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86 Cards in this Set
- Front
- Back
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During which phase of the cell cycle does DNA replication occur?
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S phase
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Where do initiation events occur during DNA replication?
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The origin of replication
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What are ter sites?
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In prokaryotes, sites of termination of DNA synthesis if the elongating DNA go "too far" around the circle.
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What are the four roles of DNA?
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"1 - DNA makes copies of itself
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Why is DNA replication the key to perpetuation of life?
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Life is distinguished from non-life by its ability to copy information and pass it from generation to generation.
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Who predicted that DNA's pairing mechanism suggests a possible copying mechanism?
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Watson and Crick, 1953
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Which experiment confirmed the process of semi-conservative replication?
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Meselson and Stahl, 1958
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What is meant by semi-conservative replication?
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Each strand of the original duplex becomes one strand of another duplex
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What is a replicon?
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All of the DNA replicated from a particular origin of replication (e.g., the E.coli chromosome)
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What is an initiator?
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A sequence-specific DNA binding protein that binds to a target replicator sequence, melts the 2 DNA strands, and recruits replication proteins to the origin (e.g., helicase)
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What molecules are bound to initiator proteins?
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ATP!
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The bacterial genome is how many replicons?
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One single circular replicon that replicates bidirectionally from a single origin.
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What is the origin of replication called in E.coli and how many base pairs does it consist of?
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oriC is 245 base pairs in length.
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What are the possible steps involved in replicating the bacterial genome?
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"1 - bidirectional replication initiates at the origin
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What is meant by catenated (or concatenaded) chromsomes?
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After replication, the joining of the old and new chromosomes that must be separated for proper segregation.
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Which enzyme is responsible for the separation of concatenated DNA molecules after bacterial replication?
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Topoisomerase II
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What regulates initiation of bacterial DNA replication?
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Methylation of the bacterial origin
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What is the impact of bacterial DNA replication on oriC?
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oriC, the origin of replication in E.coli, becomes hemimethylated during replication. There is a 13 minute delay until the repeats in this region are re-methylated.
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Which enzyme is responsible for methylation of the bacterial origin of replication?
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Dam methylase.
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What is a methyl group?
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The way in which replication is _______ and linked to the ________ is a major difference btween prokaryotes and eukaryotes.
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"CONTROLLED
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In bacteria, these steps in the life of DNA are CONNECTED.
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growth, replication, and segregation
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How long does it take to replicate the bacterial chromosome at normal temperature?
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It requires 40 minutes to replicate the bacterial chromosome (at normal temperature).
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How long after replication of the bacterial chromosome does division occur?
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Completion of a replication cycle triggers a bacterial division 20 minutes later.
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What results from fast bacterial growth?
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Multiforked chromosomes
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What structure divides the dividing bacterium into two progency cells, each containing a chromosome?
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The septum.
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DNA replication in eukaryotes is dependent on the formation of ______________.
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The pre-replicative complex (a multi-protein assembly)
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Formation and activation of pre-RCs are controlled by a cell-cycle regulated ____________.
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cyclin- dependent kinase.
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The progression into the S-phase of the cell cycle is tighly controlled by __________.
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checkpoints
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The number of origins in bacterial chromosomes and eukaryotic chromosomes differ in that ___________.
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"Prokaryotes: one origin per chromosome
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Easily unwould DNA is rich in these nucleotides:
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A-T
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Replicator selection occurs in ___ of cell cycle via ordered assembly of a pre-replicative complex (Pre-RC) at each origin.
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G1
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Origin activation occurs during the ___ phase of the cell cycle.
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S
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Temporal separation of the replicator selection and activation regulates use of origins, ensuring _____________.
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each chromosome is replicated only once in each cell cycle.
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Use of origins is tightly regulated in eukaryotes. They are...
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restricted to once per cell cycle by inactivating them after activation or passive replication
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Licensing factor is required for...
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initiation of replication at each origin.
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Prior to replication, licensing factor is present in the ___.
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nucleus
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Initiation of another replication cycle becomes possible only after ...
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licensing factor re-enters the nucleus during late mitosis
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Incompletely replicated DNA leads to _________ upon anaphase segregation,
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chromosome breakage
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What are MCM proteins?
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Mini cellular maintanance proteins that make up licensing factor.
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What does licensing factor control?
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Eukaryotic re-replication
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What is cdc6 protein?
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Synthesized only in G1, binds to ORC and allows MCM proteins to bind.
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What is prevented by the degradation of cdc6?
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Reinititation for replication
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What controls the origin of eukaryotic DNA replication?
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The ORC complex
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What are the stages of DNA replication?
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"1 - Initiation
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What are the required steps for DNA replication to occur?
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"Unwinding,
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Which enzyme is responsible for the unwinding of DNA?
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Helicase
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What is the name of the reaction that joins nucleotides together with phosphodiester bonds?
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Dehydration synthesis
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Which two reactions involve the synthesis of DNA?
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semi-conservative replication and DNA repair reactions
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In bacteria, which enzyme is responsible for adding nucleotides?
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DNA polymerase III (in a complex with other proteins)
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What is required before DNA polymerase III can add nucleotides?
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a primer
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What is the direction of DNA synthesis by DNA polymerase III?
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5'-->3'
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Which end of a nucleotide does DNA polymerase III add?
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The free 3' end.
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What does DNA polymerase III use as building blocks for DNA synthesis?
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the four nucleotide triphosphates dNTPs
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What are dNTPs?
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nucleotide triphosphates; used by DNA polymerase as building blocks
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Which phosphate of nucleotides are incorporated into the backbone of DNA?
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the alpha phosphate
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What is a DNA:primer junction?
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The transition between dsDNA and ssDNA; the primer required for DNA synthesis
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What is an incorporation assay?
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"a means of determining how much DNA has been synthesized after replication
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What is the similar structure that DNA polymerases share?
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A large cleft and are composed of three domains that resemble a hand.
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DNA polymerases have more than one ______.
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active sites
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What are sliding DNA clamps?
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associated with DNA polymerases and give them high processivity
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Whare loads and removes sliding DNA clamps to/from DNA?
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sliding clamp loaders
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What is a replisome consisted of?
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Association of helicase and primase with the DNApol III holoenzyme at the replication fork (E. coli)
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How many types of DNA polymerases are required at the replication fork?
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three
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What is the speed of DNA polymerase adding nucleotides? How many nucleotides are added per binding event?
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up to 1,000 nt/sec, and 50,000 nt added per binding event
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Why do DNA polymerases have more than one active site?
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One for DNA synthesis and one for DNA proofreading (exonuclease activity)
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In which direction is the exonuclease activity of DNA polymerase?
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3--> 5 ???
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Which strand of DNA is synthesized continuously by DNA polymerase?
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The leading strand.
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Which strand of DNA is synthesized in fragments by DNA polymerase?
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The lagging strand.
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What are the fragments synthezied on the lagging strand called?
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Okazaki fragments
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How do the sizes of Okazaki fragments differ in prokaryotes and eukaryotes?
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Okazaki fragments are 1,000-2,000 nt long in bacteria, but only 100-400 nt in eukaryotes.
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What is the name and class of the enzyme that adds the first nucleotide in DNA synthesis?
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Primase, an RNA polymerase.
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What is required for priming of replication on double-stranded DNA?
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"1- primase
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What is the name of the helicase that unwinds dsDNA to form the replication fork for the replication in E.coli?
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DnaB
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What is the name of the primase in E.coli?
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dnaG
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Which enzyme joins Okazaki fragments?
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"DNA ligase
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Which enzyme replaces the primer with DNA in E.coli?
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DNA primase I
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What removes the RNA primer from DNA?
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RNase H and an exonuclease (for the last nt)
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The overall process of DNA replication is known as _________ because the leading strand is built in a continuous manner, while the lagging strand is built discontinuously.
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semi-discontinuous
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What is the purpose of ssb proteins?
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Stabilize single strands to prevent re-annealing during replication.
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What is the purpose of topoisomerases? Give an example.
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They relieve torque in coiled DNA. More specifically, they remove positive supercoils by DNA unwinding at the replication fork. ex) DNA gyrase
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What are ALL the enzymes that comprise the replication machinery of E.coli?
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What are the classes of enzymes that function at the replication fork in all organisms? How do the specifics differ?
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Which DNA polymerase synthesizes DNA from the lagging strand in eukaryotes?
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DNA polymerase (delta)
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Which DNA polymerase synthesizes the leading strand of DNA in eukaryotes?
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DNA polymerase (epsilon)
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How does DNA polymerase "switch" in DNA synthesis of eukaryotes?
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"DNA Pol alpha/ primase complex is responsible for initiation (~10 bases RNA then 20-30 nt
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