B2.060 DNA Replication Flashcards
what phase of the cell cycle does DNA replication take place during?
S
synthesis
what does semi conservative mean?
parental strands are the template for synthesis of new DNA
what direction is DNA synthesized in?
nucleotides added to the 3’ end of growing DNA polymer
what are the steps of DNA synthesis
- Identify origins of replication
- Unwind dsDNA
- Initiate (primer synthesis)
- Elongate (fix over-winding)
- ligate newly synthesizes DNAs on lagging strand
- terminate replication
what is the function of the origin recognition complex?
ORC binds to origin DNA at late M/early G1 phase
what is the function of cdc6 +cdt1 in replication?
recruit/load six subunit MCM helicase complex onto origin during late M/ early G1 phase
what happens when cdc6 and cdt1 are release?
exit nucleus and are degraded until after mitosis
this makes sure only one replication event occurs per cell cycle (licensing replication)
what are the components of the pre-replicative complex?
ORC
cdt1
cdc5
MCM
what occurs in the transition from pre-replicative to replicative complex?
CDK phosphorylated MCM, activating helicase
MCM unwinds DNA
ORC leaves
ssDNA coated by replication protein A (RPA)
how are primers synthesized?
- DNA primare makes a 10-15 nt RNA
2. DNA pol alpha adds 30 nt onto RNA
how many primers are at the beginning of the leading strand?
one
how many primers are required on the lagging stand?
many for ongoing synthesis
what enzyme synthesizes DNA?
DNA pol d/e
PCNA (proliferating cell nuclear antigen) clamos the DNA pol d/e onto the DNA
what are okazaki fragments?
new DNA on lagging strand
1000 bp long
each has RNA primer
must be joined together
what is the over winding problem?
strand separation at the replication fork causes positive supercoiling of the double helix downstream
what enzyme helps w the over winding problem and how?
topisomerase 1 (DNA gyrase in bacteria) introduces negative supercoils ahead of fork by creating a single stranded nick which allows DNA to spin
describe the process of okazaki fragment maturation
- DNA pol d/e initiates at primer, elongates DNA to next primer
- DNA pol d/e displaces primer and endonuclease cuts off “flap”
- DNA pol d/e fills in DNA, leaves single nick
- DNA ligase seals the nick w AT{
what is a nick?
absence of a phosphodiester bond in a DNA backbone
what is telomerase and what is its function?
reverse transcriptase enzyme
helps lengthen telomeres that tend to shorten w successive replication events
what is the Hayflick limit?
approx. 50 cell divisions before senescence
due to shortening of chromosomes
which types of cells express telomerase?
stem cells cancer cells (immortality)
what are some mechanisms as using DNA replication as a drug target?
block/reduce synthesis of nucleotides
inhibit DNA polymerase
cross link DNA strands; no separation
incorporation of DNA analogues into growing chains
what types of diseases can be targeted at DNA replication?
viral/microbial infections
neoplastic diseases
how do drugs like acyclovir /foscarnet select for viruses and not destroy all cells?
- viral enzyme needed to phosphorylate/activate drug
2. drug blocks viral DNA polymerase specifically
what are the 3 checkpoints of cell cycle control?
G1-S: integrity of DNA
-R: restriction point (late G1)
G2-M: DNA duplication
M: attachment of kinetochores to spindles
what 2 key proteins are involved in cell cycle control?
p53- genome integrity/replication
RB-conditions are “pro-division”
describe the cyclin-CDK-RB process
cyclin proteins are expressed at specific points in the cell cycle
CDKs are activated when bound to cyclins
phosphorylation of RB allows cell cycle to proceed past checkpoints
what happens to p53 under normal conditions?
p53 degrades during cell cycle
allows for progression past chekpoints
what happens to p53 during cell stress/DNA damage?
p53 stabilizes, halts cell cycle
cells recover>transverse cell cycle
damage too severe>apoptosis
what are the 3 classes of regulators?
cyclins
CDKs
CDK inhibitors
how are CDK inhibitors controlled?
signaling events indicating cell is not ready to divide
p53 increases translation of CDK inhibitors too
what phosphorylates cdc6 and cdt1?
CDKs
