MCB 250 Exam 1 Study Guide • Replication proceeds 5' to 3' (DNA adds on in that direction) • All DNA polymerases require a primer and a template • 3'hydroxyl of the growing chain carries out a nucleophilic attack on the alpha phosphate of the incoming NTP forming a new phosphodiester bond • The cleavage of pyrophosphate contribute tto the energy requirements of DNA synthesis because it reduces the concetration of one of the products of the reaction (Le Chatelier) • Proteins required for Replication ◦ Helicase (DnaB) • Melts parental DNA, interacts with PolyIII and Primase• Hexamer - 6 identical subinits • Wraps completely arund the lagging stran • Requires DnaC to load • Travels 5' to 3'• Uses ATP to unwind the helix ◦ Primase (DnaG)• Synthesizes RNA primers • Recruited by helicase • Starts preferntially at 5'-CTG-3' and lays down a 10-20 nucleotides primer • Then dissociates --> not processive • Must act every 1-2kb or so on lagging strand ◦ DNA Polymerase III • Synthesizes DNA (requires RNA primer) • Requires a primer to increase the fidelity of laying down the next base (laying down a base de novo is a low fidelity event --> no stacking, geometry not defined• Cell has chosen to lay down RNA de novo and then replace it with high fidelity• Holoenzyme is composed of 14 subunits • Core polymerase▪ Alpha - DNA polymerase ▪ Sigma - 3' to 5 exonuclease▪ Theta- stimulates exonuclease • Trimerization (weird t looking symbol)• Clamp loader or gamma complex ▪ I clamp loader per replisome ▪ Doesn't dissociate (integral part of the polymerase complex ▪ Recognizes 3' end of the primer/DNA hydrid ▪ Uses ATP to open the clamp and load it onto the double stranded region ▪ Acts one on leading strand and many times on lagging strand• Clamp B ▪ Doesn't bind to DNA, DNA is free to slide through the hole ▪ Tether the PolIII core to the DNA (Acts as a clamp)▪ Confers processsivity- enzyme adds a very large number of dNTP's before it dissociates◦ Single Strand Binding Protein (SSB) • Binds single stranded DNA template cooperatively, prevents reannealing and hairpins• Binding of SSB is highly cooperative (binding one makes the others more likely to bind)◦ RNAse H • Removes RNA primers from RNA-DNA Hybrids (H stands for Hybrids) • Can only cleave bonds between ribonucleotides ultimatley leaveing one ribonucleotide ◦ DNA Polymerase I • Single polypeptide- 3 domains ▪ Polymerase activity ▪ 3' to 5' Exonuclease - Fix errors ▪ 5' to 3' Exonuclease- Remove RNA or DNA in front of the primer • Starts at the end of the PolIII synthesized stand and removes RNAprimer replacing it with DNA ▪ Not very processive - doesn't have to go far • Leaves a nick (break in backbone in one strand) • Removes RNA primers and replaces RNA with DNA◦ DNA ligase• Seals nicks between Okazaki fragments ◦ Topoisomerases • Relaxes DNA in front of the replication fork• The polymerization reaction ◦ 2 divalent cations bound to the enzyme participate in the reaction ◦ One activated the 3'-OH of the primer and the other binds to andhelps position the dNTP and to neutralize its charge ◦ Polymerase samples all the bases until it finds the right one◦ PolyII designed to have low frequency of misincorporations and to fix them if they do occur • w/o correct base pairing the incoming dNTP is not positioned correctly for reaction to occur • The 2'-OH of ribonucleotides doesn't fit in the active site • Has a 3' --> 5' exonuclease activity that removes misincorporated bases • Protein Interactions at the replication fork ◦ Helicase can contact both primase and trimerization proteins ◦ Helicase recruits primase to the open origin ◦ Primase lays down 5-10 nucleotide primer ◦ Clamp loader recognizes primer and loads clamp◦ Clamp recruits PolIII core and initiates leading strand synthesis ◦ Helicase recruits primase repeatedly to initiate lagging strand synthesis • Topoisomerases Relax DNA (prevent or remove positive supercoils) in Front of Replication Fork ◦ As helicase unwinds DNA, it decreases the twist, therefore thr writhe increases in fron tog the replisome (Lk remains constant) ◦ If this problem isn't solved DNA replication will stop◦ Both type I and II topoisomerases can act to remove the positive supercoils and allow replication to proceed◦ Gyrase is the most important enzyme for relazing DNA in fron tof a replication fork in E. coli• Replication Initiation ◦ The E. coli chromosome has a single initiation site, oriC that’s recognized by a specific initiator proteins DnaA◦ DnaA must be bound to ATP in order to act as an initiator ◦ Binding of DnaA-ATP begins a series of events that leads to establishment of replication forks ◦ Replication initiation is tightly controlledB IS LEADING STRAND Template of the right fork• Replication Initiation ◦ The model on the previous slide explains how replication can be intiated using only lagging strand intiation ◦ The leading strand is synthessized as a single continuous molecule starting at the origin in the model (not true)• A replication fork will collapse & needs to be reassembled well beyond thr origin (there's machinery in cell that does this • Replicsome must be capable of reinitiation the leading strand • E coli DNA is methylated ◦ Dam (DNA Adenine Methyltransferase) recognized the sequence (5' GATC 3' and 3' CTAG 5') and adds a methyl group to the A's◦ The methyl group protrudes into the major groove ◦ Methylation is slower than replication (takes time fro newly replicated DNA to become methylated) ◦ Methylation tells the cells: • The DNA is newly synthesized• Which strand is the newly synthesized one? --> one that’s NOT methylated (old one is the one that’s methylated) • Replication Initiation is Tightly Controlled 1. Control of DnaA-ATP levels i. The rate limiting step in initiation is the binding of DnaA-ATP to OriCii. The concentration of DnaaA-ATP is tightly controlled 1. The amount of DnaA-ATP is proportional to cell mass (small new cells don't have enough to initiate replication) 2. As the replication fork passes, DnaA-ATP is convertedto DnaA-ADP and it dissociates from its DNA binding sites 3. DnaA-ADP doesn't work as an initiator and is very slowly converted to DnaA-ATP. This is one of the ways a new round of initiation is blocked until the time is right 2. Control of access to oriC i. There