LF130 Cellular and Molecular Biology Lecture 9, 2024 PDF

LF130 Cellular and Molecular Biology Lecture 9, 2024. Enzymology of DNA replication Part 1. Okazaki fragments Dr Robert Spooner  Summary, so far...

LF130 Cellular and Molecular Biology Lecture 9, 2024. Enzymology of DNA replication Part 1. Okazaki fragments Dr Robert Spooner  Summary, so far 3’ 5’ The 1950s were a busy time The Watson-Crick model was tested by others 3’ DNA replication is semi-conservative DNA strands are anti-parallel 5’ leading Watson-Crick base pairing underlies strand these synthesis New DNA is synthesised in the 5’ → 3’ Lagging 3’ strand direction synthesis 5’ DNA synthesis is semi-continuous, with a leading and a lagging strand 5’ 3’ 5’ DNA polymerase has a proof-reading 3’ 3’ → 5’ exonuclease activity 1968, the Okazakis : how does DNA replication begin? DNA replication needs an available 3’-OH on a primer. How does it start? Reiji and Tsuneko Okazaki established the directionality of synthesis: There is a leading strand, synthesised continuously: here 5’ → 3’ synthesis proceeds in the SAME direction as that of the replication fork (RF). There is a lagging strand, synthesised discontinuously, where 5’ → 3’ synthesis proceeds in the OPPOSITE direction to that of the RF. 5’ 3’ Lagging strand, away 5’ 3’ from the replication 5’ 3’ 5’ fork 3’ Leading strand, towards the replication fork 5’ 3’ The Okazakis and the Kornbergs  Okazaki fragments 5’ Okazaki fragments on the 3’ lagging strand, away from the 5’ 3’ replication fork 3’ 5’ 5’ 3’ Okazaki fragments are: ~100-200 nucleotides in eukaryotes ~1000-2000 5’ nucleotides in E. coli. 3’ Okazaki fragments are a consequence of synthesis of new DNA in ONE direction only: new DNA is built in the 5’ → 3’ direction. Why is there is no 3’ → 5’ synthesis of new DNA? Why is there is no 3’ → 5’ synthesis of new DNA? primer strand Hypothetical 3’ → 5’ 5 3 Real life 5’ → 3’ ’ ’ growth growth misincorporation 5 3 misincorporation ’ ’ proofreading: one nucleotide proofreading: one nucleotide removed, leaving a 5’ phosphate removed, leaving a 3’ -OH 5 3 … but a 5’ triphosphate is ’ ’ required here 5 3incorporation of correct ’ ’nucleotide and chain extension No 5’ triphosphate available for hydrolysis: Hydrolysis of the incoming nucleotide no energy for polymerisation provides the energy for polymerisation Tsuneko Okazaki. 5’ → 3’ synthesis permits EDITING: 3’ → 5’ does not M13 bacteriophage has a single-stranded DNA genome Escherichia coli ~ 1-2 μm long ~ 0.5 μm wide Infected E. coli secreting M13 M13 phage ~ 900 nm long ~ 6 nm thick  M13 life cycle M13 RF cpm Infection Progeny … phage molecular weight … via the F pilus. See Dr Wallis later E. coli ssDNA on horizontal cell viral packagin gene transfer ssDNA genomes g genom e Replicative Replication by ‘rolling form (RF) circle’. See Dr Wallis dsDNA later on horizontal gene replicatio transfer n  How does DNA replication begin? Arthur Kornberg, 1971: The replication of M13 phage DNA from single-stranded (ss) infective form to double-stranded (ds) replicative form (RF) by an E. coli extract is prevented by rifampicin. Rifampicin is an inhibitor of E. coli RNA polymerase Tsuneko Okazaki found that DNase cannot completely destroy Okazaki fragments. ss ds It left little pieces of RNA, 10-12 bases long In vivo, the primer for an Okazaki fragment is RNA, not DNA!  RNA primer synthesis RNA nucleotides DNA primase DNA primase is a rifampicin-sensitive DNA- 5’ 3’ directed RNA polymerase. 3’ HO - It synthesizes an RNA primer to initiate 5’ 3’ DNA synthesis on the lagging strand. RNA primer 3’ HO - 5’ synthesised 5’ 3’ (RNA polymerases do not require a primer).  Lagging strand synthesis (1) 5’ lagging strand primers 3’ 5’ ① 3’ ① New RNA primers are synthesised by DNA 3’ 5’ 5’ primase 3’ 5’ leading strand primer 3’ ② ② DNA Pol III extends the RNA primer using 5’ ① dNTPs to make Okazaki fragments on the 3’ 5’ 5’ 3’ 3’ lagging strands 3’ 5’ 3’ 5’ On the leading strand, DNA synthesis is 5’ 3’ continuous As the replication fork separates more DNA, new primers are laid down by DNA primase  Lagging strand synthesis (2) ③ ② 5’ ① ③ The old primers are erased by the 5’ → 3’ 3’ 5’ exonuclease activity of Pol I and are replaced with new DNA 3’ ④ ③ ② 5’ ① 3’ ④ The gap/nick is sealed by DNA 5’ ligase, joining the Okazaki fragment to 3’ the growing chain ④ ③ ② 5’ ① 3’ 5’ ⑤ And so on. 3’ Detail: Okazaki fragment joining by DNA ligase 5’ 3’ 5’ 3’ A 5 3 5 3 5 3 ’ ’ ① ’ ’ ② ’ ’ 3 5 3 5 3 5 ’ ’ ’ ’ 5 OH A OH ’ ’ ’ 3’ A Step ①: DNA ligase uses ATP as Step ②: AMP is released and a an energy source, releasing phosphodiester bond is formed pyrophosphate and attaching AMP between the 3’-OH of the upstream to the 5’ phosphate of the Okazaki fragment and the 5’ phosphate downstream fragment of the downstream fragment  The simplest model would be… DNA pol III binds here… 5’ 3’ 3’ 5’ 5’ 3’ … and DNA pol III binds here, with the two Pol IIIs moving in opposite directions 5’ 3’ 3’ 5’ 5’ 3’ … but this is wrong, because …  … because the two Pol III molecules are held together … Loo 5’ p 5’ 3’ 3’ 3’ 3’ 5’ 5’ Clamp Clamp holder holder 5’ 5’ 3’ 3’ … facing the same way  And YES! There is a loop … DNA Pol III complex Loo p Electron micrograph of a replication fork and associated proteins  LF130 Cellular and Molecular Biology Lecture 9, 2024. Enzymology of DNA replication Part 2. The enzymes involved and their coordination Dr Robert Spooner … and DNA There replication is beautyisin beautifully choreography… choreographed PRINCIPAL DANCERS … and TROUPE …  … and DNA replication is beautifully choreographed Pol III core DNA primase DNA Pol I spare clamp DNA ligase halves helicase single-stranded DNA binding clamp loader protein β clamp PRINCIPAL DANCERS … and TROUPE …  Leading strand synthesis is straightforward (1) ① DNA helicase unwinds the DNA helix, separating the strands 5’ 3’ 5’ ② DNA primase manufactures an RNA primer on the leading strand template 5’ 3’ 5’ 3’ 3’ ③ The primed duplex is captured by Pol III  Leading strand synthesis is straightforward (2) 5’ 3’ 5’ 3’ When you think of DNA polymerase, think of a hand flexing and grasping 5’ 3’ And … when it opens, it can let go of the duplex. Pol III has low PROCESSIVITY: it can only make short stretches of DNA before it falls off the DNA. Processivity is a measure of an enzyme's ability to catalyse consecutive reactions without releasing its substrate. When clamped, DNA Pol III can replicate a new  Leading strand synthesis is straightforward (3) ⑤ New clamp halves maintain the clamp holder in a state of readiness 5’ 3’ 5’ 3’ ④ The clamp holder transfers the two halves of the β clamp to Pol III Clamping converts Pol III to HIGH PROCESSIVITY: it can now replicate long stretches of DNA. 5’ 3’ 3’ 5’ ⑥ Helicase continues to unwind, and Pol III replicates the leading strand continuously  Lagging strand synthesis is more complex (1) ① DNA primase manufactures an RNA primer on the 3’ lagging strand template 5’ 3’ 5’ 5’ 5’ 5’ 3’ 5’ 3’ ② The primed duplex is captured by Pol III and clamped. This forces the lagging strand template into a loop Note: each primed duplex has the 5’ end of the RNA primer facing AWAY from the replication fork Lagging strand synthesis is more complex (2) old Okazaki fragment 5’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ ③ The helicase continues to unwind, Pol III replicates the leading strand continuously and extends the new primer on the lagging strand … Lagging strand synthesis is more complex (3) helix extruded in this direction old Okazaki fragment pulled in 5’ 3’ 5’ 5’ 3’ 3’ 5’ helix extruded in this direction ④ … until the old Okazaki fragment has been pulled back to Pol III.  Lagging strand synthesis is more complex (4) 5’ 3’ 5’ 3’ 5’ ⑤ The lagging strand and template are unclamped 5’ 3’ 3’ 5’ And this is why DNA Pol III HAS to have low processivity. If it was a highly processive enzyme, it could not release the new Okazaki fragment easily. Lagging strand synthesis is more complex (5) 5’ 3’ 5’ 3’ 5’ ⑦ DNA Pol I and DNA ligase repair the gap 3’ ⑥ DNA primase primes the lagging strand template… 5’ 3’ 3’ 5’ DNA Pol I does not require high ⑧ … and the process restarts by processivity. clamping the new lagging strand primer  The trombone model… this loop goes out 5’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ this loop goes out  … and now the electron micrograph makes sense newly synthesised Okazaki fragment on the lagging strand parental DNA helix Pol III complex Electron micrograph of a replication fork and associated proteins loop newly synthesised leading strand  … and in real time NB! I:47 – 2:50http://www.youtube.com/watch?v=yqESR7E4b_8 What about eukaryotic DNA replication? Eukaryotic DNA Pols polymerise at ~ 50 nucleotides/s, ~20 times more slowly than E. coli Pol III. It would take ~50 h to replicate an average human chromosome at this rate. DNA replication in eukaryotes proceeds bi-directionally from multiple origins of replication. Remarkably similar complex: Same process/mechanism  What about SSB? NNTAGCGATTTCCGTCGCTANN Stem-loop structures can be formed if DNA is denatured and fails to re-anneal properly. The same is true for single-stranded DNA. TC T C T G DNA replication requires a supporting cast of SSB – AT GC single stranded DNA binding protein. CG GC AT 5’-NNT ANN-3’ SSB prevents inappropriate base pairing in ssDNA during replication  The role of SSB SSB 5’ 3’ 5’ 5’ 3’ 3’ 5’ SSB SSB 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  But there is a torsional problem 5’ 3’ 5’ 5’ 3’ 3’ 5’ unwinding here … causes tightening … here… … and that stimulates SUPERCOILING  LF130 Cellular and Molecular Biology Lecture 9, 2024. Enzymology of DNA replication Part 3. Topoisomerases Dr Robert Spooner DNA supercoiling (b) (c) Most DNA is negatively supercoiled Positive supercoiling results from overwinding of DNA: and so can occur upstream of the replication fork. It makes strand separation difficult. Negative supercoiling arises from the unwinding/ underwinding of DNA. Negatively supercoiled DNA is easier to replicate, so TOPOISOMERASES are used to regulate the degree and type of supercoiling.  Type II topoisomerases (gyrase in bacteria)… … convert overwound positively supercoiled DNA into underwound negatively supercoiled DNA ① Topo II binds the positive supercoil … ② … makes a nick in both DNA strands … ds cut negative ③ … passes the DNA loop supercoil through the break and re- ligates it. Type I topoisomerase… … relaxes negatively supercoiled DNA. ① Topo I binds the negative supercoil … ② … makes a nick in one DNA strand … nick ③ … unwinds the DNA and re-ligates it. relaxed DNA  Bacterial DNA polymerisation is bi-directional leading lagging Two Pol III complexes enter the DNA at an ORIGIN OF REPLICATION. Replication proceeds in both directions at the same time. lagging leading What happens when the two forks meet? Topo IV bacterial chromosome Topoisomerase IV (a type II topoisomerase) separates the catenated catenated separated daughter chromosomes by a double chromosomes chromosom stranded break and religation. es  LF130 Cellular and Molecular Biology Lecture 9, 2024. Enzymology of DNA replication Part 4. Telomeres Dr Robert Spooner The major problem for eukaryotes: telomeres telomer e ① the lagging strand template can be primed at or near the telomere (and then extended) ② the DNA polymerase ③ + + complex falls off  The major problem for eukaryotes: telomeres ④ + the primers are erased this gap is filled by a DNA polymerase and repaired by a DNA ligase ⑤ + this gap on the lagging strand cannot be filled by a DNA polymerase as there is no primer Do chromosomes get shorter with each replication? On the whole…YES. It’s an inbuilt ageing mechanism that stops cells dividing forever. Some cells express TELOMERASE that extend telomeres.  Telomerase: a reverse transcriptase Telomerase provides an RNA template to synthesise a DNA copy of the template at the 3’ end of the parental lagging strand template. The result is that telomeres are built of repetitive motifs.  Telomerase: ageing and cancer Telomere length is correlated with cellular ageing. Telomerase is active in: some germline cells epithelial cells haematopoietic cells and in > 90% of cancer cell lines. Telomerase is responsible for the immortal phenotype of cancer cells.