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
