lec 8 Flashcards
what are the main problems of replicatio
1) dna polymerase cant break the hbonds between things so need other proteins to break apart strands
2)DNa pol cant start chains, to make chains we add a 5’ phosphate from base to 3] hydroxyl of nucleotide, so need hydroyl group at one end to initiate elongation
3) can only add nucleiotides to 3] hydroxyl end but chain always grows in 5’ to 3’ this is problem bc dna is double stranded, antiparallel and semiconservative
3oh problem solution
dna pol cant replicate cuz
1) RNA primer(polymerase) comes in on 5’ end and makes rna and then there is a 3oh end and then the dna polymerase comes in and makes dna so there willbe a
2) nicked dna: when no rna pol there, nick the duplex dna to make a 3’ end; happens in rolly circle replication
3)Priming nucleotide: some virsues have the ability to have a portein that binds to dna that has a oh group sticking out so that lallows dna pol to bind
Two strands replicated in two different ways
1) leading stradn: CONTINOUSLY synthesized from a single primer on the leading strand template, grows 5’ to 3’
-pol wis working in the direction tht the strand is opening
2) Lagging strand: DISCOUNTINOUSLY synthesized from MULTIPLE primes on the template in several steps
-these discountous strands are okazaki fragments
-pol is not working in the direction that the pol is opening so this causes it to have these okazaki fragments and (discountinous)
because the strands are being made in opposite directions u need
two different polymerases, they are tied together to mkae sure that it is together and that they are not at different lengths at different times but the lagging causes lag then
dna pol is on for longer then
one replication buble = twosreplaction forks
one strand replicates, s leading and lagging start from the origin but there will be two leading two laggin becauseit will move out from original of replication since it is bidirectional so two forks occur from one origin
Initiaion of replication
important decision: whether and when to replicate dna
dna rep controlled at initiaion
control of initiaion: DNA - protein interactions
TWo functions involed in the replication of dna
cis elements: sites on dna
trans factors: proteins, diffuse throuhg cell and nucles, recognize cis elenets and bind to them, goes to whereverit needs to floats anywhere
What is OriC, At rigions, nad DNa A boxes
Origin of replication in e coli
-it is recognized by proteins, it is AT rich (3 repeats 13 bp sequences), 4 9pb DNA boxes as well
Another AT rich area next to oriC to help denaturatiomn
-AT rich reagios help to seperate the strands
DNa Aboxes: proteins bind to here and help with seperation as well, protein only binds here if there is a signmal for replication
What marks an origin as being functional
methylation of it
N6 of Adenine is methylated in sequence GATC
if u dont want it to be replicated, then take the methyl off
DnaA
protein that initiates replication in ecoli OriC
-recognizes the 4 dnaA boxes (pbp) in oriC
INITIAL COMPLEX: consists of 10-20 DnaA proteins and 4 DnaA bozes
proteins fold the boxes in a way that causes the at rich regions to like remove from eachother since these are weaker (only have 2 hydrogen bonds between eachother),
once this occurs then we have single stranded regions (opening usually happens 13 down)
What does the dna strand have to be for dnaA to initiate replication
negatively supercoiled
DnaB: Helicases
Once the DNa A started breaking the bonds, helicase comes in and unwindes it further, this enzyme requires atp
-Hexamer helps the dnaB helicase bind to the strand
DnaC is what dnaB needs to get to the dna side ; helps it catch up to dna a. carries it to the region
DNA Bis Processive= so it wont fall off until it reaches end or is removed by another protein
Single Standed DNa binding oroteins
-bind to Singly stranded part of dna
-cooperative binding: so bidning f one of the proteins causes more ot bind
-prevents double helix and INTERNAL pairing and nucleases from degraded it
DNaG : Dna primase
-need to make the short stretch of rna so that dna pol has the 3’ end
-Dna primase = dnaG = Rna polymerase
100s-1000s primers are neede for the lagging strand because each dna pol needs a 3] oh to bind and an existing strand to elongate to to make each okazaki fragment, so need equally as much primers to do so
SO when does dna pol come in
after…
dna A binds to A box
Dna B helicasse binds using dna C to get thre an hexamer to latch on, openign strands more
small binding proteins latch on to steand
dna primase (dna g) makes the rna strand needed to latch dna pol
wht do we need that strethc of rna (called rna primer) before we dcn MAKE DNA
DNA pol can only elongate an existing nucleotide strand so dna primer must make it so that pol can elongate that strand
-rna primer is small, and made by dna primase
thats why for okazaki theres sm because each strand made needs a primer to prime it so that pol can bind
What is the primosome
dna G and Dna B working together to make next prime site
What i the important dna pol
pol 3, syntehsizes dna from rna prikmers
pol 2 is for repairing dna
3’ to 5’ exonyclolytic activity
pol can go back and check its mistake
it can travel 3’ to 5’ (baclward) if it needs to fix its mistake, hydrolyzes it and expells the misrake
how to get rid of rna primer patch
RNase H
Rna primer we dont want so this enzymes comes in and degrades this primer patch, except for the last nucldeotide
-worls with dna pol 1
Dna pol 1 comes in and fills in the patch that removing the rna primer left, so it removes that last nucleotide and then fills it in with dna
dna ligase goes adn fills in the last patch
DNA pol 1
can have two exonuclease activities:
1) proofreading (nmove 3’ and 5’)
2) remove rna primer (5’ to 3’)
NEED FREE 3’ OH FOR POLYMERIZATION
DNA 1 VS 3
3 does synthesis and repair (does both together, repairs as it is synthesizing dna)
1 does patch work by reading and repair, and fills in rna primer gaps
Dna ligase
-enzyme that links dna fragements on the lagging strand following primer removal and pol 1 synthesis
-createa phosphodiester between the 3]oh and the 5’ phosphate of adjacent nucleotides
-atp is needed
-amp binds to dna quite easily so it helps to rejoin the binds
A-P provides thing
Topoisomerase
-thin that when u unwind dna, it gets more postivitely supercoiled in the front so it stops u from opening up strands so need to reduce tension
-their role is to turn pos supercoils into neg by nicking
-these enzymes regulate and recognize sueercoiling
-theycut it and reattach it to release the knotting
2 types
topoisomeraise 2 (gyrase) changes the negative supercoils (which cause more knotting) into positive supercoils
replicating bacterial genome (circular dna)
problem: get a bubble structure
so two rings are stuck together like a ollympic ring structure, only way to get the new strand seperate from the old strand, u need to nick it with toperisomeraise 2 (in vitro) in irl its topisomeriase iV
when the rings are together its called catenated, when not its decatenated
unreplicated terminus is denatured after supercoiling
i
euk replication
has multiple origins
why bc we have sm more bp of genomes
In yeast we have autonomous replication sequeces
organized in the same way
