topic 5 - DNA replication Flashcards
(34 cards)
how often do Eukaryotic Chromosomes Replicate? what is a challenge of this?
- once per cell cycle
- multiple origins of replication
What is a requirement of deoxynucleoside
triphosphate
primer:template junction
what is processivity? Name an example of a processive enzyme and its function
an enzyme’s ability to catalyze “consecutive reactions without releasing its substrate”
- average number of nucleotides added each time when an
enzyme binds a primer:template junction
- DNA polymerase
- catalyzes DNA synthesis using a single active site for any dNTP
functions of DNA polymerase
- sterically distinguish between dNTPs (3’-OH) and its rNTPs (2’-OH + 3’-OH) precursors
- show kinetic selectivity for adding correct base-paired dNTP (i.e., A-T and G-C)
- initiate synthesis using either RNA or DNA primer annealed to the template
describe the DNA polymerase palm analogy
finger - bind + enclose dNTP with
catalytic metal ions in palm
thumb - maintaining: correct position of primer and active site; and strong association b/ween DNA pol and its substrate
palm - catalytic site, monitor bp-ing of the most recently added nucleotides
What removes RNA primers from RNA:DNA hybrid to complete DNA
synthesis?
RNase H
replacement of DNA Pol α with
DNA Pol ε at the ______ strand
and Pol δ at the ______ strand
polymerase switching:
replacement of DNA Pol α with
DNA Pol ε at the leading strand
and Pol δ at the lagging strand
what is a replicon
DNA replicated from a particular origin of replication
how many proteins does the Origin Recognition Complex have and what is its function
- 6-protein complex
- ## recognizes replicators such as DNA helicase
sequence of Replicator inactivation by DNA Replication
- 3 and 5 activate
- 2 and 4 activate by extension of 3 and 5
- 1 turns on
- they each become inactive after they’ve replicated to prevent repeats
in what phase does origin activation occur? What does it trigger?
S phase
-triggers replicator associated
complex to initiate DNA unwinding and DNA Pol recruitment
In what phase does replicator selection occur? what does this process do?
- G1 phase
- identify sequences that will direct initiation of replication
describe the steps in helicase loading:
What stage of the cell cycle does this occur?
- recognition of the replicator by the eukaryotic initiator, ORC
- ORC recruits helicase loading proteins (Cdc6 and Cdt1) and Mcm2-7 helicase (costs ATP)
- during G1
describe helicase activation:
- 2 kinases (CDK and DDK) phosphorylate Sld2/3 and Mcm2-7
- CMG complex forms (Cdc45/Mvm2-7/GINS)
- DNA Pol ε (leading strand) recruited BEFORE unwinding
- DNA Pol α/primase and DNA Pol
δ recruited after unwinding
impact of high vs low cdk
low CDK: loading of euk DNA helicase
high CDK: initiate DNA replication
relative amounts of CDK in each stage of the cell cycle:
G1: CDK = low, helicase is loaded, BUT can’t be activated
S: CDK = high, helicase activated to initiate DNA replication
* used helicase is disassembled
after DNA replication
S, G2, and M: CDK stays high, prevents loading new helicase; restricts 1 DNA replication/cell cycle
What recognizes the replicator?
initiator protein
Which phase:
ORC + helicase loading proteins → assembly of DNA helicase on the replicator
G1 phase
Which phase:
activation of helicase → DNA unwinding and assembly of DNA Pols to synthesize RNA primers
S phase
circular DNA vs linear DNA replication
- circular DNA can be completely replicated
- separation of the two daughter DNA molecules by topo II
why does a little bit of DNA get lost during replication?
since a primer is needed for replication, the end segment doesn’t have anything to use as its primer so that section doesn’t get replicated.
solutions to prevent ends from not being replicated:
- protein as primer instead of RNA at each end
- use of telomere sequence
How do proteins substitute primers at the ends of chromosomes?
- OH of an amino acid substitutes for 3’-OH of RNA primer
- protein allows priming at last nucleotide -> no sequence is lost
how does the telomere sequence substitute primers?
- heat-to-tail repeats of a TG-rich DNA sequence
- serve as origin of replication for 3’
- use telomerase as the DNA pol
- e.g., human telomeres have head-to-tail repeats of 5’-TTAGGGTTAGGGTTAGGG-3’
