Accessory factors summary 1.DNA polymerase can’t replicate a genome. SolutionATP? No single stranded templateHelicase + The ss template is unstableSSB (RPA (euks)) - No primerPrimase (+) No 3’-->5’ polymeraseReplication fork Too slow and distributiveSSB and sliding clamp - Sliding clamp can’t get onClamp loader (  /RFC) + Lagging strand contains RNAPol I 5’-->3’ exo, RNAseH - Lagging strand is nickedDNA ligase + Helicase introduces + supercoils Topoisomerase II + and products tangled 2.DNA replication is fast and processive

DNA polymerase holoenzyme

Maturation of Okazaki fragments

Topoisomerases control chromosome topology Catenanes/knots Relaxed/disentangled Major therapeutic target - chemotherapeutics/antibacterials Type II topos transport one DNA through another Topos

Starting and stopping summary 1.DNA replication is controlled at the initiation step. 2.DNA replication starts at specific sites in E. coli and yeast. 3.In E. coli, DnaA recognizes OriC and promotes loading of the DnaB helicase by DnaC (helicase loader) 4.DnaA and DnaC reactions are coupled to ATP hydrolysis. 5.Bacterial chromosomes are circular, and termination occurs opposite OriC. 6.In E. coli, the helicase inhibitor protein, tus, binds 7 ter DNA sites to trap the replisome at the end. 7.Eukaryotic chromosomes are linear, and the chromosome ends cannot be replicated by the replisome. 8.Telomerase extends the leading strand at the end. 9.Telomerase is a ribonucleoprotein (RNP) with RNA (template) and reverse-transcriptase subunits.

Isolating DNA sequences that mediate initiation

Different origin sequences in different organisms E. Coli (bacteria) OriC Yeast ARS (Autonomously Replicating Sequences) Metazoans ????

Initiation in prokaryotes and eukaryotes Bacteria Eukaryotes ORC + other proteins load MCM hexameric helicases MCM (helicase) + RPA (ssbp) Primase + DNA pol  PCNA:pol   MCM (helicase) + RPA (ssbp) PCNA:pol   (clamp loader) Primase + DNA pol  PCNA:pol   DNA ligase

Crystal structure of DnaA:ATP revealed mechanism of origin assembly 1. DnaA monomer (a) forms a polar filament (b). 2. DNA binding sites occur on the outside of the filament (model). 1.2.

Crystal structure of DnaA:ATP revealed mechanism of origin assembly 1. The arrangement of DNA binding sites introduces positive supercoils by wrapping DNA on the outside. Compensating negative supercoils melt the replication bubble at the end. 2. Clamp deposition recruits Had, which promotes ATP hydrolysis and progressive disassembly of the DnaA filament (hypothesis). 1.2.

Initiation mechanism in bacteria -- 1

Initiation mechanism in bacteria -- 2

Initiation proteins in E. coli (bacteria)

10 ter sites opposite oriC coordinate the end game The ter/tus system is not essential in E. coli. Tus protein binds Ter sites and inhibits the DnaB helicase Origin Counterclockwise fork Clockwise fork Clockwise fork trap Counterclockwise fork trap

Unwinding ter from the “nonpermissive” direction springs a “molecular mousetrap” Releasing C6 springs the trap DNAHalf life (s) K d (nM) 130 (2 min) (<1 min, FAST/ 53 permissive) 6900 (115 min, SLOW/ 0.4 nonpermissive) terB C6 Mulcair et al. (2006) Cell 125,

Unwinding ter from the “nonpermissive” direction springs a “molecular mousetrap” Releasing C6 springs the trap DNAHalf life (s) K d (nM) 130 (2 min) (<1 min, FAST/ 53 permissive) 6900 (115 min, SLOW/ 0.4 nonpermissive) terB C6 5’ 3’ Mulcair et al. (2006) Cell 125,

Unwinding ter from the “nonpermissive” direction springs a “molecular mousetrap” Releasing C6 springs the trap Mulcair et al. (2006) Cell 125,

Unwinding ter from the nonpermissive direction springs a “molecular mousetrap” Releasing C6 springs the trap Mulcair et al. (2006) Cell 125,

Topoisomerase II unlinks the replicated chromosomes Topoisomerase II - Cuts DNA and passes one duplex through the other. Class II topoisomerases include: Topo IV and DNA gyrase

Summary: What problems do these proteins solve? Tyr OH attacks PO4 and forms a covalent intermediate Structural changes in the protein open the gap by 20 Å!

FunctionE. coliSV40 (simian virus 40) HelicaseDnaBT antigen Primase Primer removal Primase (DnaG) pol I’s 5’-3’exo pol  primase FEN 1 (also RNaseH) Polymerase Corepol III ( , ,  subunits ) pol ,  Clamp loader  complex RF-C Sliding clamp  PCNA ssDNA bindingSSBRF-A Remove +sc at fork (swivel) gyrasetopo I or topo II Decatenationtopo IVtopo II LigaseDNA ligaseDNA ligase I … other model systems include bacteriophage T4 and yeast Summary: What problems do these proteins solve?

The ends of (linear) eukaryotic chromosomes cannot be replicated by the replisome. Not enough nucleotides for primase to start last lagging strand fragment Chromosome ends shorten every generation!

Telomere shortening signals trouble! 1. Telomere shortening releases telomere binding proteins (TBPs) 2. Further shortening affects expression of telomere- shortening sensitive genes 3. Further shortening leads to DNA damage and mutations. Telomere binding proteins (TBPs)

Telomerase replicates the ends (telomeres) Telomere ssDNA Telomerase extends the leading strand! Synthesis is in the 5’-->3’ direction. Telomerase is a ribonucleoprotein (RNP). The enzyme contains RNA and proteins. The RNA templates DNA synthesis. The proteins include the telomerase reverse transcriptase TERT.

Telomerase cycles at the telomeres Telomere ssDNA TERT protein TER RNA template

Telomerase extends a chromosome 3’ overhang

Conserved structures in TER and TERT Core secondary structures shared in ciliate and vertebrate telomerase RNAs (TERs). (Sequences highly variable.) nucleotides 1000s of nucleotides TERT protein sequences conserved 1300 nucleotides

Starting and stopping summary 1.DNA replication is controlled at the initiation step. 2.DNA replication starts at specific sites in E. coli and yeast. 3.In E. coli, DnaA recognizes OriC and promotes loading of the DnaB helicase by DnaC (helicase loader) 4.DnaA and DnaC reactions are coupled to ATP hydrolysis. 5.Bacterial chromosomes are circular, and termination occurs opposite OriC. 6.In E. coli, the helicase inhibitor protein, tus, binds 7 ter DNA sites to trap the replisome at the end. 7.Eukaryotic chromosomes are linear, and the chromosome ends cannot be replicated by the replisome. 8.Telomerase extends the leading strand at the end. 9.Telomerase is a ribonucleoprotein (RNP) with RNA (template) and reverse-transcriptase subunits.