar
damage.
Chemical damage is stimulated by mutagens and carcinogens, although similar damage also occurs naturally including deamination and alkylation. Procarcinogens do not damage DNA in their original form but can be activated by metabolic processes into an active form, the ultimate carcinogen, which damages DNA. This process is known as metabolic activation.
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Advance Topics: Molecular Biology Lab
DNA Repair
DNA can be repaired by several mechanisms: excision repair, mismatch repair, MGMT, photoreactivation, daughter strand gap repair and translesion synthesis.
Excision repair acts on many types of damage. DNA damage on one strand is cut out and replaced by newly synthesized DNA. The information in the complementary strand is used to direct the re-synthesis. There are two mechanisms of excision repair that differ in the removal of DNA damage: base excision repair and nucleotide excision repair.
Base excision repair has two steps for the removal of damage. First the damage base is excised from the backbone by DNA glycosylase, leaving an AP site. Specific DNA glycosylases recognize different types of damage. The AP site is then excised by AP endonuclease (cuts the backbone) and AP ligase (removes the sugar).
Nucleotide excision repair removes damage in one step. A complex of DNA repair enzymes makes two nicks, one on each side of the damage, releasing the oligonucleotide containing damage bases. In both base and nucleotide excision repairs, DNA polymerase fills in the gap and ligase seals the nick.
Mismatch repair can remove replication errors and mismatches due to recombination. In this case there is no damage base, but a wrong base pairing, causing distortion of the helix. To correct such replication errors is important to distinguish parental from newly synthesized strands. This is done by detecting transient differences in methylation between the methylated parent strand and the non-methylated daughter strand (takes a few minutes for the new strand to be methylated). Certain bases are normally methylated in specific positions as a regulatory mechanism. For example, the sequence GATC in E. coli is methylated, while in humans is normally CG.
The mismatch repair mechanism starts with the binding of MutS to the unmethylated strand. MutH binds to the methylated strand and forms a complex with MutS that cleaves the DNA. Then an exonuclease working together with an helicase (to separate the strands) will remove the mispaired nucleotide from the unmethylated strand in a process that requires ATP. Finally, polymerase binds and synthesize 5’ to 3’ to fill the gap, and there is ligation of the nick. Shortly after the repair is done, a maintenance methylase will methylate the new strand.
O6-methylguanine is so mutagenic that cells have evolved a specific enzyme to fix it: O6-methylguanine methyltransferase (MGMT). Active MGMT finds O6-methylguanine in DNA and transfers the methyl group to a cysteine in itself. Methylated MGMT is inactive, so only one methyl group can be removed per MGMT molecule.
Photoreactivation is a repair mechanism specific for cyclobutane pyrimidine dimmers. It is a direct reversal of the damage, using the photoreactive enzyme photolyase. Photolyase binds to the cyclobutane pyrimidine dimmer, then absorbs blue light as it splits the dimmer. Photoreactivation is not an important repair mechanism in humans.
Daughter strand gap repair (i.e. recombination repair) fixes gaps left in newly replicated DNA where a replication fork was halted by a lesion of the parental strand. An isopolar parental strand (i.e. from the opposite side of the fork) can fill the gap in the daughter strand. The newly formed gap in the parental strand can be filled since there is an intact, newly replicated strand by it. The original gap is repaired, but the lesion remains to be fixed later by excision repair.
Translesion synthesis refers to the case were some DNA polymerase can insert nucleotides opposite to lesions in the template. The damaged base (s) do not code properly due to relaxing of the proofreading. Errors and potential mutations are introduced at a higher frequency.
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Advance Topics: Molecular Biology Lab
DNA Replication/Repair and Carcinogenesis
Damage DNA can be lethal if a cell tries to replicate. This may affect cells as the attempt to replicate or during transcription of gene products needed fot survival.
Different types of DNA injury will hinder replication. Damage nucleotides may block polymerase, and even if polymerase can synthesize pass the damaged bases, they may not code properly. Cross-linked template DNA cannot be separated for replication. Single and double strand breaks can lead to chromosomal breaks. Thus attempting to replicate damaged DNA is likely to lead to cell death.
Damage DNA causes the cell to postpone entry into the S-phase and mitosis until damage is repaired. This is regulated at several “checkpoints” in the cell cycle. The tumor suppressor gene p53 is a central protein in this process. Cells in S-phase at the time of damage are more susceptible to being killed (one reason why DNA damaging agents are used for cancer chemotherapy).
In a non-dividing cell, inability to replicate may not be a problem, but damage can be lethal if a key gene product cannot be made due to inactivation of the corresponding gene. Damaged DNA can prevent proper transcription of critical genes. RNA polymerases, like their DNA counterparts, are often blocked by lesions in the template, and mispairing can lead to production of faulty proteins.
Transcription-coupled repair means that cells preferentially repair the template strand of genes that are being actively transcribed at the time of damage. Repairing the active regions before the rest of the genome allows prompt restoration of functional templates needed for maintenance of the cell’s metabolic functions.
Xerodermata pigmentosum (XP) was the first disease recognized to be caused by defective DNA repair mechanisms. Patients are photosensitive and highly susceptible to skin cancer in sun-exposed areas. Many patients also have neurological problems. XP is a rare autosomal recessive disease that can be caused by defects in 8 different genes. Defects in 7 of them lead to defects in the initial incision step of nucleotide excision repair at cyclobutane pyrimidine dimmers, hence the extreme sun sensitivity that characterizes this disease.
Loss of mismatch repair is a primary step in the development of hereditary non-polyposis colon cancer (HNPCC). HNPCC is inherited as an autosomal dominant disease. Inheritance of one defective copy of the mismatch repair genes leads to tumors after the somatic loss or inactivation of the remaining gene. Some of the genes involved are hMSH2, a MutS homologue, and the MutL homologues hHLM1 and hPMS2. Defects on any of these genes predisposes a patient to HNPCC.
Tumor suppressor genes are those whose activation leads to tumors. For a tumor to arise, both copies of a tumor suppressor gene must be inactivated in a single cell, so the cell can keep dividing uncontrolled. Inheritance of a single defective allele in a tumor suppressor gene leads to high cancer susceptibility because the probability that the other good copy will be inactivated in one cell at some time during the lifetime of the individual is high. Therefore cancer susceptibility is inherited in a dominant pattern. Examples of tumor suppressor genes are Rb-1, genes encoding the enzymes for mismatch repair, and p53.
In normal individuals, all cells start with two functional copies of Rb-1. No tumor occurs if an occasional cell has one Rb-1 gene inactivated. But if that cell continues to proliferate and a mutation of the second allele occurs, the person may develop a tumor. This occurs in non-hereditary retinoblastoma. And individual with an inherited Rb-1 mutant allele has a higher probability of developing a second somatic mutation leading to a tumor, as is the case in hereditary retinoblastoma.
Li-Fraumeni syndrome is a hereditary cancer susceptibility syndrome caused by inheriting a defective p53 gene. Defects in p53 can allow cells to avoid cell cycle checkpoints and therefore replicate damaged DNA. p53 defects can also prevent apoptosis after DNA damage, leaving mutated cells that give rise to tumors.
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Advance Topics: Molecular Biology Lab
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