DNA repliaction

DNA replcation overview

Semi conservative, synthesized by DNA polymerase, requires pre-existing nt (open 3’OH), always need template

DNA polymerase

use template to build DNA, add nt to 3’OH, wrong dNTP incorporation inhibits extension, proofreading remove mismatch (3‘-5’ exonuclease activity)

Processivity

number of nt incorporated during single template binding event (before dissociating), overall efficieny increase as processivity increase

Fidelity

Ability of DNA polymerase to accurately replicate

Replication initiation in bacteria

DanA bind at oriC stimulating melting, DnaB (helicase) open complexs and unwinds dsDNA, SSBs keeps strands apart

DnaA

binds dsDNA at oriC at DnaA boxes(AT rich) using ATP, stimulates melting

DnaB (helicase)

Binds open complex, unwinds dsDNA complex, uses ATP

SSBs

Kepps strand apart (prevent annealing), forms 2 replication forks

Replication elongation in bacteria

Primase makes RNA primer, leading strand which synthesis towards repication fork (continuous), Lagging strand which synthesis towards oriC (discontinuous)

Primase in bacteria

makes RNA primers in 5’-3’ direction, complementary to template, serves as 3’OH for DNA polymerase, DnaG

Leading strand synthesis in bacteria

template is oriC (3’) to replication fork (5’), synthesis towards replication forks, continuous

Lagging strand synthesis in bacteria

template is oriC (5’) to replcation fork (3’), synthesis towards OriC/RNA primer, discontinuous

DNA pol I in bacteria

binds to DNA/RNA nick, 5’ to 3’ exonuclease degrades RNA primer (low processivity so doesn’t eat to much DNA), 5’ to 3’ DNA polymerase extends 3’ Okazaki fragment, fills gap creates DNA/DNA nick

DNA ligase in bacteria

seals DNA/DNA nicks, requires ATP (AMP+PPi)

DNA pol 3 holoenzyme in bacteria

2 core enzymes (alpha, epsilon, theta), Beta clamps, gamma complex, Helicase (DnaB), DNA gyrase

holoenzyme core enzymes

Catalyze synthesis, 3 subunit, Alpha subunit - DnaE, Epsilon - Dna Q (proofreading), Theta - HolE

Holoenzymes beta clamps

tethers core enzymes to template strand, DnaN

Holoenzyme gamma complex

loads beta clamps onto template (DnaX), tau subunit connects core enzymes(3xDnaX/HolE/HolB)

DNA gyrase

topoisomerase II, relieves supercoiling, 2xGyrA/2xGyrB

DNA pol III actobactic

lagging strand needs to have primase make new primer and dissociate before gamma complex loads new beta clamp, when okazaki near completion separates old beta clamp and associates with new, all happening while leading strand is continuously

Replication termination in bacteria

at terminator sequence, Tus protein binds, allows replication complex to only pass in one direction

Rolling circle repliaction

synthesis of single strand at a time, newly synthesizes displaces parental by SSBs, displaced strand nicked and complementary synthesized, all leading strand synthesis

Theta replication

typical circular replication, bidirectional movement of replication forks, leading and lagging strand

PCNA - proliferating cell nuclear antigen

eukaryotic equivalent to beta/sliding clamp

Replication protein A (RPA)

eukarotic equivalent to SSBs

Eukaryote replication

no sequence specific, multiple different origins of replication, replication factory where concnetrated, telomerase

Replication initiation Eukaryotes

Origin regions AT rich and nucleosome frees (some G form DNA), influenced by chromatin state, tightly regulation to prevent rereplication, ORC complex loads helicase duplex, more sequence specific in lower eukaryotes (ORC encircles and bind A element)

Replication elongation in eukaryotes

Pol Alpha(prime) - syntehsizes RNA-DNA primer (low processivity and slow), Pol Delta - synthesize leading strand to catch up to helicase (intermediate speed/processivity), Pol Eplison - takes over leading strand synthesis (high processivity), lagging strand only uses Pol delta and displaces Okazaki fragment edge partially to produce flap

FEN1 and RNaseH

FEN1finds okazaki fragment flaps and initiates degrading, RNaseH removes rest of primer

Replication Termination in Eukaryotes

Pol eplison replaced by Pol delta to slow down replication, sequence independent, converganes of replication forks, enzymes dissociates, gap filling occurs

Telomerase

elongates 3’ of chromosome, catylitic activity carried out by proteins, has RNA template (TER) and reverse transcriptase (TERT), technically reverse transcriptase but extremly limited (needs very specific to work)

Telomerase processes

adds base to 3’ end of chromosomes using TERC as template, Telomerase transloactes, telomere elongates again

Similarites between Prokaryotes and Eukaryotes in replication

need template, bidirectional, continuously in leading and discontinuously in lagging(Okazaki fragment), RNA primer (3’OH), high fidelity, termination from fork converging

DNA pol in lab

T7 DNA polymerase, Klenow fragment of DNA pol I, Taq DNA polymerase

T7 DNA polymerase

genetically engineered, no 3’ to 5’ exonuclease activity, ideal for sanger sequencing

Klenow fragment of DNA pol I

original used in PCR, synthesis dsDNA from ssDNA templates, receded 3’ end to male blunt (digest 3’ overhang)

Taq DNA polymerase

Thermostable DNA polymerase I, from Thermus aquaticus, opitmal at high temperatures, able to withstand high temp require in PCR without denaturing