DnaB (helicase) and DnaC (6 molecules) join the complex, and creates 2 bidirectional replication forks Initiation of DNA Chains with RNA Primers - RNA polymerase initiates synthesis of new RNA chains at specific sites on DNA, opening DNA so it can be used as template for RNA synthesis - Each DNA chain is initiated by a short RNA primer synthesized by DNA o DNA primase : type of RNA polymerase, catalyzes synthesis of short RNA primers - In prokaryotes, primers are longer than in eukaryotes - RNA primers provide the free 3-OH required by DNA polymerase
- DNA polymerase III catalyzes addition of deoxyribonucleotides (dNTPs) to RNA primers on both leading and lagging strand. o produces Okazaki fragments by elongating RNA primers these unique to lagging strand - DNA polymerase I replaces RNA primers with DNA chains, connecting the Okazaki fragments only on lagging strand 5 DNA polymerases in E. coli - 1 and 3: chromosomal DNA replication, replicative polymerases - 2, 4, and 5: DNA repair functions Replicative polymerases - 1 aids in removal of RNA primers, not highly processive o removal of RNA primer allows DNA ligase to work o 5’ 3’ polymerase activity o two exonuclease activities: 5’ 3’ and 3’ 5’ o three distinct activities - 3 is main replicative polymerase, highly processive o has 5’ 3’ polymerase and 3’ 5’ exonuclease (primer removal, corrects mistakes) o on leading AND lagging strand Leading and lagging strand DNA - DNA ligase accomplishes the closing of the nicks in DNA lagging strand (missing phosphodiester bonds, not missing bases) by using free energy from NAD o Plays a role in replication but also repair and recombination - Lagging strand DNA synthesis requires DNA ligase to maintain strand continuity - AMP = adenosine monophosphate Unwinding DNA - DNA helicase: unwinds DNA using energy from ATP - SSBs: once unwound, the strands are kept in an extended (straight) form - DNA topoisomerase: nick the DNA and release strain/positive supercoils Replication fork - Requirements: o Topoisomerase o Helicase o SSB o DNA polymerase 3: produces Okazaki fragments by elongating RNA primers o DNA polymerase 1 o DNA ligase - Okazaki fragments: short lagging strand DNA fragments
Other enzymes: - Primase - RNA polymerase: catalyzes the synthesis of RNA molecules from DNA templates (transcription) Unique Aspects of eukaryotic chromosome replication - Shorter RNA primers and Okazaki fragments - Linear DNA - DNA replication only during S phase - Multiple polymerases (at least 15) replicate the chromosomes - Multiple origins of replication - Nucleosomes (two each of histones H2A, H2B, H3, H4 plus H1) - Telomeres o Shelterin complex protects telomeres of mammalian chromosomes o Protein complexes also protect yeast telomeres o Telomere length and aging o Most human somatic cells have low telomerase activity
Telomerase adds a repeat sequence to the 3’ end of telomeres o Shorter telomeres are associated with death, some cancer cells have high telomerase activity which is thought to promote their growth o
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- Fall '13
- DNA, RNA Primers