Lecture 9 – Enzymology of DNA Replication (DNA Replication)

Lecture 9 – Enzymology of DNA replication Part 1 – Okazaki fragments 1968 – the Okazaki's: how does DNA replication begin? Reiji and Tsuneko Okazaki established the directionality of synthesis. Leading strand: Synthesized continuously in the 5' to 3' direction, moving towards the r...

Lecture 9 – Enzymology of DNA replication Part 1 – Okazaki fragments 1968 – the Okazaki's: how does DNA replication begin? Reiji and Tsuneko Okazaki established the directionality of synthesis. Leading strand: Synthesized continuously in the 5' to 3' direction, moving towards the replication fork (RF). Lagging strand: Synthesized discontinuously in the opposite direction (away from RF), forming short DNA segments called Okazaki fragments, which are later joined together. Okazaki fragments Okazaki fragments on the lagging strand, away from the replication fork Are consequences of synthesis of new DNA in one direction only: new DNA is built in the 5’ --> 3’ direction. Why is there no 3’ --> 5’ synthesis of new DNA? Tsuneko Okazaki. 5’ --> 3’ synthesis permits Editing: 3’ --> 5’ does not M13 bacteriophage has single stranded DNA genome Life cycle DNA replication begin Arthur Kornberg 1971 – the replication of M13 phage DNA from single-stranded (ss) infective form to double-stranded (ds) replication form (RF) by an E.coli extract is prevented by rifampicin (an inhibitor of E. coli RNA polymerase). Tsuneko Okazaki found that DNase cannot completely destroy Okazaki fragements. It left little pieces of RNA, 10-12 bases long RNA primer synthesis Lagging strand synthesis (1)  New RNA primers are synthesized by DNA primase DNA polymerase III extends RNA primers using dNTPs to form Okazaki fragments on the lagging strand. DNA synthesis on the leading strand is continuous. As the replication fork opens, DNA primase lays down new primers for further synthesis. The old primers are erased by the 5’ --> 3’ exonuclease activity of Pol I and are replaced with new DNA The gap/nick is sealed by DNA ligas, joining the Okazaki fragment to the growing chain And so on. Part 2 – The enzyme involved and their coordination Overview of Leading Strand Synthesis Leading strand synthesis is a crucial process in DNA replication, characterized by straightforward mechanisms that ensure accurate and efficient copying of genetic material. Step 1: Unwinding the DNA Helix DNA Helicase: o Function: Unwinds the DNA helix, separating the two strands. o Direction: Moves along the DNA from the 5' to 3' end. Step 2: Primer Formation DNA Primase: o Function: Synthesizes an RNA primer on the leading strand template. o Importance: Provides a starting point for DNA synthesis. Step 3: Primer Capture Pol III: o Function: The primed duplex is captured by DNA Polymerase III.  o Role: Initiates DNA synthesis by extending the RNA primer. Understanding DNA Polymerase III Processivity: o Definition: Refers to the ability of an enzyme to catalyze consecutive reactions without releasing its substrate. o Implication: DNA Pol III exhibits low processivity; it can only synthesize short stretches of DNA before it detaches from the DNA strand. Clamping Mechanism: o When DNA Pol III is clamped onto the DNA, it can replicate at a rate of approximately 1000 bases per second. This high speed is crucial for efficient DNA replication. Overview of Lagging Strand Synthesis Lagging strand synthesis is more complex than leading strand synthesis due to the nature of DNA replication direction. Lagging strand synthesis is more complex (1) (2) Step 1: RNA Primer Formation Role of DNA Primase: o Synthesizes an RNA primer on the lagging strand template. o Positioned so that the 5' end of the RNA primer faces away from the replication fork. Step 2: Primer Capture DNA Polymerase III: o The primed duplex is captured and clamped by DNA Pol III. o This action forces the lagging strand template into a loop formation, allowing for efficient synthesis despite the opposite direction of replication. Lagging strand synthesis is more complex (3) Direction of Helix Extrusion:  o The DNA helix is extruded in a specific direction while the old Okazaki fragment is pulled in the opposite direction. Okazaki Fragments: o Short segments of DNA synthesized on the lagging strand. o Each fragment is initiated by an RNA primer synthesized by DNA primase. Key Concept The process requires coordination between the synthesis of new fragments and the removal of old ones, ensuring that the lagging strand can be synthesized efficiently. Lagging strand synthesis is more complex (4) Lagging Strand Unclamping: o As the lagging strand is synthesized, it becomes unclamped, allowing for further synthesis. Processivity of DNA Polymerase III: o DNA Pol III must have low processivity on the lagging strand. o This is crucial because if it were more processive, it would not be able to release the new Okazaki fragment after synthesis. Key Takeaway The balance between processivity and the ability to release newly synthesized fragments is essential for effective lagging strand synthesis. Eukaryotic DNA replication Eukaryotic DNA Pols polymerise at ~50 nucleotides/s, ~20 times more slowly than E. coli Pol III. It would take ~50 hours to replicate an average human chromosome at this rate. DNA replication in eukaryotic proceeds bi-directionally from multiple origins of replication. SSB and its role Stem-loop structures can be formed if DNA is denature and fails to re-anneal properly. The same is true for single –stranded DNA DNA replication req a supporting cast of SSB – single stranded DNA binding protein  It protects the ssDNA from base pairing and from nuclease. It is constantly being displaced by Pol III and being replaced as the helix is unwound. Part 3 – Topoisomerases DNA supercoiling Most DNA is negatively supercoiled Positive supercoiling: Results from overwinding of DNA Occur upstream of the replication fork – makes strand separation difficult Negative supercoiling: Arise from the unwinding/underwinding of DNA Easier replication Role of Topoisomerases: Regulate the degree and type of supercoiling to maintain DNA functionality during replication Converted positive supercoil to negative supercoil Part 4 – Telomeres Slide 1: The Major Problem for Eukaryotes – Telomeres Telomeres: o Special structures at the ends of linear chromosomes.  Lagging Strand Synthesis: o The lagging strand template can be primed at or near the telomere and then extended. o After synthesis, the DNA polymerase complex can fall off due to the end of the template. Slide 2: Implications of Telomere Shortening Primer Erasure: o Primers used for lagging strand synthesis are erased, leaving gaps. Filling the Gap: o The gap is filled by DNA polymerase, but the segment at the telomere may remain unfilled because there is no RNA primer. Chromosome Shortening: o Do chromosomes get shorter with each replication? Yes. This is an inherent aging mechanism that prevents indefinite replication. Telomerase: o Some cells express telomerase, an enzyme that extends telomeres, allowing for continued replication without shortening.