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This 5 page Class Notes was uploaded by Udbluehen03 on Friday October 7, 2016. The Class Notes belongs to BISC401 at University of Delaware taught by Lachke,Salil in Fall 2016. Since its upload, it has received 6 views. For similar materials see Molecular Biology of the Cell in Biology at University of Delaware.
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Date Created: 10/07/16
DNA is the genetic material and not the proteins New DNA strands are synthesized by sing the existing (parent) strands as templates in the formation of new daughter strands that are complimentary to the parent strands The pairing of complementary bases in DNA through hydrogen bonding means that the information contained within each strand is redundant. The nucleotides on a single strand can therefore be used to reconstruct nucleotides on a newly synthesized partner strand Three Models of DNA Replication - Dispersive o New double helix: A composite of Parent and Daughter regions - Conservative o The two daughter strands would form a new double-stranded DNA molecule and the parent duplex would remain intact o Would never generate H-L DNA (Heavy-light) (hybrid DNA) - Semi conservative o The parent strands would be permanently separated and each would form a duplex molecule with the newly synthesized daughter strand base-paired to it o DNA replication is semi conservative Parent strand –> heavy Generation 1 –> 2 Heavy – Light Generation 2 –> 2 Heavy – Light and 2 Light DNA is synthesis occurs only in the 5’ to 3’ direction Directionality of DNA (antiparallel strands) has consequences for DNA synthesis, because DNA polymerase can synthesize DNA in only one direction by adding nucleotides to the 3' end (i.e. extending the 3’ end) DNA polymerase cannot initiate chain synthesis de novo - They require a short, preexisting RNA or DNA strand, called a primer, to begin growth - With a primer base-paired to the template strand, a DNA polymerase adds deoxy ribonucleotides to the free hydroxyl group at the 3’ end of the primer as directed by the sequence of the template strand DNA Replication - In order for duplex DNA to function as a template during replication, the two interwined strands must be unwound or melted to make their bases available for pairing with the bases of the dNTPs that are polymerized into the newly synthesized daughter strands - The unwinding of the parent DNA strands is performed by enzymes called helicases - Unwinding begins at segments in a DNA molecule called replication origins or simply origins - Once helicases have unwound the parent DNA at an origin, a specialized RNNA polymerase called primase forms a short RNA primer complementary to the unwound template strands - The primer is elongated by DNA polymerase α for another 25 nucleotides or so, forming a primer made of RNA at the 5’ end and DNA at the 3’ end Replication Fork - The DNA region at which all proteins come to together to carry out the synthesis of daughter strands - As replication proceeds, the replication fork and the associated proteins move away from the origin - Local unwinding of duplex DNA produces torsional stress, which is relieved by topoisomerase I o In order for DNA polymerase to move along and copy a duplex DNA, helicase must sequentially unwind the duplex and the topoisomerase must remove the supercoils that form - Synthesis of one daughter strand, called the leading strand, can proceed continuously from a single RNA primer in the 5’ to 3’ direction, the same different as movement of the replication fork - Problem in synthesis of the other daughter strand, lagging strand o Growth of the lagging strand must occur in the 5’ -> 3’ direction, copying of its template strand must somehow proceed in the opposite direction from the movement of the replication fork o A cell accomplished this feat by synthesizing a new primer every 100 to 200 nucleotides on that template strand as more of the strand is exposed by unwinding o Each of these primers, base-paired to the template strand, is elongated in the 5’ -> 3’ direction, forming discontinuous segments named Okazaki fragments o The RNA primer of each Okazaki fragment is removed and replaced by DNA chain growth from the neighboring Okazaki fragment o Finally, an enzyme called DNA ligase joins the adjacent fragments The challenges of replicating DNA 1. DNA needs to be unwound, i.e. the two strands need to be separated. Answer: Initiator, Helicase proteins 2. The separated DNA strands need to be kept separated for some time. Answer: SSB Proteins (Single-Strand Binding Proteins) 3. Supercoils induced in upstream DNA need to be removed. Answer: Topoisomerase, gyrase proteins 4. DNA cannot be synthesized without a “primer” sequence. Answer: Primase protein 5. DNA needs to be synthesized by adding nucleotides… Answer: DNA Polymerase protein complex Bidirectional DNA replication mechanism - In theory, DNA replication from a single origin could involve one replication fork that moves in one direction - Alternatively, two replication forks might assemble at a single origin and then move in opposite directions, leading to directional growth of both daughter strand - Eukaryotic DNA contains multiple replication origins separated by tens to hundreds of kilobases. - A six-subunit protein origin recognition complex (ORC) binds to each replication origin and associates with other proteins required to load two hexameric helicases, composed of six homologous MCM proteins, oriented in opposite directions. RPA proteins bind to separated parent strands at the origin. - Replication at the origins is coordinated by DDK (kinase) activation of MCM helicase. DDK is regulated by S-phase cyclin-dependent kinases. - Two large T-antigen hexameric helicases bind at the replication origin in opposite orientations. • Step 1: Helicases use ATP hydrolysis energy to move in opposite directions, unwinding the parent DNA and generating single-stranded templates, which are bound by RPA proteins. • Step 2: Primase–Pol α complexes synthesize short primers (red) base-paired to each of the separated parent strands. • Step 3: PCNA-Rfc–Pol ε complexes replace the primase–Pol α complexes and extend the short primers, generating the leading strands (dark green) at each replication fork. • Step 4: The helicases continue to unwind the parent strands, and RPA proteins bind to the newly exposed single-stranded regions. • Step 5: PCNA-Rfc–Pol ε complexes extend the leading strands. • Step 6: Concurrently, primase–Pol α complexes synthesize primers for lagging- strand synthesis at each replication fork. • Step 7: PCNA-Rfc–Pol δ complexes displace the primase–Pol α complexes and extend the lagging-strand Okazaki fragments (light green), which are ligated to the 5′ ends of the leading strands. - Unwinding and synthesis of leading and lagging strands occur concurrently SV40 DNA - Circular genome of a virus that infects monkeys - The molecular machine that replicates SV40 DNA contains only one Viral protein; all other proteins involved in SV40 DNA replication are provided by the host cell - The viral protein is large T antigen - Forms a hexameric replicative helicase - A protein that uses energy from ATP hydrolysis to unwind the parent strands at a replication fork - Primers for the leading and lagging daughter strands are synthesized by a complex of primase, which synthesizes a short RNA primer and DNA polymerase α (Pol α) o (Pol α) extends the RNA primer with deoxy ribonucleotides for another 25 nucleotides or so, forming a mixed RNA and DNA primer - The primer is extended into daughter-strand DNA by DNA polymerase δ (Pol δ) o (Pol δ) is less likely to make errors during copying of the template strand than is Pol α because of its proofread mechanism - During the replication of cellular DNA Pol δ synthesis lagging strand DNA, while DNA polymerase £ (Pol £) synthesis most of the length of the leading strand - Pol δ and Pol £ each form a complex with PCNA (proliferating cell nuclear antigen) following primer synthesis - PCNA o A homotrimeric protein that has a central hole through which the daughter duplex DNA passes, thereby preventing the PCNA-Pol δ and PCNA-Pol £ completes from dissociating from the template - RFC o Replication factor C o A pentameric protein o Opens the PCNA ring so that it can encircle the short region of double-stranded DNA synthesized by Pol α - Leading strand is extended by Pol £ which can extend the growing strand up to the replication fork - Lagging strand is bound by multiple copies of RPA (replication protein A) o A heterotrimeric protein o Maintains the template in a uniform conformation that is optimal for copying by Pol δ - Bound RPA proteins are dislodged from the protein strand by Pol δ as it synthesizes the complementary strand base paired with the parent strand
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