Modes of replication 2.

Some common circular modes of replication

Mitochondrial DNA replication Sigma () replication (the rolling circle)

Looped rolling circle replication Theta () replication

Mitochondrial DNA Replication - essentially a modification of bacterial theta replication that forms catenanes (however it is a continuously replicating system- Why?)

(From M. L. DePamphilis, 1999)

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

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 never leaves 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 never leaves 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 singlestranded phages

Phage strategies II: Three different ways of priming during 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 most common bacterial mode of DNA synthesis

Eukaryotic Replication - another very complex process

Besides the numerous holoenzyme protein complexes listed 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 the DNA 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

Eukaryotic replication is characterized by switching of DNA polymerases

RF-C replaces Primase and attracts PCNA

Combined Pol/primase product is ~ 50-100 bp Low processivity Further extension by Pol or Pol is between 100 and 10,000 bp Fig. 8.16

Proliferating Cell Nuclear Antigen

The critical nature of the sliding clamp (PCNA) in eukaryotic processivity

Without the PCNA clamp the polymerase would dissociate and diffuse away from the template about every 20-100 bases

Fig. 8.18

Possible organization at a Eukaryotic replication fork

(From M. L. DePamphilis, 1999)

Probably a more realistic approximation of the eukaryotic replication fork and replisome structure

A probable Eukaryotic Replication Center in the nucleoplasm

Entire set of cis-acting sequences that is sufficient to direct initiation of replication (the origin is part of replicator)

Initiating Replication: The Replicon Model (Jacob, Brenner and Cuzin) and its implications for starting replication

Recognizes an element in replicator to activate initiation of replication

Fig. 8.23

General model of the replicon concept in prokaryotes

Initiator is the only sequence-specific DNA-binding protein involved in initiation

Fig. 8.25

We shall look at this in more detail

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

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

Replication origins are generally similar in overall structure

Fig. 8.24

Overview of Initiation of Replication in E. coli

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)
TTATNCANA Binding

GATCTNTTNTTTT Unwinding