DNA Replication.pdf

MCB.2. Understand the structure and properties of nucleic acids, the organization of genetic information, and its inheritance by successive generations Dr. John Th’ng GC11 [email protected] LEARNING OBJECTIVE: By the end of this module, students should be able...

MCB.2. Understand the structure and properties of nucleic acids, the organization of genetic information, and its inheritance by successive generations Dr. John Th’ng GC11 [email protected] LEARNING OBJECTIVE: By the end of this module, students should be able to: MCB.2.3. Describe the processes involved in the replication of DNA in bacteria and humans, highlighting the importance of the directionality of replication, requirement for primers, the role of Okazaki fragments, and the key enzymes involved  DNA Replication DNA Replication - how is DNA replicated? - process is similar between bacteria and human cells - enzymes are different DNA Replication  DNA Replication - when does it happen The Eukaryotic Cell Cycle: DNA & Histone G1 Synthesis S G2 M  DNA Replication Basic steps - base pairing according to Chargaff’s rule - polymerases can only work in 5’-3’ direction - DNA polymerases cannot initiate synthesis - needs a primer (DNA or RNA) at 5’ end - RNA primer synthesized by a primase - for DNA replication - DNA primer - for polymerase chain reaction (PCR) DNA Replication - New DNA strands are synthesized by using the parental strands as templates in the formation of daughter strands complementary to the parental strands - Synthesis occurs simultaneously on both strands of DNA - High degree of fidelity - Daughter strands are always synthesized in 5’ to 3’ direction - DNA replication is bidirectional - Semi-conservative DNA Replication  DNA Replication Bidirectional synthesis (bacteria and human) - single origin (bacteria) vs multiple origins (human)  DNA Replication 1. Bidirectional synthesis 2. Multiple origins in each human chromosome - generate “Replication Bubbles”  DNA Replication 1. Bidirectional synthesis - double-stranded DNA is antiparallel - DNA synthesis in 5’-3’ direction - how does replication fork progress?  DNA Replication 1. Bidirectional synthesis - double-stranded DNA is antiparallel - DNA synthesis in 5’-3’ direction - how does replication fork progress?  DNA Replication Overall process: Double stranded DNA DNA strand separation DNA synthesis Replicated DNA  DNA Replication Proteins involved: 1. Helicase - opens up dsDNA 2. Single strand binding protein (SSB) - maintains single strands 3. Primase - synthesizes primer 4. DNA polymerase - DNA synthesis - Okazaki fragments in lagging strand 5. Rnase - removal of RNA primer - filling in gaps 6. DNA Ligase - joins DNA fragments  DNA Replication Requirements: - Single stranded DNA Templates - Deoxyribonucleotides (dATP, dGTP,dCTP,dTTP) - Enzymes to polymerize bases - DNA polymerases - polymerizes ONLY in 5’ to 3’ direction - cannot initiate polymerization on its own - needs short RNA primer to initiate - Editing (proofreading & correction) - DNA Ligase to join DNA fragments  DNA Replication Initiation…. - DNA strand separation - by helicase  DNA Replication Initiation…. - DNA strand separation - binding of single-stranded binding protein (SSB)  DNA Replication Initiation…. - Synthesis of RNA primer Leading strand Lagging strand DNA Primase Pyrophosphorylase NTP NMP + PPi 2Pi  DNA Replication Initiation…. Figure 5-8. The structure of a DNA replication fork. Because both daughter DNA strands are polymerized in the 5 -to-3 direction, the DNA synthesized on the lagging strand must be made initially as a series of short DNA molecules, called Okazaki fragments.  DNA Replication Initiation…. Antiparallel direction - DNA polymerase can only add in the 5’ 3’ direction - Synthesize new fragments in lagging strand - OKAZAKI FRAGMENTS  DNA Replication Initiation…. DNA Polymerase dNTP dNMP + PPi  DNA Replication DNA Replication - DNA Polymerases - synthesize DNA in the 5’-3’ direction - never in 3’-5’ direction - template-directed enzymes - requires RNA primer - common properties: - DNA elongation - proofreading - check for errors (eg. misincorporation) - correct/repair errors - 3’-5’ exonuclease  DNA Replication Proofreading - 3’-5’ exonuclease - in DNA polymerases for elongation DNA Replication  DNA Replication Termination (strand level) - DNA synthesis stops at RNA primers - lagging strands - removal of RNA primers - 5’-3’ exonuclease in DNA polymerase I in bacteria cells - RNase H in human cells - refilling of gaps left by RNA removal - by DNA polymerase I in bacteria - by DNA polymerases  or  in humans - resealing of DNA strands by DNA ligase  DNA Replication Termination (strand level)  DNA Replication Termination (strand level)  DNA Replication Termination (replication forks) - when replication forks meet - topoisomerase II removes tangled replicated DNA strands DNA Replication - overall process is similar between bacteria and human cells - enzymes are similar, and have different names  DNA Replication (Bacteria) Helicase - separate double stranded DNA Primase - synthesize RNA primer DNA polymerase III - take over from primase to synthesize the DNA strands from the 3’ OH of the RNA primer DNA polymerase I - has nuclease activity to remove RNA primer - DNA polymerase activity fills in resulting gap Ligase I - join DNA fragments  DNA Replication (Human) Helicase - separate double stranded DNA Primase - synthesize RNA primer DNA polymerase α - synthesis of short DNA fragment from primer - Okazaki fragments DNA polymerase δ and ε - take over polymerase  to elongate DNA RNAse H - removes RNA primers Ligase I - join DNA fragments DNA Replication *  DNA Replication 1. Bidirectional synthesis 2. Replication bubbles cause torsional stress - due to strand separation of double-stranded DNA  DNA Replication DNA is supercoiled in chromosomes - separation of DNA double strands for replication or transcription will introduce supercoils & increase tension on DNA strands  DNA Replication DNA supercoils - increased twisting of DNA strands caused by unwinding of DNA for replication - create topoisomers - increase torsional stress - topoisomers released by topoisomerases  DNA Replication DNA Topoisomerases - relax supercoils - unwind supercoiled DNA - during transcription - ahead of the replication fork - untangle replicated DNA after replication in preparation for cell division - in bacteria & human cells  DNA Replication DNA supercoils - relaxation by topoisomerases - nick DNA strand(s) - unwind the supercoils - reseal the nick  DNA Replication DNA supercoils - package DNA to fit into nuclei - topoisomerases - relaxes superhelical coils of DNA - during replication and transcription - Types I and II topoisomerases - replication, transcription  DNA Replication DNA supercoils - topoisomerases - Type I - cleaves one strand DNA - relaxes supercoil - ATP not required  DNA Replication DNA supercoils - topoisomerases - Type II - cleaves both strands DNA - requires ATP  DNA Replication DNA supercoils - topoisomerases - Type II - cleaves both strands DNA - separate interlocked DNA after replication - separate entangled chromosomes - prepare for cell division - human & bacteria - requires ATP  DNA Replication Mammalian chromosomes are linear - have to replicate to the end of linear DNA - how??? DNA Replication - at ends of linear DNA  DNA Replication Telomeres - ends of chromosomes in eukaryotes - DNA is linear - cannot replicate right to the end of DNA molecule - lose repeats after each round of replication - loss of telomeres limit replicative life-span of cells - lead to cellular aging and senescence - synthesized by telomerase - contain RNA as template - reverse transcriptase - addition of telomere repeat sequences at ends - not expressed in most cells in the body - only in some cells, eg. germ & stem cells, lymphocytes  SUMMARY Understand the storage of genetic information, and how it is faithfully passed down to successive generations. - how is DNA organized in cells - how is DNA replicated