1. There are three general features of DNA replication that must be accommodated in models of the replication process in all organisms:
A. Semiconservative replication
B. Bidirectional growth of new strands
C. Growth of new strands starting from a common site (replication origin)
2. Figure 12-4: Demonstration of bidirectional chain growth from a single origin in viral DNA. At increasing times, samples of infected cells are taken, the circular viral DNA is extracted and cut with a restriction enzyme (EcoRI) that makes only one specific cut in the viral DNA. The results are then viewed by electron microscopy. The distance from the ends of the cut DNA to the center of the replication 'bubble' was found to be constant at all times even though the bubble grew in size around this center as replication progressed, indicating that replication is bidirectional from a common origin.
Animation 12.2 (Lodish CD): Bidirectional Replication of DNA.
3. Figure 12-5: Consensus sequence of the minimal bacterial replication origin based on analysis of genomes from six bacterial species.
We can define a replication origin as a segment of DNA in the genome that is necessary and sufficient for replication of a circular DNA molecule, such as a plasmid or virus, in an appropriate host cell. The E. coli replication origin has been intensively studied. It consists of about 240 base pairs of DNA at the start site for replication. E. coli oriC and a number of other bacterial origins of replication contain two types of repetitive sequences, a 9-bp sequence (9-mer, DnaA protein binding site) and an AT-rich 13-bp sequence (13-mer, easily dissociated DNA double helix). The high AT content facilitates local melting of the double helix to allow the DNA replication complex to assemble on the single stranded DNA.
4. We can generalize to identify three common features of replication origins:
A. Unique DNA segments that contain multiple short, repeated sequences.
B. Short repeat units are recognized by multimeric origin-binding proteins. These proteins control the initiation of DNA replication by directing assembly of the replication machinery to specific sites on the chromosome.
C. Usually contain an AT-rich stretch to facilitate unwinding of double stranded DNA.
5. Although the concept of copying a DNA molecule is relatively straightforward, the enzymology required for this process in the cell is actually quite complex. As it turns out, a large number of proteins and enzymes are required for DNA replication. Many proteins are required for replication because DNA polymerases, which add nucleotides to the 3' end of a DNA molecule, cannot perform a number of other steps required for replication (listed below).
A. DNA polymerases cannot melt double stranded DNA (dsDNA).
B. DNA polymerases cannot initiate DNA synthesis.
C. DNA polymerases cannot extend a DNA molecule from the 5' end.
6. To accommodate the inability of DNA polymerase to perform the above three functions, the cell requires enzymes to melt the DNA and initiate DNA synthesis. In addition, a rather awkward mechanism is employed by the replication machinery to accommodate the inability of polymerase to extend a 5' end of a DNA molecule. In this lecture we will discuss these reactions in more detail.
7. Replication, step-by-step:
A. Since most is known about the replication process in E. coli, this process will be covered first. As we shall see, corresponding events occur in eukaryotic cells.
B. Figure 12-7: Model of initiation of replication at E. coli ori C. Since DNA polymerase cannot melt double stranded DNA, a set of proteins is required to melt DNA at the origin of replication. This is accomplished in E. coli as follows:
(1) The DnaA protein binds DNA at the origin of replication, binding to 9-mer repetitive segments described above. DnaA has the ability to melt a small region of DNA at the origin (provided that the DNA is negatively super-coiled). These are the 13-mer repeats described above.
(2) The small region of DNA melting generated by DnaA is then extended by the DnaB protein, which is delivered by DnaC protein. One DnaB hexamer clamps to each DNA single strand at oriC. DnaB is a "helicase", a protein that uses the energy of ATP hydrolysis to separate DNA strands.
(3) Figure 12-8. Stabilization of single-stranded DNA. The DNA melted by DnaA and DnaB is stabilized in a single-stranded form by a protein called single strand DNA binding protein (SSB). SSB essentially coats the single stranded DNA (ssDNA), and prevents it from reannealing.
(4) Figure 12-11. Schematic model of the relationships among replication proteins at a growing replication fork for E. coli:
a. DnaB helicase melts the double helix as it moves along lagging strand template toward its 3' end. Each segment of lagging strand template uncovered becomes coated with SSB and forms a loop.
b. Pol III (labeled Core 1 in diagram) adds nucleotides to 3' end of leading strand using the template strand uncovered by DnaB helicase. This polymerase works processively, that is it remains bound to template via its beta-subunit clamp to continuously synthesize the leading strand.
c. A second Pol III (labeled Core 2 here) synthesizes the Okazaki fragments that will eventually become the lagging strand (discontinuous synthesis. The two core polymerases are linked by the dimeric tau protein.
When an Okazaki fragment is completed, the Core 2 polymerase detaches from the DNA template but remains bound to tau protein. The released core 2 rebinds the DNA template in the region of the RNA primer for the next Okazaki fragment using another beta-clamp protein. This process repeats itself many times to produce the lagging strand.
Animation 12.3 (Lodish CD): Nucleotide polymerization by DNA polymerase.
Figure 12-10. Beta-subunit clamp of DNA polymerase III.
Animation 12.1 (Lodish CD): Coordination of leading and lagging strand synthesis.
8. Summary: There are three DNA polymerases in E. coli. Each has a distinct role:
A. DNA polymerase I: Removes RNA primers and fills in resulting gaps. DNA polymerase I also has a 3' to 5' exonuclease activity (to correct mistakes), and additionally has a 5' to 3' exonuclease activity. The 5' to 3' activity is used to erase RNA primers generated by the primase. DNA polymerase I will then go on to fill the resulting gap. Note: There are different types of DNA exonucleases. A 5' exonuclease binds to the end of a dsDNA molecule and degrades one of the DNA strands in a 5' to 3' direction. Conversely, a 3' exonuclease binds to a DNA end and degrades one strand in a 3' to 5' direction.
B. DNA polymerase II: Involved in DNA repair. (Won't be discussed further.)
C. DNA polymerase III: The fastest most processive polymerase. Performs the bulk of synthesis for DNA replication. DNA polymerase III wraps around the DNA, making this interaction stable and highly processive. DNA polymerase III has a 3' to 5' exonuclease activity. This activity allows the polymerase to reverse direction and to erase mistakes it has made during DNA synthesis (which it could then replace with the correct base).