DNA Replication
Similarities of DNA and RNA
-Bidirectional -DNA polymerase works in 5'-3' -Leading and lagging strands -Primers are required
DNA Replication Machinery
Helicase Primase DNA Polymerase Ligase
lagging strand
The strand that is synthesized in fragments using individual sections called Okazaki fragments
Clamp
hold dna plymerase to lagging strand when dissociates the dna polymerase will release the lagging strand
Where does DNA replication initiate?
Origin(oriC in prokaryotes) Where it proceeds away formthe origin Bidirectionally Creates 2 replication forks that make up the replication bubble
exonuclease activity
Part of DNA polymerase error proofing -If DNA polymerase attaches to a non-complementary nucleotide, the DNA will "bubble out" -DNA Polymerase will stall -3'-5' exonuclease acitvity will be activated (backspace) to remove the mismatched nucleotide -DNA polymerase will then continue to add complementary nucleotides in the 5' to 3' direction.
Why is DNA antiparallel?
Since DNA polymerase requires a free 3' OH group for initiation of synthesis, it can synthesize in only one direction by extending the 3' end of the preexisting nucleotide chain. Hence, DNA polymerase moves along the template strand in a 3'-5' direction, and the daughter strand is formed in a 5'-3' direction. This difference enables the resultant double-strand DNA formed to be composed of two DNA strands that are antiparallel to each other.
What is required to give DNA Polymerase a starting 3' -OH(hydroxyl) end?
A Primer
What is the result of DNA synthesis starting at the 3' end(OH hydroxyl)?
DNA can only grow (synthesize) in the 5' to 3' direction
enzymes in replication
Enzymes unwind the two strands DNA polymerase attaches complementary nucleotides DNA ligase fills in gaps Enzymes wind two strands together helicase, DNA polymerase, ligase, Primase, Gyrase
semiconservative replication
Method of DNA replication in which parental strands separate, act as templates, and produce molecules of DNA with one parental DNA strand and one new DNA strand
Okazaki fragments
Small fragments of DNA produced on the lagging strand during DNA replication, joined later by DNA ligase to form a complete strand.
DNA damage
Ultraviolet (UV) Radiation Alkylating Agents Free Radicals Thermal energy Gamma and X-rays
Senescence
cellular old age when the cell stops dividing
thymine dimers
covalent link of adjacent thymine bases in DNA
Strand invasion and exchange
RecA helps 3' end invade dsDNA This forms a D loop Branch migration can displace the original strand ATP-dependent
Base Excision Repair (BER)
Removes modified bases, damaged area, by endonucleases and phosphodiesterase. the gap is then filled by DNA polymerase and ligated by ligase.
Telomeres
Repeated (GGGGTTA) DNA sequences (Junk DNA) at the ends of eukaryotic chromosomes. DNA "caps" act as caps that protect the internal regions of the chromosomes, and they're worn down a small amount in each round of DNA replication. lost after 50-100 replication events and cell stops dividing and enters senescence (cellular old age)
Supercoiling
superhelical tension in DNA leading to twisting and tangling of DNA strand
What can mutations in DNA cause?
-Defective proteins -Disease (sickle cell-anemia) -Cancer by accumulation of mutations
What problems of replication are unique for eukaryotes?
-Linear chromosome with ends, These ends pose a problem for DNA replication. The DNA at the very end of the chromosome cannot be fully copied in each round of replication, resulting in a slow, gradual shortening of the chromosome. -Much more genetic material (avrg. 50x more) -More packaging (nucleosomes) -DNA polymerase work much slower
DNA replication steps
1) Helicase- unwinds the parental double helix 2) DNA topoisomerase - upstream of helices alleviating torsional strain 3) Single-strand binding proteins (SSBP) stabilize unwound DNA, aided by DNA gyrase (topoisomerase). 4) Primase synthesizes a short RNA primer for DNA polymerase to bind to in the 5' to 3' direction to start replication on each strand. 5) DNA polymerase synthesizes the leading strand in 5' to 3' direction while the lagging strand is made discontinuously by primase making short pieces and then DNA polymerase extending these to make Okazaki fragments. 6) DNA ligase joins the Okazaki fragments together
replication fork
A Y-shaped region on a replicating DNA molecule where the parental strands are being unwound and new strands are being synthesized.
nonhomologous end joining
A quick-and-dirty mechanism for repairing double-strand breaks in DNA that involves quickly bringing together, trimming, and rejoining the two broken ends; results in a loss of information at the site of repair.
thermal energy
Depurination (loss of A or G bases from their surgars) is common in mamal due to the generation of thermal energy Results in apurinic site, which if left unrepaired generate mutations during DNA replication because they cannot specify appropriate paired base
telomerase
Needed on Lagging strand end an enzyme in eukaryotes germ cells that lengthen telomeres to full length, absent in body cells. contains built in RNA Primer Complementary to GGGGTTA repeated telomeric sequence Increases the length of the 3' strand to enable primer to hybridize and thus make a full-length 5'strand reverse transcriptase enzyme.... carries its own RNA. May also be (re)activated in cancer cells to make them "immortal"
Ultraviolet (UV) radiation
Non-ionizing radiation that can crosslink adjacent thymine bases Forms Pyrimidine Dimers, This lesion impairs base pairing and stalls replication
Prokaryotes 3 DNA polymerase (all contain 3'-5' exonuclease activity)
Pol I 1--DNAPolymerase I, approx 400 molecules per cell, required to remove RNA primers and replace them with DNA, has exonuclease capability Pol II 2-DNA Polymerase II, approx. 40 per cell, involved in DNA Repair Pol III 3-DNA Polymerase III, approx. 10 molecules per cell, Responsible for synthesis of both the leading and lagging strands, Main enzyme for replication, Also proofreading and editing
semidiscontinuous replication
The mode of replication in which one new strand is synthesized continuously while the other is synthesized discontinuously. The two strands of a double helix are synthesized by a different sequence of events, one growing toward the replication fork and the other growing away from it
leading strand
The new continuous complementary DNA strand synthesized along the template strand in the mandatory 5' to 3' direction.
dispersive replication
a disproved model of DNA synthesis suggesting more or less random interspersion of parental and new segments in daughter DNA molecules
origin of replication
a particular sequence in a genome at which replication is initiated
Gyrase (topoisomerase II)
introduces double-stranded break ahead of replication fork and swivels the cleaved ends to relieve stress of helix unwinding prevents supercoiling [DNA replication]
mismatch repair proteins
nucleus has mismatch repair complexes to scan DNA for mismatch errors not caught by DNA Polymerase error-proofing Newly made strands are likely to have nicks that enable the repair machinery to identify the strand that needs repair Generating normal DNA molecule (if not will be mutated) Proteins remove newly synthesized strand, repair of gap by polymerase and ligase
homologous recombination
process by which a cell replaces a stretch of DNA with a segment that has a similar nucleotide sequence Can be used to accurately repair Double stand breaks and increase genetic variability of offspring during meiosis (Cross over) 1)Nuclease digestion--nuclease digests 5' end of broken strand 2)Strand invasion--by complementary base-pairing 3)DNA synthesis--Repair polymerase synthesizes DNA using undamaged complementary DNA as template. Invading strand released, and broken double helix is reformed. DNA synthesis continues using complementary strands from damaged DNA as template Ligation--After Ligation Double strand break is accurately repaired
RNA primer
short segment of RNA used to initiate synthesis of a new strand of DNA during replication these RNA segments are subsequently removed and filled in with DNA
conservative replication
A disproved model of DNA synthesis suggesting that one-half of the daughter DNA molecules should have both strands composed of newly polymerized nucleotides.
single strand binding proteins
A protein that binds to the unpaired DNA strands during DNA replication, stabilizing them and holding them apart while they serve as templates for the synthesis of complementary strands of DNA. on lagging strand
Primase
An enzyme that joins RNA nucleotides to make the primer using the parental DNA strand as a template, from which polymerization can initiate.
Helicase
An enzyme that untwists the double helix at the replication forks, separating the two parental strands and making them available as template strands. used to separate strands of a DNA double helix or a self-annealed RNA molecule using the energy from ATP hydrolysis, a process characterized by the breaking of hydrogen bonds between annealed nucleotide bases.
Meselson-Stahl Experiment
Used isotope of nitrogen to change the weight of DNA N15 & N14, demonstrated that the semi-conservative model and dispersive theories are best description of replication.
Topoisomerase at the replication fork
relieve supercoiling in DNA by cutting the DNA helix and allowing one section to rotate; the enzyme then reseals the DNA break Free rotation of double helix about phosphodiester bond relieves torsional stress ahead of helicase, after which single-strand bread is sealed in absence of topoisomerase the DNA cannot rapidly rotate and torsional stress builds up
what is the purpose of nucleotide pairing?
Base pairing is fundamental to for two main aspects of the DNA molecule: 1--Structure: hydrogen bonds between nitrogen bases are critical to stabilize DNA double helix structure. 2--Complementarity: Watson-Crick base pairing (A-G, C-T) allows complementarity between DNA double strands. This is vital in replication processes, when each DNA strand serves as a template for the synthesis of a new strand.
Depurination/Depyrimidation
Base removed due to covalent bond breakage--> causes point mutations (substitution, indel) - Depurination: removal of purine base - Depyrimidation: removal of pyrimidine base
Double Strand Breaks
DNA damage often resulting from gamma and x-rays ionizing radiation water molecules to form free radicals involving a fracture of both strands of the double helix by the DNA backbone. these can be debilitating to a cell and at least two distinct (major pathways) repair systems are devoted to their repair. -non-homologous end-joining ----------quick and dirty -homologous recombination ----------perfect repair
Deamination
Hydrolytic removal of amino groups from cytosine to generate uracil. This may be why DNA contains no thymine and not uracil. If DNA contained uracil, cells could not tell the difference between these spontaneous uracils and those that would belong.
DNA polymerase
Synthesizes DNA, Replicates with high fidelity(1 in every 10^7 nucleotides) Adds new nucleotide pairs with a base in the template strand. Catalyzes covalent linkage of nucleotides into growing new strand ATP to ADP Enzyme involved in DNA replication that joins individual nucleotides to produce a DNA molecule
template strand
The DNA strand that provides the template for ordering the sequence of nucleotides in an mRNA transcript.
ligation
The newly synthesized 3' end of the invading strand is then able to anneal to the other 3' overhang in the damaged chromosome through complementary base pairing. After the strands anneal, a small flap of DNA can sometimes remain. Any such flaps are removed, and the SDSA pathway finishes with the resealing, also known as ligation, of any remaining single-stranded gaps.
What happens when the wrong nucleotide is inserted into the new strand?
The strand has a tendency to separate,"bubble out", from the template, causing polymerase to stall and activating the 3' to 5' nuclease activity
What happens at the replication fork?
Two DNA polymerases collaberate to copy the leading strad template and the lagging stand template of DNA --DNA polymerase produces okazaki fragment on lagging strand, clamp dissociates and polymerase temporarily releases the lagging strand --as the helicase unwinds and seperates the DNA template primase synthesizes primers at the newly revealed 3'end --proteins maintain unpaired single strand unraveled --Polyerase attaches to lagging strand and gets locked in with the clamp --as the polymerase adds nucleotides and creates base pairs the proteins fall off, polymerase stops when it encounters the primer of the previous okazaki fragment --Ligase joins 3' (OH) end of one okazaki fragment with 5'(phosphate) end of other
Synthesis of lagging strand
1--Helicase unwinds the DNA 2--Primase adds RNA primers are laid down at intervals along at the newly exposed, single stranded stretch. foundation for DNA polymerase to start at available 3' end closest to the fork ------In Eukaryotes, RNA primers are make at intervals of about 200 nucleotides on the lagging strand and each RNA primer is about 10 nucleotides long. ------Primes are removed by nucleases that recognize an RNA stand in an RNA/DNA helix and degrade it; this leaves gaps that are filled by a repair DNA polymerase that can proofread as it fills in the gaps. 3--DNA polymerase synthesizes new stand using the DNA template until it runs into the next RNA primer 4--The completed fragments are finally joined together by an enzyme called DNA Ligase, which catalyzes the formation of a phosphodiester bond between the 3'-OH end of one fragment and the 5'-phosphate end of the next, thus lining up the sugar-phosphate backbone. This nick sealing reaction requires input of energy in the form of ATP
DNA repair
Collective term for the enzymatic processes that correct deleterious changes affecting the continuity or sequence of a DNA molecule.
Why is it that the primer of the last Okazaki fragment that does get made can't be replaced with DNA like other primers?
DNA polymerases can't start a new strand of DNA from scratch, even if they are given a template. That's because the polymerization reaction they catalyze involves attaching the phosphate group of an incoming nucleotide to the hydroxyl group of an existing nucleotide (one that's already part of the strand), as shown at right. Without this hydroxyl group to use as a "hook," a DNA polymerase has nothing to attach nucleotides to and cannot catalyze its reaction to make new DNA.
Events at the Replication Fork in E. coli
DnaA proteins bind oriC (origin of replication) causing bending and separation of strands DnaB and other helicases separate strands, SSB attach Primase synthesizes RNA primer Lagging and leading strand is synthesized DNA polymerase I removes RNA primers, fills gaps with DNA Okazaki fragments are joined by DNA ligase
Ligase
Enzyme that reseals nicks that arise in the backbone of a DNA molecule, in the laboratory, can be used to join together tow DNA fragments
replication bubble
Segment of a DNA molecule that is unwinding and undergoing replication. a region of DNA, in front of the replication fork, where helicase has unwound the double helix
How does gyrase prevent supercoiling?
The unique ability of gyrase to introduce negative supercoils into DNA at the expense of ATP hydrolysis[1] is what allows bacterial DNA to have free negative supercoils. The ability of gyrase to relax positive supercoils comes into play during DNA replication and prokaryotic transcription. The right-handed nature of the DNA double helix causes positive supercoils to accumulate ahead of a translocating enzyme, in the case of DNA replication, a DNA polymerase. The ability of gyrase (and topoisomerase IV) to relax positive supercoils allows superhelical tension ahead of the polymerase to be released so that replication can continue.
