DNA Replication Flashcards
(90 cards)
1
Q
DNA REPLICATION MECHANISM
A
Semi-conservative
- It produces two copies of the original DNA molecule, each of which contains one of original strand, and one newly synthesized strand.
2
Q
Components required for synthesis of DNA
A
- All four dNTPs (deoxynucleoside triphosphate)- bldg blocks of DNA molecule (guanosine, adenosine, thymidine, cytidine)
- DNA template
- DNA polymerase
- Co-factor: Magnesium ions- required for DNA polymerase activity
- RNA primer providing a free 3’ OH group. This primer is attached to the template SS DNA sequence
3
Q
DNA CHAIN ELONGATION CATALYZED BY
A
DNA Polymerase
4
Q
DNA POLYMERASE
DNA synthesis in both eukaryotes and prokaryotes
A
- Catalyzes the formation of a phosphodiester bond between the 3’ OH group of the deoxyribose in the last nucleotide to the 5’ phosphate of the dNTP precursor ( the incoming deoxynucleotide)
- Deoxynucleoside 5’ triphosphate provides the energy source for the reaction ( cleavage of a nucleoside triphosphate to form a nucleoside monophosphate and Pyrophosphate).
5
Q
Anti-viral Nucleoside analogs- viral reverse transcriptase inhibitors
A
- AZT- Azidothymidine (Zidovudine)-deoxythymidine
- Didanosine (ddl)—-deoxyadenosine
- lack the 3’ OH
- Utilized by the viral reverse transcriptase enzyme
- Upon incorporation into the ds DNA chain, termination occurs due to the lack of 3’ OH
6
Q
Anti-Viral Nucleoside analogues- Viral DNA polymerase inhibitor
A
Acyclovir——deoxyguanosine
-Only activated in infected cells. Only the infected cells possess the viral kinase
-Causes chain termination d/t lack of 3’ OH
7
Q
Anti-viral Nucleotide Base analogues
A
TENOFOVIR- adenosine analog
- competes with its natural nucleotide counterpart, deoxyadenosine 5’- triphosphate for incorporation into newly forming HIV DNA
- Once successfully incorporated, termination of the elongating DNA chain ensues and DNA synthesis is interrupted
8
Q
Cytosine arabinose (araC)= cytosine nucleoside analogues
- Adenosine Arabinose also acts in a similar fashion.
- AraC has a 3’ OH group to act as an acceptor for further chain elongation. however, the OH group at position 2 prevents polymerase adding the next dNTP due to stereo restraints which results in chain termination.
A
- Ribose sugar replaced by arabinose
* In cells it is rapidly converted to cytosine arabinose triphosphate- acts as a substrate for several human DNA pols
9
Q
3’ to 5’ exonuclease activity
A
*DNA POLYMERASE I-as it synthesizes DNA in the 5’-3’ direction, it proofreads using the 3’-5’ exonuclease activity to remove errors. Note: Pol I uses its 5’-3’ polymerase activity to filll in gaps generated during most types of DNA repair
**only removes a nucleotide if its base is not hydrogen bonded to the template
- BOTH DNA POLYMERASE I AND III have 3’ to 5’ exonuclease activity
10
Q
5’-3’ exonuclease activity
A
- DNA polymerase I-also removal of the primer and replacing it with DNA. Substrate must be vase paired.
- DNA polymerase III
11
Q
Bacterial genomes
A
- Usually circular
- Contain single ORI
- THETA REPLICATION at distinct sites called origins of replication (ORI)
12
Q
Eukaryotic genomes/chromosomes
A
- Many replication origins
- No replication termini
- LINEAR REPLICATION
13
Q
Bacteriophage (bacterial virus) and plasmids that have a DNA genome utilize
A
ROLLING CIRCLE REPLICATION
14
Q
OriC
A
Origin of replication in E.Coli
- Has a length of 245 no
- contains a tandem array of three nearly identical 13-nucleotide sequences and four 9-nucleotide sequences that act as binding sites for DNA protein
- contains DNA sequences that are A-T rich (weak bonding compared to G-C bonding). Thus the OriC facilitates easier melting and strand separation of the DNA molecule.
15
Q
Initiation of DNA replication ( prokaryotes)
A
- Initiator proteins ( DNaA protein)- binds to origins of replication and breaks hydrogen bonds between bases
- DNA helicase(DNaB)- opens helix and binds primase to form primosome.
- Helicase inhibitor (DNaC)-delivers helicase to DNA template
- DNA Primase: an RNA polymerase that synthesizes the RNA primer on the lagging strand to enable DNA polymerase to syntesize DNA strand.
- DNA polymerase I: removes the RNA primer and replace with DNA
- DNA polymerase III: synthesis of leading and lagging strands.Synthesizes the Okazaki fragments
Note: Leading strand synthesis requires one priming event
*Single stranded DNA binding protein (ssb): binds SS DNA in the replication bubble and prevents it from annealing or forming secondary structure
16
Q
Single-strand binding proteins (SSB) proteins
A
Binds to SS DNA in the replication fork to prevent reannealing and straightens out the DNA template to facilitate the DNA polymerization process.
17
Q
RNAseH and FEN-1 protein
A
Removes the RNA primer in eukaryotes
18
Q
Helicase (DNaB)
A
Unwinds DNA in opposite directions away from the origin
19
Q
DNA LIGASE
A
- Joins the Okazaki fragments together
- LIGASE generates a phosphodiester bond between adjacent Okazaki fragments. This produces a continuous DNA strand.
- Each Okazaki fragments requires a separate primer
-Ligase utilizes ATP as the energy source for this reaction
20
Q
CLAMP PROTEIN
A
Tightly holds the DNA polymerase onto the template for synthesis of long template. There4 it increases expressively
21
Q
DNA GYRASE ( type II topoisomerase)
A
- Introduces negative supercoils into the DNA.
- This reduces the positive super coils introduced by the opening of the DNA.
- Also aids in the separation of the DNA during replication and transcription.
22
Q
CIPROFLOXACIN
A
- A Quinolone drug that inhibits bacterial DNA gyrase ( Topoisomerase II). This results in a build up of positive supercooling ahead of the replication fork during DNA replication.
- Used in the tx of resp and UTI infections and can also be used to treat anthrax.
23
Q
- Polymerase-alpha
- synthesizes RNA primer on leading and lagging strand
- low processitivity-falls off after synthesizing a short DNA segment
- No exonuclease activity
- Polymerase- sigma
- main replicative enzyme
- synthesizes DNA from leading and lagging strands
- high processitivity-can syntesize long stretches of DNA.
- 3’-5’ exonuclease proofreading activity.
A
Eukaryotic DNA polymerase
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Q
- CAMPTOTHECIN
- Anti-cancer drug
- Binds to and inhibits topoisomerase I activity. This results in DNA damage.
- ETOPOSIDE
- Another anti-cancer drug
- inhibits the activity of topoisomerase II. It prevents the re-ligation of DNA. This ultimately leads to DNA damage and apoptosis.
A
Inhibitors of Eukaryotic replication
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Actinomycin D (Dactinomycin)
-An antibiotic derived from Streptomyces.
-Inhibitor of replication
-Planar phenoxazone ring- intercalated between adjacent guanine-cytosine bases
-polypeptide chains extend along the minor groove of the helix, thereby stabilizing the drug-DNA complex
-prevents DNA replication by: preventing the formation of regions of ssDNA.
- Binds tightly to double stranded DNA and inhibits both DNA transcription and replication.
It is effective in both prokaryotes and eukaryotes
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HOW DOES DNA POLYMERASE READ A THE TEMPLATE STRAND?
3’-5’ direction
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DNA PRIMASE
- Addition of an RNA primer to to the template DNA
- Synthesis of RNA primer that’s complimentary to the DNA template. The primer provides the free 3’-OH group for the synthesis of DNA.
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TELOMERES
A special complex containing enzymatic proteins and an RNA molecule.
- Possesses several thousand non-coding repeated sequences- typically AGGGTT. Lengthening of DNA strand.
- Has a protein that acts as a reverse transcriptase, as well as a short piece of RNA that can act as a template for DNA synthesis.
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Polycistronic
PROKARYOTIC MRNA
* MRNA that encodes several proteins
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In Eukaryotes the initial product of transcription must be processed, and modified to form the mature mRNA. How is it processed or modified?
- 5’ cap -7-methylguanosine cap
- Polyadenylation of the 3’ end of the mRNA
- Splicing of Econ’s and the removal of introns
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RNA POLYMERASE II
Carries a set of pre-mRNA processing proteins on its c-terminal tail
-Close up of the 5’ cap on mRNA transcripts produced
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Capping of the 5’ end performed by the following four enzymes
- Phosphatase: removes a phosphate from the 5’ end of the RNA
- THE CAPPING IS IMPORTANT BECAUSE it helps distinguish between different RNA in the cell.
- Guanylyl transferase: adds a GMP in a reverse linkage ( 5’to 5’ instead of 5’ to 3’)
- Guanine-7-methyl transferase: adds a methyl group to the 7 position of the terminal guanine.
- 2’-O-methyl transferase: adds a methyl group to the 2’-O position to the next to last base on the 5th end
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5’-methyl cap has important roles in the regulation of mRNA
* Processing- prevents 5’ degradation. The CBC & elF-4E/elf-4G block the access of de-capping enzymes to then cap. This increases the half-life of the mRNA in the cytosol.
* Transport- regulates export of mRNA out of the nucleus. mRNA is transported in complexes that contain a Cap Binding Complex (CBC) at the 5’ end and RNA binding proteins along the rest of the sequence.
* Translation- efficient translation of the mRNA into protein. The CBC is replaced b the trajnslayion factors elF-4E and elF-4G which facilitates binding of mRNA to the ribosome.
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Modification of the 3’end of the RNA is accomplished by several enzymes associated with RNA polymerase II that bind to specific sequences on the RNA.These are:
* CPSF- Cleavage and Polyadenylation Specificity Factor which binds to the hexamer AAUAAA ( the Polyadenylation signal)
* CstF- Cleavage stimulating factor F binds the G-U rich element beyond the cleavage site.
* Cleavage factors bind to the CA sequence at the cleavage site
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PABP
| Poly-A Binding Proteins
-Binds to the poly-A tail and assist in directing translation by the ribosome.
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Functions of 3’ mRNA polyadenylation
* Increases the half-life of the mRNA by protecting it from degradation in the cytoplasm.
* PABP binds to poly A tracts protecting mRNAs from ribonuclease attack.
* PABP interacts with e-IF 4G and this interaction is thought too lead to the stimulation of translation of eukaryotic mRNAs
* Aids in transcription termination
* AIDS in the export of the mRNA from the nucleus
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Econ’s
Protein coding sequences
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Introns
Non-coding sequences
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GU
5’ end of the intron consensus sequence
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AG
3’ end of the intron consensus sequence
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Small nuclear ribonucleoprotein particles (SnRNP) and numerous other splicing factors mediate splicing. These are;
U1
U2
U4
U5
U6
-They form base pairs with consensus sequences at each end of the intron.
-After the 5 snRNPs bind the primary transcript, U1 and U4 leave, this activating the spliceosome.
- U2,U5 and U6 interact to bring neighboring Exons into correct alignment allowing for two trans-esterification reactions to occur.
42
Lariat structure
- The 2nd-OH of the branch site A attacks the 5’ phosphate (p) at the splice donor site ( 5’ end of the intron) forming a 2’—-5’ unusual phosphodiester bond.
- This reaction also leaves a free 3’ hydroxyl at the end of exon 1 which will participate in the next reaction.
43
Systemic Lupus Erythematosus
An autoimmune disease with multiple symptoms; extreme fatigue, arthritis, fever, skin rashes, and kidney problems.
- It is a condn in which individuals antibodies of a person react against their own nuclear proteins such as the U1 RNA component of the spliceosome, his tones, and topoisomerase.
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Alternative Splicing
- Helps to increase biological diversity
| - Allows a number of different proteins to be produced from the same gene
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Alpha- Tropomyosin
A protein with many isoforms; from regulating actin filaments in muscle cells (muscle contraction) and non- muscle cells ( cell division and shape).
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RNA EDITING
Chemical modification of mRNA is known as RNA editing.
* RNA editing is a process in which information changes at the level of mRNA
* Coding properties of transcript are altered. Information changes at the level of mRNA
* Results in the alteration in the amino acid sequence of the specified protein and generation of functionally different proteins
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TWO DIFFERENT TYPES OF RNA EDITING
*Adenosine to Inosine ( A-I)
(Editing of the Glutamate receptor ) which results in a codon change within the from glutamine to arginine.
* The editing affects an amino acid located in the inner wall of the ion channel and alters calcium ion permeability.
* This editing is a requirement for proper brain development.
* Enzyme: Adenosine deaminase
* Cytidine to Uridine ( C-U)
Creates a stop codon (CAA-UAA)
* Enzyme: Cytidine deaminase enzyme
Ex; Apo-B gene encodes two alternative forms of the apolipoprotein B: Apo-B100 ( liver) and Apo-48 ( intestine)
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Metaphase of Mitosis
* Chromosomes maximally condensed
* karyotyping
* Chromosomes align at equator
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Anaphase of mitosis
Sister chromatids separate
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Prohase I of Meiosis I
Homologous recombination/crossover; Oocytes arrested in prophase I- UNTIL ovulation
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Anaphase I of meiosis I
Homologous chromosomes randomlysegregate
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Anaphase II
Sister chromatids of a chromosome separate
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NON-DISJUNCTION
Non-separation of the homologous chromosomes/sister chromatids in the meiotic I and II respectively
* Increased with paternal or maternal age
* Results in a higher risk of trisomy (chromosomal abnormality) in the fetus.
* Higher paternal age, greater the risk of development of errors during replication ( new dominant single gene mutations)
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Turner syndrome
45X
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Klinefelter syndrome
XXY
*Male with an extra X chromosome
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Maternal uniparental disobey
When both copies of a pair of homologous chromosomes are derived from mom
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Paternal uniparental disomy
When both copies of a homologous chromosome pair are derived from dad
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Types of cells
* Labile cells- multiply throughout life; skin, GIT, epithelial cells. Most susceptible to cancer
* Stable cells- G0 phase ( QUIESCENT) phase. Can divide appropriately if stimulated. Ex. Liver cells.
* Permanent cells: Have lost ability to divide, But still able to perform their functions. Permanently in G0. Ex. neurons and cardiac muscle cells
* Permanent cells
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P53
Tumor Suppressor
- ‘Guardian of the genome’
- DNA damage results in increased levels of p53 protein which causes;
* Cell cycle arrest or activation of GI-S checkpoint
* Activates DNA repair systems
* If damage is extensive, may cause Apoptosis
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Mosaicism
Presence of two or more populations of cells with different genotypes in an individual
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Lariat structure
During intron removal from a pre-mRNA molecule;
-The 2’-OH of the branch site A attacks the 5’ phosphate (p) at the splice donor site ( 5’ end of the intron) forming a 2’ to 5’ unusual phosphodiester bond. This reaction produces a Lariat structure.
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Alternative Splicing
Can produce different forms of a protein from the same gene
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Beta-thalassemia
Many individuals with this disorder have mutations in the beta globin gene that generates additional splice sites within the mRNA
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Limb Girdle Muscular Dystrophy
Some pts with this disorder possess a mutation in the calpain-3 gene that generates a new splice site within exon 16
* This results in a shorter calpain-3 mRNA that is missing many base pairs in exon 16
* mutation interrupts a codon causing downstream codons to be out of frame and thus producing a defective protein.
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Two types of mosaicism
1. The mixed somatic/Germaine mosaic; the karyotype analysis shows a mixture of cells; Normal cells ( 46XX); and cells with aneuploidy ( 47, XX+21)
2. Confined germ line mosaic where all somatic cells are from one genotype
- Gametes have a different genotype so there is a high risk of having children with a genetic disorder
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Meiosis
Takes place in the germ cell lines ( cells of the ovary or testis)
- Gametes are haploid ( n; 23 chromosomes)
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Meiosis 1
- Crossing over-genetic diversity
- homologous chromosomes separate ( reduction division) (n; 2d)
- 23 chromosomes and 46 sister chromatids
- Begins with one diploid parent cell and ends with two haploid daughter cells halving the number of chromosomes in each cell.
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Meiosis II
Sister chromatids of chromosomes separate ( n;d)
- starts with two haploid parent cells and ends with with four haploid daughter cells, maintaining the number of chromosomes in each cell.
- so there are 23 chromosomes, and 23 chromatids
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FISH
Fluorescence in situation hybridization can be used to detect small deletions or identify chromosomes involved in complex rearrangements
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Cycling
A set of proteins that regulate the events of the cell cycle. They are expresses at specific points in the cell cycle. Cyclins interact with and activate cyclin-dependent kinases (Cdk proteins), enabling them to phosphorylate other proteins required to proceed through the cell cycle. The cell commits at the restriction point at which time Cyclin D accumulates and interacts with Cdks 4 and 6. this leads to phosphorylation of the Rb protein, which releases transcription factors from inhibition, in turn leading to synthesis of Cyclins A and E, which interact with Cdk2 to initiate DNA synthesis.
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Variable Number Tandem Repeat (VNTR)
This is a location in a genome where a short nucleotide sequence is organized as a tandem repeat. These can be found on many chromosomes, and often show variations in length ( number of repeats) among individuals.
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What are the four types of polymorphisms?
1. SNP- Single Nucleotide Polymorphism; consist of a single base pair change, and may occur anywhere in a gene, including an exon, as well as between genes.SNPs are usually detected by a variety of DNA- sequencing- based techniques.
2. Short Sequence Repeats ( SSR);stretches of DNA in which a di, tri, or tetranucleotide sequence is repeated multiple times. Vary from individual to individual. Can be measured by polymerase chain reaction (PCR) amplification, electrophoresis.
3. Short tandem repeats or VNTR ( Variable Number Tandem Repeat); repeats of tens to hundreds of bases, with the exact number of repeats being polymorphic.
4. Copy Number Variation or LCR- Low Copy Repeat; which consists of different numbers of copies of a specific genetic sequence.
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Xist gene
Involved in x-inactivation ( Lyonization)
* this gene produces an RNA that coats the X-chromosome
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Chromosome Nomenclature
MSA
- Metacentric Chromosome; p and q arms are of equal length. Centromere in the center; chromosome 1
- Submetacentric Chromosome; p arm is shorter than q arm; chromosome 4
- Acrocentric Chromosome; p arm contains little genetic information. Chromosome 13,14,15,21,22; involved in Robertsonian translocation.
75
What are the different modes of inheritance?
* Autosomal dominant and Autosomal recessive
* X-linked dominant and X- linked recessive
*Y- linked inheritance
THE ABOVE ARE CONSIDERED MENDELIAN MODES OF INHERITANCE
* Mitochondrial inheritance- NON-MENDELIAN INHERITANCE
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Autosomal Disorders
- Familial hypercholesteromia d/t LDL receptor deficiency
- Marfan syndrome
- Osteogenesis Imperfecta-brittle bone disease
- Achondroplasia
- NF-I; Neurofibromatosis type 1
- Acute intermittent porphyria-a heme synthesis deficiency.
-Huntington disease
-Myotonic dystrophy
These are triplet repeat expansion disorders.
77
Trinucleotide Repeat Disorders; Triplet Repeat Expansion Disorders
-Huntington’s Disease-CAG
-Myotonic Dystrophy-CTG
These are Autosomal Dominant disorders
-Fragile X - is a X-linked disorder-CGG abt 200 plus no
-Fredrich’s Ataxia-GAA
-
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PLEIOTROPIC
Producing or having multiple effects from a single gene
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Gangliosides
Glycolipids containing NANA ( N- acetyl neuraminic acid)
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Glycolipids
Gangliosides
Globosides
Cerebrosides
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Glycerophospholipids
Phosphatidylethanolamine
Phosphatidylinositol
Cardio lip in
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Sphingomyelin
Sphingophospholipid
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Triaglycerol
Storage lipid and is present in cell membrane
84
Two essential fatty acids
Alpha- linolenic acid- Omega 3 fatty acid
| Linoleic acid- Omega 6 fatty acid
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Omega 3 fatty acids
Alpha- Linolenic
DHA
EPA
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Omega 6 fatty acids
linoleic acid
| ARA- Arachnidonic acid
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Saturated fatty acids
Palmitic acid
| Stearic acid
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Lipids; fixative and stain
Osmium tetroxide
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Claudins and Occluding
Tight junctions
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Integrins
Hemidesomosomes
