Molecular Biology Lecture 2 - DNA Replication - PDF

Molecular Biology Dr Nzar Shwan MLT department Lecture 02: Monday, 23rd September 2024 4th Year (Semester 7) DNA Replication As discussed in the previous lectures the genetic material is transmitted from parent to offs...

Molecular Biology Dr Nzar Shwan MLT department Lecture 02: Monday, 23rd September 2024 4th Year (Semester 7) DNA Replication As discussed in the previous lectures the genetic material is transmitted from parent to offspring and from cell to cell. For transmission to occur, the genetic material must be copied. During this process, known as DNA replication, the original DNA strands are used as templates for the synthesis of new DNA strands. In other words, DNA replication is the process of duplication of the entire genome prior to cell division. Biological significance Extreme accuracy of DNA replication is necessary in order to preserve the integrity of the genome in successive generations. In eukaryotes, replication only occurs during the S phase of the cell cycle. Replication rate in eukaryotes is slower resulting in a higher fidelity/accuracy of replication in eukaryotes. In humans 3000 nucleotides are added per minute. In bacteria about 30,000 nucleotides are added per minute. 1 Existing DNA Strands Act as Templates for the Synthesis of New Strands DNA replication relies on the complementarity of DNA strands according to the AT/GC rule. During the replication process: The two complementary strands of DNA come apart and serve as template strands, or parental strands, for the synthesis of two new strands of DNA. After the double helix has separated, individual nucleotides pair with nucleotides on the template strands via hydrogen bonding (must obey the AT/GC rule). A covalent bond is formed between the phosphate of one nucleotide and the sugar of the previous nucleotide. The two newly made strands are referred to as the daughter strands (Note that the base sequences are identical in both double-stranded molecules after replication). 2 DNA Replication is Semiconservative Proposed DNA Replication Models: a) Conservative model: Both strands of parental DNA remain together following DNA replication. In this model, the original arrangement of parental strands is completely conserved. b) Dispersive model: Proposes that segments of parental DNA and newly made DNA are interspersed in both strands following the replication process. c) Semiconservative model: The double stranded DNA is half conserved following the replication process. In other words, the newly made double-stranded DNA contains one parental strand and one daughter strand. Components of Replication DNA polymerases: Deoxynucleotide polymerization. Helicase: Processive unwinding of DNA. Topoisomerases: Relieve torsional strain that results from helicase-induced unwinding. RNA primase: Initiates synthesis of RNA primers. Single-strand binding proteins: Prevent premature reannealing of dsDNA. 3  DNA ligase: Seals the single strand nick between the nascent chain and Okazaki fragments on lagging strand The mechanism of DNA replication 1. Initiation Proteins bind to DNA and open up double helix. Prepare DNA for complementary base pairing. 2. Elongation Proteins connect the correct sequences of nucleotides into a continuous new strand of DNA. 3. Termination Proteins release the replication complex. Initiation: Unwinding the DNA double helix Replication starts at origin of replication Initiator proteins identify specific base sequences on DNA called sites of origin (origin of replication). o Prokaryotes – single origin site e.g. E. coli -oriC. o Eukaryotes – multiple sites of origin (replicator) e.g. yeast - ARS (autonomously replicating sequences). Enzyme Helicase unwinds and separates the ds DNA by breaking the weak hydrogen bonds, producing replication fork. Single-Strand Binding Proteins attach and keep the two strands of DNA separate. 4  Enzyme Topoisomerase attaches to the fork to relieve stress on the DNA molecule as it separates. Elongation of replication Synthesis always in the 5´-3´ direction Before new DNA strands can form, there must be RNA primers present to start the addition of new nucleotides. Primase is the enzyme that synthesizes the RNA primer, DNA polymerase can then add the new nucleotides. DNA polymerase can only add nucleotides to the 3´ end of the DNA This causes the NEW strand to be built in a 5´ to 3´ direction. Because the DNA strands are antiparallel, the DNA polymerase functions asymmetrically. 5  o The Leading Strand is synthesized as a single strand from the point of origin toward the opening replication fork. o The Lagging Strand is synthesized discontinuously against overall direction of replication o The Lagging Strand is made in MANY short segments, 1000–2000 bp in prokaryotic and 100–200 bp in eukaryotic. (called Okazaki Fragments), o Okazaki Fragments is replicated from the replication fork toward the origin of replication. The enzyme Ligase joins the Okazaki fragments together to make one strand. 6 Formation of Replication Bubbles Replication occurs in both directions along the length of DNA and both strands are replicated simultaneously. This replication process generates "replication bubbles". The DNA Polymerase Complex A number of different DNA polymerase molecules engage in DNA replication. All DNA polymerases have 2 properties; 1. Only synthesize DNA in one direction 5´ to 3´ 2. Only add to the end of existing double stranded DNA Therefore, they cannot start synthesis of DNA from scratch (RNA polymerases can, but not DNA polymerases) Bacteria have 3 types DNA Pol’s I, II, and III DNA Pol III involved in replication of DNA, DNA Pol I involved in repair Humans have 4 types DNA Pol’s alpha, beta, delta- nuclear DNA DNA Pol gamma - mitochondrial DNA (Note: there are other DNA polymerases in eukaryotes) 7  Proofreading New DNA Although the action of DNA polymerases is very accurate, synthesis is not perfect and a noncomplementary nucleotide is occasionally inserted erroneously. DNA polymerase initially makes about 1 in 10,000 base pairing errors. Enzymes proofread and correct these mistakes, the DNA polymerases all possess 3 to 5 exonuclease activity. This property imparts the potential for them to detect and excise a mismatched nucleotide (in the 3´ to 5´ direction). After the mismatched nucleotide is removed, DNA polymerase resumes DNA synthesis in the 5ʹ to 3ʹ direction. This process, called proofreading. The new error rate for DNA that has been proofread is 1 in 1 billion base pairing errors. Termination of replication In prokaryotes the DNA replication terminates when replication forks reach specific “termination sites”. The two replication forks meet each other on the opposite end of the parental circular DNA. 8 Telomeres in eukaryotes In eukaryotic replication, following removal of RNA primer from the 5´end of lagging strand; a gap is left. This gap exposes DNA strand to attack of 5´ exonucleases. This problem is overcome by “Telomerase”. How? 9