ch. 10

DNA Replication A single-celled zygote contains 6.4 billion base pairs of DNA IF 1 error in 1 million bp = 6400 errors every time cell divides Bacteria replicates 1000 nucleotides/sec…1 in 1 billion error! Three Models for DNA replication ________________________ Taylor-Woods-Hughes – showed semiconservative replication in eukaryotes __________ Replication can occur with circular DNAs ________________ Replication Used by some viruses, and F factor Like theta, has origin of replication Linear DNA Replication with Multiple Origins Replication has many Requirements ________________ ________________ ________________ The Mechanisms of Replication Bacterial DNA replication a. ______________ Circular chromosome has one (few) origin of replication _________ proteins bind to oriC, opening dsDNA Helicase and single-stranded binding (SSB) proteins bind to strand The Mechanisms of Replication b. Unwinding __________________ - enzyme that breaks hydrogen bonds b/t base of strands, cannot initiate unwinding! Binds to the lagging strand template, moves 5’-3’ (moves fork!) ssDNA ____________- prevent ss regions from snapping back DNA ___________ - topoisomerase, reduces torsional strain that builds as fork moves ________________ – can relieve or introduce supercoiling _____________ in bacteria Priming is Required for DNA synthesis c. Priming DNA polymerases cannot initiate DNA synthesis - require a _________ group to extend _______ - synthesizes a short oligonucleotide to get DNA replication going Molecules are RNA, 10-12 nucleotides long (SO Primase is an RNA polymerase! ) Why use RNA? D. Elongation DNA polymerases then synthesize DNA E. coli has 5 DNA polymerases! DNA polymerase I - has 5’-3’ polymerase activity has 3’-5’ exonuclease activity - allows correction of errors has 5’-3’ exonuclease activity - used to remove primers! (Main function may be removal of primers!) D. Elongation DNA polymerases then synthesize DNA E. coli has 5 DNA polymerases! DNA polymerase III -large complex (holoenzyme) has 5’-3’ polymerase activity has 3’-5’ exonuclease activity - allows correction of errors DNA pol I - Arthur Kornberg <1 mistake in 1 billion bp - how? Three mechanisms - 1. DNA polymerases are choosy - they usually pick the correct nucleotide 2. Insertion of the wrong nucleotide leads to incorrect positioning of 3’-OH - stalls polymerase ___________________________ (3’-5’ exonuclease removes) 3. Mismatch repair - fixes errors after replication The Replication Fork IF synthesis occurs on both strands - Two DNA Pol III complexes are required Each fork requires: 1. Helicase 2. ssDNA BP 3. DNA Gyrase 4. DNA primase 5. DNA polymerase Termination Occurs when two forks meet Sequences in some systems bind termination protein (Tus in E. coli) - blocks helicase Replication is FAST! (almost 1000bp/second) Replisome Eukaryotic DNA Replication Differences with bacterial replication: 1. Multiple replication events at the same time on same chromosome 2. Large variety of DNA polymerases 3. Nucleosome assembly makes this more of a mess! 4. Linear Chromosomes Eukaryotic DNA Replication Eukaryotic Origins - ARS - autonomously replicating sequences - enable DNA to replicate Licensing of DNA replication With thousands of origins, how does the cell make sure replication is only initiated once/cell cycle from each? (BIG PROBLEM) a. Replication ____________ factor attaches to origin, then (AND ONLY then) initiator proteins can function! Eukaryotic DNA Replication Unwinding - ssDNA BP, topoisomerases DNA polymerases - Function in replication, recombination, repair! __________ Remodeling – necessary for replication machinery to access DNA __________________ Assembly – chromatin assembly factors (CAFs) move with replication fork Circles Do Not have Ends Linear Chromosomes Create Replication Problems ______________ is a ribonucleoprotein Do most somatic cells have telomerase activity? Telomerase uses RNA guide to carry out __________________ Progerias are associated with shortened telomeres Hutchinson-Gilford syndrome