BIOLOGY 305 SECTION 3 LECTURE NOTES ALLEN JOHNNY BORLAY (BSc, MSc) COURSE INSTRUCTOR DNA REPLICATION GENERAL OVERVEW In both prokaryotes and eukaryotes, DNA replication occurs as a prelude to cell division. This DNA replication phase is called the S phase (synthesis phase). The two daughter DNA molecules formed from replication eventually become chromosomes in their own right in the daughter cells. As with all phenomena that involve nucleic acids, the basic machinery of DNA replication depends on complementarity of DNA molecules and on the ability of proteins to form specific interactions with DNA of specific sequences GENERAL OVERVEW Copying genetic information for transmission to the next generation Process of DNA duplicating itself Process of duplication of the entire genome prior to cell division Begins with the unwinding of the double helix to expose the bases in each strand of DNA Each unpaired nucleotide will attract a complementary nucleotide from the medium – will form base pairing via hydrogen bonding Enzymes link the aligned nucleotides by phosphodiester bonds to form a continuous strand Three alternative patterns for DNA replication In semiconservative replication, each daughter duplex contains one parental and one newly synthesized strand. Dispersive replication results in daughter duplexes that consist of strands containing only segments of parental DNA and newly DNA newly synthesized DNA In conservative replication, one daughter duplex consists of two newly synthesized strands, and the parent duplex is conserved Meselson and Stahl Experiment Experiment allowed differentiation of parental and newly formed DNA Bacteria were grown in media containing either normal isotope of nitrogen (14N) or the heavy isotope (15N) DNA banded after equilibrium density gradient centrifugation at a position which matched the density of the DNA When bacteria grown in 15N were transferred to normal 14N containing medium, – the newly synthesized DNA strand had the 14N while the parental strand had 15N Meselson and Stahl Experiment cont. Meselson and Stahl Experiment cont. • They checked the composition of the resulting DNA molecules by density gradient centrifugation found an intermediate band indicating a hybrid molecule containing both 14N and 15N DNA. PROKARYOTIC DNA REPLICATION Replication in prokaryotes starts at a single origin of replication The rate of replication in prokaryotes is faster (10×) than in eukaryotes The number (3) of DNA polymerases is less than that in eukaryotes Approximately 1000 nucleotides are added per second (In E.coli) Addition of nucleotides requires energy similar to ATP PROKARYOTIC DNA REPLICATION BASIC STEPS DNA unwinds at the origin of replication Helicase opens up the DNA-forming replication forks→ these are extended bidirectionally Single-strand binding proteins coat the DNA around the replication fork to prevent rewinding of the DNA. Topoisomerase binds at the region ahead of the replication fork to prevent supercoiling. Primase synthesizes RNA primers complementary to the DNA strand. DNA polymerase starts adding nucleotides to the 3'-OH end of the primer. Elongation of both the lagging and the leading strand continues. RNA primers are removed by exonuclease activity. Gaps are filled by DNA pol adding dNTPs. The gap between the two DNA fragments is sealed by DNA ligase, which helps in the formation of phosphodiester bonds. ILLUSTRATION Enzymes/Proteins and Their Function in Prokaryotic DNA Replication Enzyme/protein Specific Function DNA pol I Exonuclease activity removes RNA primer and replaces with newly synthesized DNA DNA pol II Repair function DNA pol III Main enzyme that adds nucleotides in the 5'-3' direction Helicase Opens the DNA helix by breaking hydrogen bonds between the nitrogenous bases Ligase Seals the gaps between the Okazaki fragments to create one continuous DNA strand Primase Synthesizes RNA primers needed to start replication Sliding Clamp Helps to hold the DNA polymerase in place when nucleotides are being added Topoisomerase Helps relieve the stress on DNA when unwinding by causing breaks and then resealing the DNA Single-strand binding proteins (SSB) Binds to single-stranded DNA to avoid DNA rewinding back. POINTS TO . NOTE Leading strand →continuously synthesized strand The overall direction of the lagging strand will be 3' to 5', and that of the leading strand 5' to 3'. Lagging strand → strand that is replicated in short fragments and away from the replication fork Okazaki fragment → DNA fragment that is synthesized in short stretches on the lagging strand Replication fork → Y-shaped structure formed during initiation of replication Primer →short stretch of nucleotides that is required to initiate replication → in the case of replication → the primer has RNA nucleotides Origins of replication → specific sequences of nucleotide where replication SUMMARY Replication in prokaryotes starts from a sequence found on the chromosome called the origin of replication—the point at which the DNA opens up Helicase opens up the DNA double helix, resulting in the formation of the replication fork. Single-strand binding proteins bind to the single-stranded DNA near the replication fork to keep the fork open. Primase synthesizes an RNA primer to initiate synthesis by DNA polymerase, which can add nucleotides only in the 5' to 3' direction. One strand is synthesized continuously in the direction of the replication fork; this is called the leading strand. The other strand is synthesized in a direction away from the replication fork, in short stretches of DNA known as Okazaki fragments. This strand is known as the lagging strand. Once replication is completed, the RNA primers are replaced by DNA nucleotides and the DNA is sealed with DNA ligase, which creates phosphodiester bonds between the 3'-OH of one end and the 5' phosphate of the other strand OPTIONAL ILLUSTRATION . Depiction of DNA replication at replication fork. a: template strands, b: leading strand, c: lagging strand, d: replication fork, e: RNA primer, f: Okazaki fragment OPTIONAL ILLUSTRATION OPTIONAL ILLUSTRATION BASIC SIMILARITY IN MACHINISM Both are bi-directional processes DNA polymerases work 5’ to 3’ Leading and lagging strands Primers are required Initiator proteins/enzymes bind at the origin of replication TERIMA KASIH (THANKS) DNA replication in Eukaryotes occurs in three stages – Initiation • Proteins bind to DNA and open up double helix • Prepare DNA for complementary base pairing – Elongation • Proteins connect the correct sequences of nucleotides into a continuous new strand of DNA – Termination • Proteins release the replication complex