DNA Replication - Module 2, Class 2 - PDF

Module 2: DNA Class 2 DNA Replication  How is genetic information in the form of DNA replicated? Replication Initiation, Elongation, Termination (prokaryotes) DNA replication: Molecular mechanisms of DNA polymerase(s) molecular Replication is a...

Module 2: DNA Class 2 DNA Replication  How is genetic information in the form of DNA replicated? Replication Initiation, Elongation, Termination (prokaryotes) DNA replication: Molecular mechanisms of DNA polymerase(s) molecular Replication is a concerted effort of many enzymes mechanisms Methods for studying DNA replication From studies of replication: In research: PCR (polymerase chain reaction) DNA sequencing (sequencing by synthesis) Molecular cloning Clinical: Chemotherapeutic drugs Antivirals; some antibiotics CDK4/6 inhibitors (cancer therapeutics) Complementary strands provide the templates for duplicating the genetic information Properties of DNA replication  Accurate (1 mistake in ~1010 bp synthesized) replication mistakes have (bad) consequences!  Complete (every base pair is replicated)  Fast (~1000 bp/sec)  Chemistry of DNA replication 3′ end of strand O Catalyzed by DNA polymerase (DNA Pol) 5′ end of strand O o Nucleotides added to the 3’ end _ CH2 O P O O C G O O O P (synthesis in 5’-3’ direction; substrates are dNTPs) O H 2C O PRIMER TEMPLATE STRAND O STRAND _ O PO O A T O CH2 O o Nucleotides are added based on pairing with the H2C O O P O complementary strand Primer-Template Junction(PTJ) OH 3′ end of strand C G O CH2 O o DNA Pol can add nucleotides to existing 3’ end – O O O _ O P O P O P O CH2 O _ _ _ O P O cannot start de novo synthesis O O pyrophosphate O needs a primer (short RNA sequence; can also use OH A O CH2 O DNA primer) to extend incoming deoxyribonucleoside triphosphate O P O o The 3’ end of the primer (site of extension): T O CH2 O Primer-Template Junction (PTJ) O P O 5′ end of strand  Anatomy of a replication fork replication fork replication fork o DNA replication is semi-conservative each new DNA molecule contains one strand of the original helix + newly synthesized complementary strand o DNA replication is semi-discontinuous DNA Pol can only work in 5’→ 3’ direction one strand can be copied continuously – leading strand one strand has to be copied in pieces (Okazaki fragments) – lagging strand o DNA replication is bidirectional two replication forks go in opposite directions from replication start site  Anatomy of a replication fork o DNA strands need to be separated (unwound) for replication fork to progress Helicase unwinds the DNA o Local unwinding of the DNA causes supercoils (overwinding) ahead of the replication fork Topoisomerases resolve the supercoiling o DNA Pol requires a primer to extend new strands Primase synthesizes short (8-10) RNA primers for leading and lagging strands o RNA primers have to be removed; Okazaki fragments have to be connected (ligated) to complete replication DNA ligase connects Okazaki fragments DNA replication is initiated at specific sites: origins of replication A A A A Origin of replication AT-rich region Initiator binding region Genes (proteins) essential for bacterial replication were identified in a genetic screen (DNA replication sensitive mutants – DnaA, DnaB, etc) DnaA – initiator; binds to dsDNA, separates the strands DnaB – helicase; unwinds the DNA during replication DnaC – helicase loader; positions the helicase on ssDNA; inhibits the helicase from working DnaG – primase; makes the RNA primer DNA replication is initiated at specific sites: origins of replication o DnaA (with ATP) binds to the initiator-binding region o DnaA coats the DNA, destabilizes the helix, wrapping around 1 strand o DnaC/B complex interacts w DnaA, opens DnaB ring, places is it on ssDNA. Oposite strand is also loaded. Bleichert, Borchan & Berger (2017), Science 355, eaah6317 DNA replication is initiated at specific sites: origins of replication primase o DnaC/B complex interacts w DnaA, opens DnaB ring, places is it on ssDNA. Oposite strand is also loaded. o DnaB recruits primase (DnaG) to start making the RNA primer o When RNA primer is synthesized, DnaC is released (disintegrates), helicase can start working. SSB (ssDNA-binding proteins) coat unwound DNA (ssDNA). DNA Pol protein complex joins and DNA synthesis starts from the 3’ end of the primer. Chemistry of DNA replication: molecular mechanisms of DNA Pol Federley and Romano, 2010  How is accuracy of replication ensured? Primer-Template Junction(PTJ) Palm region interacts with PTJ and newly synthesized DNA. As nucleotides are added to the nascent strand, the strands slide so that the new PTJ is in the active site. How do you think DNA pol interacts with DNA? Through minor/major groove? Backbone? How is accuracy of replication ensured? When correct dNTP enters the active site: o dNTP base pairs with the template o Finger domain closes around the bound dNTP o Tyr from finger domain stacks with the paired base o Lys/Arg interact with beta and gamma phosphate (also coordinated by Mg ions) o Release of pyrophosphate releases the fingers, it pops back to open position o DNA template moves one position down What if wrong nucleotide is incorporated? Overall accuracy of replication: 1 mistake per ~1010 bp synthesized Accuracy of DNA Pol: 1 mistake per ~105 bp o When the template base is in a rare tautomeric form, it will pair improperly o The template base switches back to common tautomer (more stable) now there is a mismatch o Geometry of the helix doesn’t fit anymore the latest pair is not positioned (lack of correct base pairing) DNA Pol cannot continue – cannot position the next dNTP Proofreading 3’-5’ exonuclease activity it can remove the mismatched base and fix the mistake What if wrong nucleotide is incorporated? o Non-paired end has low affinity for DNA Pol active site o Higher affinity for exonuclease active site o Typically, 3-5 nucleotides are removed; the rest reanneals, and shifts back to the polymerase active site o Proofreading 3’-5’ exonuclease increases accuracy ~ 100x  How can we assay DNA Pol activity? Incorporation assay (can also be used for transcription, other polymers) o Collect samples at certain time points o Separate free dNTPs from DNA o Check incorporation of labeled dNTPs in DNA - 5000 b o Filter binding: positively charged paper (binds DNA); wash (template) nucleotides off; measure signal. Quantitative and fast – but no information on product length o Separation on gel (electrophoresis): same reaction; separate products on denaturing gel. Slower and less quantitative, but gives info on product length 20 b (primer) + o Primer extension assay: primer labeled instead of dNTP 0 30 60 90 120 time (min) How can we assay DNA Pol activity? Processivity of DNA Pol number of dNTPs added per single binding to primer-template junction different for different polymerases repair polymerases have low processivity (10-20bp) replicative polymerases have high processivity; >50000 bp Template challenge assay labeled - 5000 b (template) + 2x DNA Pol (+ dNTPs + 1000x unlabeled PTJ) o Let reaction run long enough to make full- length product not labeled o Separate on denaturing gel 20 b (primer) + High Low processivity processivity Helicase assay labeled + helicase + ATP + buffer w Mg o Active helicase will displace the primer - o Run on non-denaturing gel 5000 b (template) 20 b (primer) + no helicase helicase Different DNA Pol in bacteria serve different functions DNA Pol III Holoenzyme: o DNA Pol III Core enzyme o sliding DNA clamps ring-shaped, around DNA DNA Pol processivity factor o sliding clamp loader (tau complex)  Resolving Okazaki fragments o RNAse H removes RNA primer (only 1 rNMP left) o DNA Pol I fill in the gap between Okazaki fragments has 3’-5’ exonuclease activity (proofreading) has 5’-3’ exonuclease activity removes the last ribonucleotide (and usually a couple of deoxynucleotides) o DNA ligase seals the nick (covalently links the Okazaki fragments)  Replication of leading and lagging strand is coupled at the replication fork Many proteins at the replication fork work together for optimal replication o Lagging strand is folded back o Clamp loader (tau) stimulates helicase o Helicase recruits and stimulates primase o Sliding clamp keeps DNA Pol on DNA From: Yao and O’Donell, 2020  Part 2: Workshop Please sit with your PBL groups (or people who you want to work with)