DNA REPLICATION TOPIC 3.4 & 7.2

Assessment Statements Explain DNA replication in terms of unwinding the double helix and separation of the strands by helicase, followed by formation of the new complementary strands by DNA polymerase Explain the significance of complementary base pairing in the conservation of the base sequence of DNA State that DNA replication is semi- conservative

DNA replication Cells must prepare for cell division by doubling the DNA content of the cell in a process called DNA replication Needed: –Enzymes (helicase and polymerase) –Free nucleotides

Helicase Initiates separation of complementary base pairs Begins at a point in or at the end of a DNA molecule Moves one base at a time breaking the hydrogen bonds Double-stranded DNA becomes two separate strands Unpaired nucleotides can then be used as a template

Formation of two complementary strands Free-floating nucleotides form the complementary pair with one of the single- stranded unzipped molecule Two nucleotides become covalently bonded together Formation is catalyzed by DNA polymerase Same happens to the other unzipped strand, but in the opposite direction

TOK Who should decide how fast and how far humans should go with our study of DNA and the technology that is rapidly emerging?

Significance of complementary base pairing The pattern of DNA replication ensures that two identical copies of DNA are produced from one

No ‘new’ DNA Every DNA molecule after replication consists of a strand that was ‘old’ now paired with a strand that is ‘new’ Considered semi-conservative b/c half of a pre-existing DNA molecule is always conserved

Assessment Statements State that DNA replication occurs in a 5’ to 3’ direction Explain the process of DNA replication in prokaryotes, including the role of enzymes (helicase, DNA polymerase, RNA primase and DNA ligase), Okazaki fragments and deoxynucleoside triphosphates State that DNA replication is initiated at many points in eukaryotic chromosomes

Semiconservative Model 1958 – Meselsohn and Stahl carried out experiments involving the bacterium E. coli Used two isotopes of nitrogen and determined what proportions of the isotopes were present in strands of DNA after one and two replications After one replication, each daughter molecule possessed one strand with the heavy isotope and one strand with the light isotope After a second replication, the molecules were either hybrid or without the heavy isotope Evidence showed that the replication process of DNA is semiconservative

Prokaryotic DNA vs. Eukaryotic DNA Prokaryotic DNA is circular and has a single origin of replication Eukaryotic DNA is linear and has thousands of origins Presence of multiple replication origins greatly accelerates the copying of large eukaryotic chromosomes

Detailed DNA replication Detailed DNA replication (continuous synthesis) 1.Replication begins at a sequence of nucleotides called the origin of replication 2.Helicase unwinds the double-stranded DNA helix and single-strand binding proteins react with the single-stranded regions of the DNA and stabilize them 3.DNA polymerase III adds nucleotides to the 3’ end of a pre-existing chain of nucleotides

4.RNA polymerase or primase constructs a RNA primer complementary to the parent DNA 5.DNA polymerase III then allows the addition of DNA nucleotides in a 5’ to 3’ direction to produce the growing DNA strand 6.DNA polymerase I removes the primer from the 5’ end and replaces it with DNA nucleotides

dNTP Each nucleotide that is added is actually a deoxyribose triphosphate As it is added, two phosphates are lost providing the energy necessary for the chemical bonding of the nucleotides

Discontinuous synthesis DNA strands can only be assemble 5’ to 3’ direction due to the action of DNA polymerase III The lagging strand elongates away from the replication fork and is synthesized discontinuously as a series of short segments called Okazaki fragments When the DNA polymerase III reaches the RNA primer on the lagging strand it is replaced with DNA polymerase I, which removes the RNA and replaces it with DNA

DNA ligase then attaches and forms phosphodiester bonds The DNA is further unwound, new primers are made, and DNA polymerase III jumps ahead to begin synthesizing another Okazaki fragment Overview

ProteinRole HelicaseUnwinds the double helix at replication forks PrimaseSynthesizes RNA primer DNA polymerase IIISynthesizes new strand by adding nucleotides onto the primer (5’ to 3’) DNA polymerase IRemoves the primer and replaces it with DNA DNA ligaseJoins the ends of DNA segments and Okazaki fragments