Some common circular modes of replication

Mitochondrial DNA replication

Sigma (σ) replication (the rolling circle)

Looped rolling circle replication

Theta (θ) replication

Modes of replication 2.

(From M. L. DePamphilis, 1999)

Mitochondrial DNAReplication - essentially a modification of bacterial theta replication that formscatenanes (however it is a continuously replicating system- Why?)

Sigma replication: How many circular phage particles rapidly replicate their DNA (can be single- or double-stranded molecules) (From Genes VIII)

Some critical aspects of rolling circle replication1. Leading strand is covalently linked to parental

template for the lagging strand (if double stranded)

2. Prior to precursor synthesis, the linear branch has a 5’ terminus (“A” protein must have acted there)

3. Rolling circle continues unabated forming concatemers (either as ds or ss products)

4. Circular template for leading strand synthesis neverleaves the circular part of the molecule

5. DNA can only be nicked to start the process if it is supercoiled

Some critical aspects of rolling circle replication

1. Leading strand is covalently linked to parental template for the lagging strand (if double stranded)

2. Prior to precursor synthesis, the linear branch has a 5’ terminus (“A” protein must have acted there)

3. Rolling circle continues unabated forming concatemers (either as ds or ss products)

4. Circular template for leading strand synthesis neverleaves the circular part of the molecule

5. DNA can only be nicked to start the process if it is supercoiled

Phage strategies I:Generating the minus strand in single-stranded phages

Phage strategies II:Three different ways of primingduring replication of the minus strand

Looped rolling circle replication:How a single stranded circular phage that has formed the minus (-) strand can end DNA synthesis andregenerate its plus (+) strand

Theta replication: the mostcommon bacterial modeof DNA synthesis

Eukaryotic Replication - another verycomplex process

Besides the numerous holoenzyme protein complexeslisted in the next slide, there are all of the subunits of each enzyme that must be organized together

Furthermore, one has to be able to replicate all of theDNA only a single time. This is problematic because of all of the origins of replication that are necessary;this requires considerable regulation about when/where replication will begin (we will deal with this aspect a bit later)

This table is less than 10 years old and look at the paucity of DNA polymerases as compared to Table 8.2 in your text; there has literally been an entirely new group of eukaryotic DNA polymerases discovered, the TLS repair polymerases

Eukaryoticreplication is characterized by switching of DNApolymerases

Combined Polα/primaseproduct is ~ 50-100 bpLow processivity

Further extension by Polε or Polδ is between100 and 10,000 bp

Fig. 8.16

RF-C replacesPrimase and attracts PCNA

The critical natureof the sliding clamp(PCNA) in eukaryotic processivity

Without the PCNA clampthe polymerase woulddissociate and diffuse away from the template aboutevery 20-100 bases

Fig. 8.18

Proliferating Cell Nuclear Antigen

Possible organization at a Eukaryotic replication fork

(From M. L. DePamphilis, 1999)

Probably a more realisticapproximation of the eukaryotic replication fork and replisomestructure

A probable Eukaryotic Replication Center in the nucleoplasm

Recognizes an element in replicator to activate initiation of replication

Entire set of cis-acting sequencesthat is sufficient to direct initiationof replication (the origin is part of replicator)

Initiating Replication: The Replicon Model (Jacob, Brenner and Cuzin) and itsimplications for startingreplication

Fig. 8.23

General model of thereplicon concept inprokaryotes

Fig. 8.25

Initiator is the onlysequence-specificDNA-binding proteininvolved in initiation

Replication origins are generally similar in overall structure

4 pentamer sites that bind T antigen + a 20 bp palindromewhere DNA unwinds

B1 and A bind to initiator ORC; B2 facilitates unwinding and binding of other factors

We shall look at this in more detail

Fig. 8.24

Single replicator in E. coli is called oriC; 9 mer (4X repeated) is binding site for DnaA (initiator); 13 mer (3X repeated) is initial site of ssDNA formation (melting)

TTATNCANABinding

GATCTNTTNTTTTUnwinding

Overview of Initiation of Replication in E. coli