Take Home Message - DNA replication is the process the duplicates the chromosomes in S-phase before the cells divide!- DNA replication has very high fidelity to ensure that the daughter cells don't get mutated DNA!- Every step of replication (initiation, elongation, end replication) is a delicately regulated process!- Replication doesn't start randomly, but only at replication origins. Proceeds bidirectionally.!- Replication of chromosomes ends needs a special enzymme called telomerase!- Replication inhibitors hit quickly dividing cells more often--> used frequently in cancer chemotherapy! Learning Objectives 1) Describe main features of DNA replication and replication fidelity!2) Describe how DNA replication is initiated!3) Explain how chromosome ends are replicated!4) Know the discussed DNA replication inhibitors used in medicine and explain their mode of inhibition!!1) Describe main features of DNA replication and replication fidelity - Genome replicated once/cell cycle during S-phase (synthesis phase). Duplicate 3.2 billion bps w/o errors!- Replication is fast, human cell= a few hours!- While cells are replicating DNA, other processes have to continue as well!- DNA replication is semiconservative: per round of replication, each of the 2 DNA strands is used as a template to synthesize a new strand!- A=T, CIIIG!- the 2 strands of a DNA double helix are always complementary to each other!- DNA polymerases synthesize the new DNA strand!- syn. onto template strand!- always always always in 5'-->3' direction!- Replication forks are asymetrical!- leading strand: strand syn. continuously from 5'->3 as DNA syn. progresses - lagging strand: syn. discontinuously (backstitching) in small Okazaki fragments (100-200 nts)!!!!!!!!!!- DNA polymerases sometimes make mistakes! !- Some nucleotides (ex. C) can undergo a tautomeric shifts to bp with A and get incorporated by DNA poly into the primer strand. But then there is a rapid tautomeric shift back to normal cytosine which destroys its bping with A... DNA poly can repair the error with its proofreading ability - Main DNA replication polymerases have 3'->5' (backwards) proofreading exonuclease activity!- the last incoming wrong nucleotide is removed and replaced with a correct one!- Has 2 catalytic sites: 1 for polymerization and one for exonuclease activity. They are separate. !- There is very high fidelity of DNA synthesis during replication!- 5'->3' poly (1 error/10^5 nts) + 3'->5' exo proof (1 error/10^2 nts) + strand directed mismatched repair (1 error/10^2 nts)= 1 error/10^9 nucleotides!- Strand directed mismatch repair - Sometimes DNA poly will overlook a mismatch and keep going, so we have mismatch repairproteins (MutS and MutL--complexes in us) that recognize the wrong bp and remove the newly synthesized DNA strand around it 2) Describe how DNA replication is initiated - DNA polys can not start DNA synthesis de novo! They need a basepaired primer with a few 3' OH.!- Primer (RNA) is synthesized by primase--> sits down on DNA and syn. an RNA primer--> now DNA poly can take over!- Primase works throughout DNA replication: only lays down 1 primer for the leading strand, but lays down an RNA primer for every single Okazaki fragment!- Later, the RNA primer is removed/chewed out, and the DNA polymerase fills in the nick!- a DNA helicase opens/unwinds the DNA double helix ahead of the replicationn fork. As it unwinds the DNA, it hydrolyzes ATP and propels itself along the DNA. Some move 5'->3' and some move 3'-> 5'.!- Single stranded DNA binding proteins help to stabilize the ssDNA (unwound DNA) and keep it straight!At the replication fork (know these) !!!!!!!!!! - For the replication fork to move, the growing tension is relieved by DNA topoisomerase I (creates ssDNA breaks) and II (creates dsDNA breaks)!- DNA replication begins at multiple origins simultaneously, not all used every round!- Diff. regions on chromosomes replicate at diff. times during S-phase!- Euchromatin= early!- Heterochromatin (telomeres and centromeres)= late!- Complicated protein assembly--> start of replication!- Origin Recognition Complex (ORC) - pre-RC (pre-replication complex) - to prevent replication from occuring again during that cell cycle, the ORC is phosphorylated! 3) Explain how chromosome ends are replicated - the telomeres (ends of the chroms) have a single stranded 3' DNA overhanng...telomeres are syn. by telomerase - cells w/o telomerase will have a 3' overhang and will lose that every cell cycle!- Telomerase= a protein with a strand/part of RNA on the inside. It uses the RNA to elongate the single strand on top until the strand is long enough to put down an RNA primer. Then, you can finish the lagging strand with DNA polymerasae.!!4) Know the discussed DNA replication inhibitors used in medicine and explain their mode of inhibition - Topoisomerase inhibitors: used in cancer treatment!- DNA crosslinkers: physically block the moving replication fork!- Thymidylate synthase inhibitors: w/o thymine, replicative synthesis stalls!- Nucleotide reductase inhibitors: delete cells of their nucleotide (A, C, T, and G) pool and cause replication fork
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