A Replisome Primase Primosome DNA Polymerase III acts here 5' 3' DNA Polymerase III acts here DNA Polymerase I extends one Okazaki fragment and removes the RNA from another. DNA Ligase then joins fragments together. ssDNA binding protein (SSB) Helicase (DnaB) Primase Gyrase S p i n g a t 1 , r m A o k y c f s v e l 5 b / u . E - Primosome

Table 20.2 in 4th edition.

Pol III* Core  complex Gamma complex loads the beta subunit (or clamp, not shown) onto DNA Pol III has a dimer of the “core subunits”, which contain the polymerizing α subunits. Fig. 21.17

 - Clamp – exists free and as subunit of Pol III holoenzyme . Donut-shaped Dimer. “Clamps” a subunit onto DNA, and makes it highly processive. Fig. 21.15 in Weaver

The effect of  subunit on the  clamp. Fig. 21.16 The effect of  subunit on the  clamp.

Can the  clamp can slide off the end of linear DNA? Plasmid DNA with nick  clamp Assay Load clamp onto circular plasmid DNA. Treat DNA further. Separate DNA-bound clamp from free clamp. Based on Fig. 21.13

Red – treated with the indicated enzyme before chromatography Fig. 21.11 Blue – control Red – treated with the indicated enzyme before chromatography First peak = protein-DNA complex Second peak = free  protein Conclusion: many  dimers can be loaded on the DNA, and it will slide off linear DNA

Based on Fig. 21.13 f,g Yellow line- control red line- experimental Load holoenzyme onto DNA with SSB, if all 4 dNTPs added, clamp falls off (control- only 1 or 2 dNTPs retains clamp) SSB can retain clamp, but linearize again after loading SSB, clamp falls off. Clamp sliding off the ends of linear DNA can be stopped by DNA binding proteins such as SSB and EBNA. Clamp will slide off SSB-coated DNA if it is part of the holoenzyme that is replicating DNA.

 (tau) subunits (2) of Pol III bind to helicase. Pol III core dimer synthesizing leading & lagging strands.  (tau) subunits (2) of Pol III bind to helicase. The dimer of Pol III core subunits point same direction, so DNA template (lagging strand) must loop around enzyme to be dragged through opposite direction of fork movement. Figure 14.19 in Genes VIII by B. Lewin

b Clamp loading g complex of Pol III holoenzyme Order of events: ( g2 , d, d’, c,  ) g complex of Pol III holoenzyme - loads b subunit dimer onto DNA (at the primer) and Pol core (and unloads it at the end of Okazaki fragment) Order of events: Uses ATP to open b dimer and position it at 3’ end of primer. “Loaded” b clamp then binds Pol III core (and releases from g). Processive DNA synthesis.

Recycling phase Once Okazaki fragment completed, b clamp releases from core. b binds to g . g unloads b clamp from DNA. b clamp recycles to next primer.

Figure 21.25

Terminating DNA synthesis in prokaryotes. Each fork stops at the Ter regions, which are 22 bp, 3 copies, and bind the Tus protein. Ter sequences must be in the same orientation as the fork they terminate (i.e., their function is orientation-dependent). Fig. 21.26

Decatenation of Daughter DNAs catenane Decatenation is performed by Topoisomerase IV in E. coli. Topo IV is a Type II topoisomerase: breaks and rejoins 2 strands of a duplex DNA. Eukaryotic chromosomes also have to be decatenated, because of the multiple replication origins, which also give two replication forks approaching each other. Fig. 21.27

DNA replication in Eukaryotes Eukaryotic DNA polymerases (5): - has primase activity d - elongates primers, highly processive, can do proofreading - DNA repair g - replication of Mitochondrial (and/or Chloroplast DNA in plants)

Eukaryotic DNA polymerases do NOT have 5' to 3' exonuclease activity Eukaryotic DNA polymerases do NOT have 5' to 3' exonuclease activity. A separate enzyme, called FEN-1, is the 5' to 3' exonuclease that removes the RNA primers. Eukaryotes also have equivalents to the: Sliding clamp – PCNA (a.k.a. proliferating cell nuclear antigen) SSB – RP-A

Problem for eukaryotes: Replicating the 5’ end of the lagging strand (because chromosomes are linear molecules) When the gapped chromosome is replicated, sequences will be lost, and it will shorten each time its replicated. Gap generated by removal of the RNA primer

Euk. chromosomes end with many copies of a special “Telomeric” sequence. (3 copies on this chromosome end) Cells can lose some copies of the telomere w/out losing genes. (Replication of this chromosome would produce 1 that is shorter by 1 telomere)

Telomeres form an unusual secondary structure. Telomere Sequences Telomeres form an unusual secondary structure. Dashes are Ts The T3G3 telomere shown in the figure may be from Paramecium. 5’ 3’

Telomerase Enzyme that adds new telomeric repeats to 3’ ends of linear chromosomes.

Proteins bind the 3’ SS overhang for protection. Fig. 21.32 Proteins bind the 3’ SS overhang for protection.

More on the importance of Telomerase Apoptosis - Cells are very sensitive to chromosome ends because they are highly recombinogenic. Telomeres don’t trigger apoptosis. Aging - There are rapid aging diseases (e.g., Werner’s Syndrome) where telomeres are shorter than normal. Cancer - Most somatic cells don’t have telomerase, but tumor cells do. Over-expression of telomerase in a normal cell, however, won’t turn it into a tumor cell. Plants - Transgenic Arabidopsis with the telomerase gene turned off developed normally up to a point, then became sick.

How is a Repl. origin selected? Priming at the oriC (Bacterial) Origin

OriC consensus sequence

Sequence of Events at the Replication Origin Hu – DNA-binding, histone-like protein in E. coli (not shown in the picture), not sequence-specific in its binding so binds many places in the genome, including OriC Primase (purple) with the first primers (arrows).

Order of events at OriC 1. Several copies of dnaA bind the four 9-mers; DNA wraps around dnaA forming “Initial Complex”. This requires ATP and a protein, Hu,that is already bound to the DNA. 2. This triggers opening of the 13-mers (Open complex). 3. Two copies of dnaB (helicase) bind the 13-mers. This requires dnaC (which does not remain with the Prepriming Complex) and ATP. 4. Primase binds to dnaB (helicase) and the DNA. 5. dnaB:primase complex moves along the template 5’ > 3’ synthesizing RNA primers for Pol III to extend.