lecture 8 & 9 Flashcards
where are primers needed at each strand
leading strand- primer required at 5’ end of newly synthesized strand
lagging strand- needed at each Okazaki
DNA helicase, called ___ in prokaryotes, do what?
DnaB
slides on DNA template for the lagging strand and in the 5’ to 3’ direction, uses ATP to separate strands (breaks hydrogen bonds)
unzips double helix
topoisomerase, also called ____ in prokaryotes, does what?
gyrase or topoisomerase II
removes positive supercoils, untwists DNA by cutting one or both strands of DNA to unwind it when helicase is breaking the hydrogen bonds, then reseals it
Primases are specialized ____
in E. coli, an RNA primer is synthesized by the ___ primase, this primase must bind ____ for activity
RNA polymerases
DnaG
DNA helicase
RNA primers initiate ____
leading strand initiated by…
RNA primers initiate each of the thousands of the Okazaki fragments on lagging strand
leading stranded initiated by primase at replication origin
what does DNA polymerase I do
removes RNA primer at the end of each Okazaki fragment and replace with DNA (b/c cannot have RNA) — 5’ to 3’ activity- “nick-translation activity”
(RNaseH can also remove RNA primer by breaking phosphodiester bonds in RNA), but not the last rNMP) — RNaseH is like a backup
what does DNA ligase do
seal the nick in the phosphodiester bond after DNA polymerase I goes through
SSB’s protect DNA from ___ , it stimulates DNA polymerase activity by melting small ____ in the single-stranded template
endonucleases
DNA hairpin structures
which DNA polymerase is the replicative enzyme in E. coli
DNA poly III
what do DNA polymerases 1, 2, 4, and 5 do in E. coli
DNA Poly I = okazaki fragment processing and DNA repair
DNA poly III, IV, V = DNA repair
name the E. coli DNA polymerases that have 3’ to 5’ exonuclease activity
Pol I
Pol II
Pol III
name the E. coli DNA polymerases that have 5’ to 3’ exonuclease activity
DNA poly I
E. coli DNA Pol III is called a ____ and has ___ subunits
holoenzyme
22
name the components of the E. coli DNA Pol III holoenzyme
3 Pol III cores
Beta sliding clamps attached to each core
clamp loader at bottom — tau proteins attach clamp loader to the 3 cores
which of the 3 Pol III core subunits in the holoenzyme has the DNA pol activity
alpha
function of beta clamps in holoenzyme
what happens without beta sliding clamp?
help DNA poly III slide across subunit — increase speed and processivity of replication
without beta clamps, DNA poly III added 10 nucleotides/second, with beta — much faster(100,000 nucleotides per binding event)
without beta sliding, DNA poly III synthesizes new DNA through distributive synthesis (associated and deassociates with template)
clamp loader complex of holoenzyme also called ___
tau complex
tau subunit of clamp loader has ___ activity and binds ___
ATPase
binds Poly III and DnaB (helicase)
clamp loader of holoenzyme does what
assembles beta clamps onto DNA
- attaches to beta clamp using energy of ATP to open the beta clamp - when B clamps opens, it is now able to encircle a single-stranded DNA with a primer, ATP hydrolysis ejects the clamp loader
what are the components of the E. coli replisome?
DNA Poly III holoenzyme, DnaB helicase, primase
DnaB helicase connects with DNA poly III holoenzyme — increases helicase activity
leading strand - Pol III - beta clamp moves continuously with DnaB helicase
lagging strand - Poly III - beta clamp moves on and off DNA to extend multiple primers
describe trombone model
model for replication of lagging strand – forms a loop
- looping template DNA grows and shortens during lagging strand synthesis
Poly III core synthesizes lagging strand, forms loop, okazaki fragments dissociate (ligase and Pol I fill in gaps)
beta clamp recycling in e. coli
1- at end of replication, poly III core dissociates from beta after completing okazaki fragment
2- pol I target beta clamp to remove primer
3- dna ligase targets beta clamp to join okazakis
4- unoccupied beta clamp is unloaded
replication fork in eukaryotes - enzymes
helicase = CMG complex (moves in 3’ to 5’, opposite to E. coli DnaB)
sliding clamp = PCNA
clamp loader = RFC
SSB’s = RPA
describe the DNA polymerases in eukaryotes
3 different enzymes:
1- DNA Pol alpha (primase) - extends each RNA primer
2- DNA Pol epsilon - synthesizes leading strand
3- DNA Pol delta - synthesizes lagging strand
initiation of replication- origin is bound by ___
origin = ___ element
initiator protein (trans-acting element)
origin = cis-acting
when initiator binds to origin…
leads to DNA melting (denaturation of strands) and recruitment of other proteins — DNA helicase binds to initiator protein, helicase opens helix and binds primase
describe prokaryotic initator protein
DnaA, binds to 9 bp repeats with ATP
- leads to melting at 13 bp repeats, required for binding of helicase (DnaB) and fork formation (helicase loader (DnaC))
describe open-complex formation after DnaA binds origin
when DnaA and ATP bind origin, DnaA oligomerizes and wraps DNA around oligomer – another protein, HU, facilitates open complex formation at 13 bp
open complex = DnaA-ATP-OriC-HU
what happens after open-complex formation
DnaC loads DnaB helicase onto each strand, ATP hydrolysis causes DnaC to be ejected, DnaB helicase expands replication bubble and primase forms RNA primers at each replication fork, RNA primers direct clamp loading and assembly of one Pol III holoenzyme
replication initiation in E. coli is controlled at multiple sites…
1- binding of DnaA at OriC
2- DNA methylation
3- nucleotide-bound state of DnaA (ATP)
describe control of initiation by methylation
only methylated origins can be replicated
OriC contains many GATC sequences that is a target for adenosine methylation by Dam methylase
there are 11 GATC sequences with 245 bp of OriC
SeqA protein binds hemimethylated DNA, preventing DnaA binding
describe control of initiation by ATP
DnaA has to be bound to ATP to oligomerize and form open complex
DnaA-ADP cannot destabilize the A=T rich region to maintain open complex — forms closed complex
termination of DNA replication
replication forks meet head on when replication is complete
forks cannot travel past Ter sites
at end – 2 daughter chromosome separated by topoisomerase II
mismatch repair
1- first protein, MutS- recognizes mismatched bp and binds to it
2- MutS-MutL - scan bidirectionally along DNA scanning for GATC sites- forms loops
- MutS-MutL recruits helicase II and degrades newly replicated DNA past the
mismatch
3- MutH - site-specific endonuclease that cleaves unmethylated GATC sites
