DNA Replication Lecture Notes PDF

o All cells undergo a division cycle during their life span. o Some cells are continually dividing (e.g. stem cells), others divide a specific number of times until cell death occurs. Replication = Duplication o During the process of cell division everything within the cell must be duplicat...

o All cells undergo a division cycle during their life span. o Some cells are continually dividing (e.g. stem cells), others divide a specific number of times until cell death occurs. Replication = Duplication o During the process of cell division everything within the cell must be duplicated in order to ensure the survival of the two resulting daughter cells. o It is the synthesis of a new DNA from the originally existing "template“ DNA. o It makes an identical copy (duplicate) of the existing one 1. DNA replication must possess a very high degree of fidelity. 2. The entire process of DNA replication is complex and involves multiple enzymatic activities. 3. Occurs during the S phase of the cell cycle 4. Starts at specific sites in DNA 5. Involves several proteins and enzymes.  The Cell Life Cycle Gap 1 - Doubling of cell size. Synthesis of DNA - Regular cellular Regular cell activities. activities cease and transcription and translation etc. S a copy of all nuclear DNA is made Gap 2 - Final G1 G2 preparation for division M Mitosis - Cell division 6. Occurs once during a cell cycle. 7. Involves the whole genome. 8. Is template dependant, directed by base pairing. 9. Requires primer for initiation 10. Is semiconservative. 11. Proceeds in 5à3 direction. 12. Occurs bidirectionally and simultaneously in both strands 13. Semidiscontinuous Replication is Bidirectional BIDIRECTIONAL REPLICATION Origin 5’ 3’ 3’ 5’ o The mechanics of DNA replication was originally proposed by Watson and Crick in (1953), o DNA replication was originally characterized in the bacterium ,E. coli o E. coli contains 3 distinct enzymes capable of catalyzing the replication of DNA, identified as DNA polymerase (pol) I, II, and III.  E.Coli DNA polymerases DNA DNA polymerase-III. polymerase-I. DNA polymerase-II  Major steps of DNA replication o Identification of origin of replication o Unwinding of double stranded DNA o Formation of the replication bubles o Establishment of replication fork. o Initiation of DNA synthesis. o Elongation of DNA. o Termination o Reconstitution of chromatin structure.  The proteins in replication include: o Processivity accessory proteins o Helicase o Single strand binding proteins o Primase o Topoisomerases o DNA polymerase (pol) I, II, and III. o DNA ligase Steps of Replication o Replication starts at specific site known as origin of replication (Ori). o The dnaA protein identifies and binds to specific sequences in the Ori (AT rich area ). o The two parent strands are unwound with the help of DNA helicases. Figure 11.5 Figure 11.6 n DNA replication is initiated by the binding of DnaA proteins to the DnaA box sequences 11-17 Figure 11.6 Composed of six subunits Travels along the DNA in the 5’ to 3’ direction Uses energy from ATP Bidirectional replication 11-18 o Single stranded DNA binding proteins (SSB) attach to the unwound strands, preventing them from winding back together. o The strands are held in position, binding easily to DNA polymerase, which catalyzes the elongation of the leading and lagging strands. ► Asthe two strands of the double helix are separated, a problem is encountered, namely, the appearance of positive supercoils (also called supertwists) in the region of DNA ahead of the replication fork ► Type I DNA topoisomerases: These enzymes reversibly cut one strand of the double helix. ► Type II DNA topoisomerases: These enzymes bind tightly to the DNA double helix and make transient breaks in both strands. ► They have both nuclease (strand-cutting) and ligase (strand-resealing) activities.  Direction of DNA replication ► The DNA polymerases responsible for copying the DNA templates are only able to “read” the parental nucleotide sequences in the 3′→5′ direction. ► They synthesize the new DNA strands in the 5′→3′ (antiparallel) direction. ► Therefore, beginning with one parental double helix, the two newly synthesized stretches of nucleotide chains must grow in opposite directions : ► One in the 5′→3′ direction toward the replication fork. ► One in the 5′→3′ direction away from the replication fork o Primase, (A specific RNA polymerase) which is one of several polypeptides bound together in a group called primosomes, helps to build the primer. o synthesizes the short stretches of RNA (approximately 10 nucleotides long) that are complementary and antiparallel to the DNA template.  Replication Overall direction 3’ of replication 3’ 5’ 5’ 3’ 5’ 3’ 5’ Figure 11.10 Innermost phosphate  Leading strand synthesis ► the leading strand is defined as the new DNA strand at the replication fork that is synthesized in the 5'→3' direction in a continuous manner. ► On the leading strand DNA polymerase III is able to synthesize DNA using the free 3' OH group donated by a single RNA primer and continuous synthesis occurs in the direction in which the replication fork is moving.  Replication Overall direction 3’ of replication 3’ 5’ 5’ Okazaki fragment 3’ 5’ 3’ 5’ 3’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.  Lagging strand synthesis ► The lagging strand is the DNA strand at the opposite side of the replication fork from the leading strand, running in the 3' to 5' direction. ► Because DNA polymerase III cannot synthesize in the 3'→5' direction, the lagging strand is synthesized in short segments known as Okazaki fragments. Along the lagging strand's template, primase builds RNA primers in short bursts.  Replication Overall direction 3’ of replication 3’ 5’ 5’ Okazaki fragment 3’ 5’ 3’5’ 3’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.  Replication 3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ 3’5’ 3’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.  Replication 3’ 3’ 5’ 5’ 3’ 5’ 3’5’ 3’5’ 3’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments. ► The RNA fragments are then removed by DNA polymerase I and new deoxyribonucleotides are added to fill the gaps where the RNA was present. ► DNA ligase then joins the deoxyribonucleotides together(phosphodiester bond), completing the synthesis of the lagging strand. DNA pol. III and I : lagging strand DNA pol. I  Keep the parental Breaks the hydrogen strands apart bonds between the Synthesizes daughter two strands DNA strands III Alleviates supercoiling Covalently links DNA fragments together Synthesizes an RNA primer Figure 11.7 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 11-28  Proofreading ► DNA polymerase III has, in addition to its 5′→3′ polymerase activity, a 3′→5′ exonuclease. ► As each nucleotide is added to the chain, DNA polymerase III checks to make certain the added nucleotide is, in fact, correctly matched ,If it is not, the 3′→5′ exonuclease activity edits the mistake. ► The 5′→3′ polymerase then replaces it with the correct nucleotide.  Termination ► 1. Leading strand closes in on other lagging strand ► 2. Ligase completes new strand ► 3. Daughter strands paired with original strands ► - semi-conservative replication Summary o It is the most abundant replicating enzyme in E. coli o Removes the primers from the lagging strand o Ensure the fidelity of replication through the repair of damaged and mismatched DNA ► DNA polymerase I also has a 5′→3′ exonuclease activity. ► DNA polymerase I removes the RNA nucleotides by the 5′→3′ exonuclease activity. ► DNA polymerase I replaces it with deoxyribonucleotides (5′→3′ polymerase activity). ► it also “proofreads” the new chain using 3′→5′ exonuclease activity o It’s primary role is to ensure the fidelity of replication through the repair of damaged and mismatched DNA (proof-reading) o This enzyme is much less abundant than pol I, however, its activity is nearly 100 times that of pol I. o It is responsible for the bulk replication of the E. coli genome. o There have been 5 distinct eukaryotic DNA polymerases identified, α, ß, γ, δ and ε. o The identity of the individual enzymes relates to its sub-cellular localization as well as its primary replicative activity o The enzyme of eukaryotic that is the equivalent of E. coli pol-III is pol-δ. o The pol-I equivalent in eukaryotes is pol-α. o Pol-γ is responsible for replication of mitochondrial DNA o Pol-II is equivalent to pol-ε and responsible for proofreading and repair. o Pol-β is responsible for DNA repair ► The ability of DNA polymerases to replicate DNA requires a number of additional accessory proteins. ► Thecombination of polymerases with several accessory proteins yields DNA polymerase holoenzyme. ► DNA helicase (which "unzips" the DNA at the hydrogen bonds) unwinding ► Topoisomeras The cell uses topoisomerases to relieve tortional stress. ► SSB's(single strand binding proteins, which hold the DNA strands apart while they are replicated ► RNA primase (which lays down RNA nucleotide "primers" on the template) ► DNA polymerase (which adds new dATP, dTTP, dGTP and dCTP to the growing strand ► DNA ligase (which joins the fragments on the lagging strand.) oWhile the DNA polymerase on the leading strand can operate in a continuous fashion, RNA primer is needed repeatedly on the lagging strand to facilitate synthesis of Okazaki fragments. Repair ► Despite the elaborate proofreading system employed during DNA synthesis, errors including incorrect base-pairing or insertion of one to a few extra nucleotides can occur. ► In addition, DNA is constantly being subjected to environmental insults that cause the alteration or removal of nucleotide bases. ► The damaging agents can be either chemicals, for example, nitrous acid, or radiation, for example, ultraviolet light ► Repair of damaged DNA is critical for maintaining genomic integrity and thereby preventing the propagation of mutations: ► Horizontally, that is DNA sequence changes in somatic cells ► Vertically, where nonrepaired lesions are present in sperm or oocyte DNA and hence can be transmitted to progeny. ► The mechanisms of DNA repair include : ► Nucleotide excision repair (NER) ► Mismatch repair (MMR) ► Base excision repair (BER) ► Homologous recombination (HR) ► Nonhomologous end-joining (NHEJ) ► HR & NHEJ used for DNA double- strand breaks (DSBs). ► Most of the repair systems involve : ► 1.Recognition of the damage (lesion) on the DNA. ► 2. Removal or excision of the damage. ► 3.Replacement or filling the gap left by excision using the sister strand as a template for DNA synthesis. ► 4.Ligation. ► Excision repair can be divided into nucleotide excision and base excision.  Methyl-directed mismatch repair (nucleotide excision repair) ► Identification of the mismatched strand: When a mismatch occurs, the Mut proteins that identify the mispaired nucleotide(s) must be able to discriminate between the correct strand and the strand with the mismatch. ► Discrimination is based on the degree of methylation. ► Thismethylation is not done immediately after synthesis, so the newly synthesized DNA is hemimethylated (that is, the parental strand is methylated but the daughter strand is not).  Repair of damaged DNA ► An endonuclease nicks the strand. ► An exonuclease remove the mismatched nucleotide(s). ► The gap left by removal of the nucleotides is filled, using the sister strand as a template, by a DNA polymerase. ► DNA ligase  Repair of damage caused by ultraviolet (UV) light ► Exposure of a cell to UV light can result in the covalent joining of two adjacent pyrimidines (usually thymines), producing a dimer. ► These thymine dimers prevent DNA polymerase from replicating the DNA strand beyond the site of dimer formation. Thymine dimer  UV radiation and cancer Xeroderma Pigmentosum § Pyrimidine dimers can be formed in the skin cells of humans exposed to unfiltered sunlight. § In the rare genetic disease xeroderma pigmentosum, the cells cannot repair the damaged DNA. ( Repair Defect ). § Extensive accumulation of mutations and, consequently, skin cancers occur.  Two ways to repair double strand breaks ► Nonhomologous end joining alters the original DNA sequence when repairing a broken chromosome. ► The initial degradation of the broken DNA ends is important because the nucleotides at the site of the initial break are often damaged and cannot be ligated. ► Nonhomologous end joining usually takes place when cells have not yet duplicated their DNA. ► Repairing double-strand breaks by homologous recombination is more difficult to accomplish but restores the original DNA sequence. ► It typically takes place after the DNA has been duplicated (when a duplex template is available) but before the cell has divided.