DNA Replication Flashcards
Direction(s) of replication
Bidirectional
Origins of replication in eukaryotes vs. prokaryotes
Prokaryotes: 1 origin of replication
Eukaryotes: multiple origins of replication
Differences between Okazaki fragment processing in eukaryotes vs. prokaryotes
Prokaryotes: Okazaki fragments get chewed up by DNA pol I
Eukaryotes: Okazaki fragments come off as flaps as synthesis continues past them- flaps get cleaved by endonucleases
Activity of all DNA replication endonucleases
3’ -> 5’ activity (like a delete key)
Replisome
Complex of DNA replication proteins
Proteins recruit, activate, and inhibit one another
Replicator
Sequence containing origin of replication
Initiator
Protein that binds to replicator, initiating replication
Gel electrophoresis mobility shift assay (GEMSA)
Method of determining protein-DNA interaction
DNA is run on gel by itself and with protein bound to it: DNA by itself migrates down the gel faster than DNA and protein together
Initiation of replication steps in E. coli
- Initiators bind to replicator
- Localized unwinding
- Recruitment of helicase and helicase loading protein
- Helicase recruits primase and kicks off initiators
- DNA pol III recognizes DNA and synthesizes off of primer
- Continues until 2 forks run into each other
How many times replicator is used per cell cycle in eukaryotes
Once: makes sure that DNA is only replicated once and is replicated completely
Telomerase
Enzyme that extends the 3’ ends of DNA
2 parts of telomerase
Telomerase RNA (TER): attaches to 3' end of ssDNA and acts as template Telomerase reverse transcriptase (TERT): synthesizes DNA off of RNA template
Inhibition of telomerase
Telomere becomes longer -> more telomere binding proteins accumulate -> telomerase is inhibited
Protein associated with yeast telomeres
Cdc 13: recruits telomerase
Protein associated with human telomeres
POT 1: inhibits telomerase
t-loop
Masks ends of telomeres (ends are folded into middle of dsDNA)
Protects telomeres from random fusion events and acts as a method of telomere length control
Major themes of initiation control in eukaryotes
- Helicase can only load onto the origin replication complex during G1 phase
- Licensing factors (kinases that control the start of replication) can only bind to helicase during S phase
- After use, licensing factors are degraded and can only be remade during G1 phase
- Once helicase splits, it can’t come back together
How elongation works
Hydroxyl group of growing strand attacks alpha-phosphate of incoming nucleotide
Incoming nucleotide binds to complementary nucleotide
Fidelity
Accuracy of polymerase
Kinetic selectivity of polymerase
Polymerase can accept the wrong base, but reaction runs faster when binding is correct
How polymerase is selective
Enzyme contains selectivity pocket: base pairing needs to be correct for ribose sugar to be correctly positioned in active site
Discriminator amino acids
Can discriminate between dNTP (DNA) and rNTP (RNA) to prevent DNA-RNA hybrids from being formed
Contained in polymerase
Polymerase structure
Situated like a hand
Fingers: alpha helices
Thumb: alpha helices
Palm: beta-pleated sheets
I helix
Thumb of polymerase: maintains correct positioning of template (specifically 3’ end) at active site
