DNA Replication

It is crucial that the genetic material must be reproduced accurately.

• Because the 2 DNA strands are joined only by hydrogen bonds, they are able to separate without breakage of covalent bonds.
• Each of the separated strands can act as a template for the synthesis of a complementary strand. Thus...the structure of DNA carries the information needed to perpetuate its sequence.
• The parental duplex is replicated to form two daughter duplexes.
• Each daughter duplex consists of one parental strand and one (newly synthesized ) daughter strand.
• The unit which is conserved from 1 generation to the next is one of the two individual strands comprising the parental duplex.

• E. coli Bacteria grown in 15N media so that the "heavy" nitrogen becomes incorporated into the nitrogenous bases.
• Allowed time for one cycle of DNA replication in 14N.
• DNA extracted and subjected to Cesium chloride - Density Gradient Centrifugation. This technique allows for the separation of molecules based upon their relative densities.
• 15N labeled DNA shows a lower band position than 14N labeled DNA following centrifugation.
•  DNA extracted from the E. coli in the above experiment showed an intermediate band position (intermediate between that of 15N and 14N) which can result only from one strand containing N15 and one strand containing N14. This result proved that replication is semi-conservative and that the new DNA is a hybrid DNA containing 1 parental strand and 1 daughter strand. If replication had been conservative, there would have been one band at the 15N position and a second band at the 14N position.

 See page 94 for figure describing Meselson-Stahl experiment.

• He actually discovered DNA polymerase I or pol 1 but pol III is the major polymerase in prokaryotic DNA replication.
•  Only 5' deoxynucleoside triphosphates can be polymerized by DNA polymerases. Therefore, DNA replication can only proceed in the 5' to 3' direction.
•  Both a pre-formed primer with a free 3' OH group [hydrogen bonded to a template] and a template are needed for DNA replication.

• In E. coli circular DNA there are two simulataneous replication forks [bi-directional replication]
•  Replication begins at a particular sequence known as the origin (ori sequence).
• The ori of E. coli is a sequence consisting of 245 base pairs.
•  From start to finish, the entire chromosome is then replicated in ~ 30 minutes.
•  An initiator protein first binds to the ORI sequence.
• Then, an enzyme known as helicase catalyzes the unwinding and separation of the 2 strands of parental DNA. This is an energy-requiring process which is coupled to the hydrolysis of ATP. The helicases must continually unwind the helix ahead of the advancing replication fork.
• Single-stranded DNA-binding proteins must then stabilize the unwound DNA by binding to the single-stranded strands and preventing them from re-hybridizing.See Figure 5.9 in text.
•  Interestingly, short primer strands of RNA are then synthesized first by a specialized enzyme known as primase. Primase synthesizes short fragments of RNA (3-10 nucleotides long). These primers are, of course, complementary to the template DNA.
•  Once the primer has been synthesized (always in the 5' to 3' direction) DNA polymerase III (pol III) can then begin to synthesize a DNA daughter strand.
•  The major DNA polymerase is DNA polymerase III (Pol III) for both leading strand and lagging strand synthesis.
•  The leading strand in DNA replication can be synthesized in a continuous manner in the 5' to 3' direction by Pol III.
•  The other daughter strand must be synthesized in a series of short segments (OKAZAKI FRAGMENTS) in the 5'-3' direction as the replication fork continues to open. Each time a new Okazaki fragment is synthesized, primase must first synthesize a short RNA primer, then pol III polymerizes the DNA fragment.
•  Naturally, the RNA primers must be completely removed prior to the conclusion of DNA replication. DNA Pol I is able to hydrolyze DNA or RNA in the 3'-5'direction or in the 5'-3' direction (exonuclease activity). Pol I uses its 5'to3' exonuclease activity to remove the primer (1 ribonucleotide at a time) and then uses its 5'to 3' polymerase activity to replace the primer with an appropriate DNA sequence. In E. coli there is an additional enzyme known as RNase H which degrades the RNA strand in RNA/DNA hybrids.
•  Once the primers have been removed, DNA ligase joins the Okazaki fragments together (formation of phosphodiester bonds). Discontinuity results during DNA replication because no known polymerase can join a 3' OH and a 5'monophosphate group. Must be performed by DNA ligase with energy provided by ATP or NAD.

• brace proteins - called the gamma complex in E. coli. These proteins recognize and bind DNA at the junction between the primer and the template, serving as a brace which helps the polymerase remain bound to the template.
•  sliding-clamp proteins- called the beta protein in E. coli . These proteins bind adjacent to the brace proteins, forming a ring around the template DNA. The ring formed by the sliding clamp maintains the association of the polymerase with its template as replication proceeds.  See figure 5.8 in text.
•  As the DNA unwinds, there is a tendency for the helix to become too tightly supercoiled. (See Figure 5.10)
•  If not relieved, eventually this would interfere with the ability of helicase to unwind the helix.
•  It is the job of the topoisomerases to relax the supercoiling by making either single-stranded breaks in the DNA (Type 1 topoisomerases) or double-stranded breaks in the DNA (Type 2 topoisomerases). The topoisomerases are also needed to unravel the newly replicated circular DNA molecule that has a tendency to become intertwined.

 See FIGURE 5.11 for complete model of the E coli replication fork.

• If an incorrect nucleotide is added during replication, the enzymes reverse direction and remove the nucleotides one nucleotide at a time.
• When incorrect nucleotides have been removed, the enzymes continue polymerization in the 5' to 3' direction.

• Single protein
• Requires template and primer
• 5' to 3' exonuclease activity combined with 5' to 3' polymerase activity allows it to nick translate which is important in removal of RNA primers.
• Does have 3' to 5' exonuclease activity for PROOF READING

• Very complex enzyme
• In its most active form it is associated with nine other proteins to form the pol III holoenzyme, referring to the fact that the enzyme is composed of several subunits
• Smallest aggregate which retains polymerizing activity is referred to as the Core Enzyme
• Pol III shares with pol I a requirement for a template and a primer
• However, pol III cannot polymerize at a nick. There must be a gap in the sequence .
• Does have 3'-5' exonuclease activity - PROOF READING FUNCTION
•  NO 5'-3'exonuclease activity.

 Quiz