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Unformatted text preview: Lecture 13
DNA Replication Allison p132 p138-142 Leading Strand & Lagging Strand Fig 6.5 Inititiation of DNA replication
Tetramer of Initiator protein (DnaA) binds to Replicator (Origin - oriC) TTATNCANA x4 GATCTNTTNTTTT x3 Local unwinding of AT-rich DNA (3x13mers) Enzymology of DNA synthesis in E. coli
1. Helicase 2. Primase 3. SSB - Single Strand Binding protein 4. Gyrase - DNA topoisomerase (I & II) 5. DNA polymerase III 6. DNA polymerase I 7. DNA ligase Enzymology is similar in eukaryotes, but there are more proteins see Fig 6.7 1. Helicase – DnaB (hexamer)
Unwinds dsDNA to create ssDNA Requires ATP During elongation, associated with Gyrase - a Topoisomerase II (see #4)] 2. Primase - DnaG
First step in Elongation Is an RNA polymerase Synthesizes 8-14 nt RNA primers de novo (no 3’ OH required) - used to prime DNA replication Fig 6.7 step 5 3. SSB
Single-strand DNA binding protein SSB binds and stabilizes ssDNA opened by helicase 4. DNA topoisomerase (DNA gyrase)
(1) tension generated by helicase and DNA polymerase (2) chromosome packaging (nucleosomes) Supercoil must be removed for replication to continue
Fig 6.7 step 4 5. DNA polymerase III
The holoenzyme of DNA PolIII consists of 10 different proteins: Catalytic Core = alpha, epsilon & theta subunits Tau (x2) – hold two PolIIIs together, one on leading strand the other on lagging strand Sliding clamp protein (x2) –helps DNA polymerase hold onto the template DNA as it traverses the template strand + 5 clamp loading proteins 5. DNA polymerase III Has two active cores
5’ → 3’ polymerase activity – DNA synthesis (6,000 nt/min) Epsilon subunit:
3’ → 5’ exonuclease activity –repairing mistakes Theta subunit: Stimulates exonuclease DNA polymerase resembles a right hand Fig 6.13 DNA polymerase III is processive - Adds many nucleotides before disengaging distributive processive DNA polymerases use a single active site to catalyze DNA synthesis - Incorrect (un-basepaired) base does not position α-phosphate correctly DNA polymerase use a single active site to distinguish dNTPs from rNTPs - 2’OH on ribose does not allow correct positioning of α-phosphate of incoming nucleotide Structure of Sliding DNA Clamp protein 5’ - Interacts with DNA polymerase closest to newly-synthesized DNA (exit channel) The Sliding Clamp Protein helps DNA polymerase to bind to the template strand rapidly after each round of lagging strand synthesis RNA primer newly synthesized Lagging strand The Sliding Clamp Protein helps DNA polymerase to bind to the template strand rapidly after each round of lagging strand synthesis 6. DNA polymerase I RNAseH DNA polI DNA polI 7. DNA ligase
Ligase uses energy from ATP to complete the phosphodiester bond 6. DNA polymerase I
DNA PolI has at least 3 different activities: 5’ → 3’ exonuclease activity - removal of RNA primers 5’ → 3’ polymerase activity –DNA synthesis (20-50 nt/min) 3’ → 5’ exonuclease activity –repairing mistakes DNA replication is accurate
DNA synthesis by DNA polymerase can incorporate a wrong nucleotide at a frequency of ~10-5 – 10-6 Mistakes in DNA replication are much lower Spontaneous mutation rate in E. coli ~ 1/109 Why? Proof reading! Proof reading of DNA polymerase is due to 3’ → 5’ exonuclease activity (epsilon subunit) The end replication problem
Telomere shortening - leaves 8-12 nt 3’-overhang Fig 6.18 A virus answer to replicating the ends of linear DNA
A protein that is covalently bound to each 5’ end of the DNA bind dCTP, which provides the 3’-OH for polymerization of new DNA e.g. Adenovirus Another answer to replicating the ends of linear DNAs TELOMERES and Telomerase
- the ends of chromosomes have a conserved repetitive 6-bp sequence - and a single-stranded 3’ overhang GT strand CA strand Fig 6.18 Telomeres have a loop structure (T-loop) TTAGGG-3’ TTAGGG-3’ |||||| AATCCC-5’ How is the top strand replicated in the absence of a template? see Fig 6.20 D-loop (base paired) Telomerase - a ribonucleoprotein Reverse Trascriptase Eukaryotes have a specific RNA template and enzyme (telomerase) to complete replication of telomeres see Fig 6.20 Defects due to Telomerase mutation
Telomere shortening - Dyskeratosis congenita - mutation of Dyskerin or Telomerase RNA causes premature aging by affecting rapidly-dividing cells e.g. skin cells intestinal cells Fig 6.20 white blood cells ...
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This note was uploaded on 04/26/2010 for the course LS 252-009-20 taught by Professor Chen during the Spring '09 term at UCLA.
- Spring '09