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DNA Replication in Eukaryotes.doc

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Kathleen Gilmour

DNA Replication in Eukaryotes Key Points -DNA structure is anti-parallel -oriented from 5' to 3' -synthesis occurs from 5' to 3' due to chemistry -if synthesizing from 5' to 3', in order to make a copy that contains a complement and is anti-parallel, the bases are read 3' to 5' -Helicase enzymes unwind DNA -topoisomerases change the topology of DNA to help it unwind -replication begins at a defined site (the origin) and is bi-directional -in bacteria, it is known as the origin (ori) -in eukaryotes, it is known as the ars (autonomously replicating sequence) -there are many ars sequences on each chromosome because of its large size -DNA polymerase cannot synthesize de novo -it cannot synthesize without something starting it off (a pre-existing primer) -it requires a primer (i.e.: RNA primer) -telomerases carries its own RNA template to finish replicating at the ends of linear DNA molecules Why do we need 3 types of DNA synthesis for a chromosome? (And multiple polymerases in the cell)? -the enzymes of DNA replication include: -helicase: unwinds the DNA -unwinds the helix to make the templates available using ATP -primase: synthesizes RNA primer (starting point for nucleotide assembly by DNA polymerases) -creates the RNA primer that DNA will extend upon -synthesize RNA primer de novo -DNA polymerases: assembles nucleotides into a chain, removes primers, fills resulting gaps -a complex of protein -has many functions (i.e.: removing primers and polymerizing activity) -DNA ligase: closes remaining single-chain nicks -DNA polymerase cannot start synthesis de novo -requires a primer to initiate synthesis -the primer can be a suitable DNA molecule (which has a 3' OH) and that can be extended -RNA polymerases can synthesize a RNA primer de novo -Assembling Antiparallel Strands -the newly synthesized strand is oriented from 5' to 3' from left to right -the template is copied in the order from 3' to 5' -there is discontinous synthesis on one strand because you have to reprime it Enzyme Activities 1) DNA Helicase -unwinds DNA at the replication fork 2) Topoisomerase -as you unwind the DNA, the super-coiling must be relieved -reduces the super-coiling before helicase fully unwinds the DNA -e.g.: DNA gyrase (changes gyration / writhing in space) 3) Single-Stranded DNA Binding Proteins -a protein that binds to single strands of DNA to prevent them from forming stem-loop structures and prevent hydrogen bonding -allows bases to be available for polymerizing complex -a.k.a. SSBs 4) Primase -removed by RNase-H 5) DNA Polymerase 6) Ligase -joins nicks in the DNA -joins 3' OH groups -segments of RNA and DNA are called Okazaki fragments 7) Telomerase What happens when you need more or faster DNA synthesis? -DNA polymerase has a maximal rate at which they can synthesize -synthesis must be started more frequently by starting at different locations -in humans, there are many ars (origins of replication) -more origins = faster rate -visible as bubbles along the chromosome -replication proceeds from the origins as two replication forks moving in opposite directions -in E.coli (bacteria): -the bacterial genome is a circular replicon -replication initiates at a site of origin of replication known as (oriC) and proceeds in a bidirectional manner until the terminus is reached (ter) -ori binds proteins that recruit polymerases to tell it to start -ter binds proteins that recruit polymerases tha
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