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4. DNA Replication.pdf

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Biology (Sci)
BIOL 200
Richard Roy

Naveen Sooknanan McGill Fall 2011 DNA Replication: DNA needs to replicate before cell division in order to produce daughter strands which are DNA duplexes identical to the parental DNA molecule.  DNA needs to replicate faithfully so as to not transfer mutations to RNA, and eventually, to proteins  Mutations can alter protein shape altering their function or rendering them useless altogether In DNA replication, either parental DNA strand can be used as a template to synthesize a corresponding daughter strand, producing two daughter strands in total which are identical to the parental duplex  Matthew Meselson and Franklin Stahl discovered that DNA replicated in a semi- conservative manner In the conservative replication mechanism, the parental DNA molecule remains intact after replication producing the original strand and a completely new daughter strand  The parental DNA duplex remains intact In the semiconservative replication mechanism, the parental duplex splits up after the first replication creating ½ new and ½ old duplexes  In the second replication, each of these strands is used as a template producing two hybrid DNA duplexes and two completely new ones Meselson and Stahl discovered that DNA replicated in a semiconservative manner by conducting the following experiment on an E. coli gene:  Parental DNA was synthesized using N (heavy nitrogen)  These parental DNA molecules were placed in a medium containing N where it was allowed to replicate twice o This assured that any daughter strands would be lighter tha14parental DNA because they would be made from N o Upon centrifugation, parental DNA duplexes would produce heavy bands, new DNA would produce a light band, and hybrid DNA would produce a medium band  The centrifugated product after 1 replication showed only medium bands, showing a semiconservative mechanism  Upon 2 replications, the centrifugated product showed medium and light band, showing the presence of only hybrid and new strands  This shows that DNA replicates in a semiconservative manner Nucleotide polymerization in replication requires DNA polymerase as was as deoxynucleoside 5’ triphosphates (dNTPs)  Replication also requires a primer which can be either DNA or RNA 1Naveen Sooknanan McGill Fall 2011 o Primers are usually RNA in vivo, whereas DNA primers are typically used in vitro o Contrasts RNA polymerization, which does not require a primer  Replication proceeds in the 5’  3’ direction like in RNA polymerization  dNTPs are added one by one to the free 3’ hydroxyl end of the primer  The primer can be very short, only 5-100 nucleotides long and it anneals to a specific sequence on the template strand  Every annealed dNTP releases 1 pyrophosphate molecule  The primer is removed after replication and replaced with DNA In order for replication to occur, the duplex must first be unwound. This is done by an enzyme called DNA helicase.  Unwinding is initiated at specific points on the DNA duplex called origins of replication o Prokaryotes usually only have one origin of replication whereas eukaryotes have multiple origins  Unwinding causes a replication “bubble” at the replication site  Origins tend to be AT rich because the weaker AT bonds are easier to break because they only have 2 H bonds The primer, which anneals to the template strand, is synthesized by a specific RNA polymerase called primase. Primase involved RNA polymerization and does not require a primer  DNA polymerase will use this primer to extend the daughter strand The unwinding of DNA by helicase can cause supercoiling ahead of the replication fork. These supercoils are relieved by an enzyme called topoisomerase  Mutations of topoisomerase can be lethal DNA replication occurs in the 5’  3’ direction and the two strands of DNA are antiparallel.  One strand is able to synthesize a daughter strand in the direction of unwinding without interruption. This strand is called the leading strand and undergoes continuous replication  The other strand, called the lagging strand, cannot synthesize a new strand in the 5’  3’ direction and must be synthesized in interrupted fragments Lagging strand synthesis must proceed by creating short fragments called Okazaki fragments  RNA primers anneal to the lagging strand  DNA polymerase elongates the strand until it hits the origin or another RNA primer o Elongation occurs opposite to the direction of unwinding  The previous primer is removed and is replaced with dNTPs from DNA polymerase o This leaves a break in the sugar phosphate backbone which must be repaired by DNA ligase  The size of the Okazaki fragments depends on the species, or whether the organism is a eukaryote or prokaryote  This process is true for both prokaryotes and eukaryotes 2(c) RPA. Single- DNA (c) RPA. Single- DNANaveen Sooknanan McGill Fall 2011 As a eukaryotic model, we will use a viral called SV40 which infects monkeys  Much of what we know about DNA replication has been learned from bacteria like E. coli and viruses like SV40  Viruses are typically small in genome size because they leech enzymes from their host cells SV40’s genes only encodes for a protein called a large T antigen, which is a hexamer (6 subunits)  The large T antigen is a viral “helicase” which replaces the monkey’s own helicase enzymes o Large T antigen is capable of replicating DNA at a much faster rate than typical helicase enzymes, which benefits the virus  This replication rate allows for the SV40 virus to hijack the host genome and cell enzymes In eukaryotes, a replication protein A (RPA) binds to single stranded DNA that has been unwound within the replication bubble  This keeps the DNA in optimal conformation for copying by DNA polymerase o Prevents the DNA from folding onto itself  RPA has specific secondary structure containing beta sheets which hook the single stranded DNA in place On the leading strand, DNA polymerase δ (Pol δ), replication factor C (RFc) and proliferating cell nuclear antigen (PCNA) carry out DNA synthesis:  RPA is displaced by this complex as replication proceeds  PCNA is a homotrimeric (contains three identical subunits) which loads and removes the RFc/ Pol δ complex and keeps it from dissociating from the template strand Lagging strand synthesis again uses the process of Okazaki fragment synthesis but works a bit differently  Primase and Pol α form a complex on the template o Primase creates an RNA primer which Pol α extends only a few nucleotides  Pol δ/RFc/PCNA complex then replaces the prior complex and extends the Okazaki fragment  Ribonuclease H and FEN-1 displace the RNA primer at the 5’ end of the Okazaki fragment o Only displaces an RNA on an RNA/DNA hybrid, regardless of location  Pol δ then replaces the primer with DNA and the broken sugar phosphate backbone is ligated with DNA ligase  This forms 1 continuous strand of DNA Using a circular viral chromosome which was cleaved into a linear form by an endonuclease called EcoR1, George C. Fareed and Norman P. Salzman discovered that DNA replication proceeds bidirectionally
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