Single and double-stranded DNA is susceptible to cleavage by nuclease. Chain cleavage by nucleases. Chain cleavage can leave the alpha (a) phosphate group attached to the 5’ carbon of the sugar or the 3’ sugar carbon. ● The alpha (a), beta (B) and gamma (y) phosphates of deoxythymidine triphosphate (dTTP) There are five DNA polymerase in E.coli, ● Polymerases I , II , III , IV , V DNA repair functions Chromosomal DNA replication -replicative polymerases Replicative Polymerases Polymerases I and III ● Pol.I - aids in removal of RNA primers - Has 5’ to 3’ polymerase and 5’ to 3’ exonuclease activity - Proofreading : has 3’ to 5’ exonuclease activity - Not highly processive; short tract synthesis ● Pol. III-main replicative polymerase; highly processive
-has 5’ to 3’ polymerase activity -lacks 5’ to 3’ exonuclease activity - Proofreading : has 3’ to 5’ exonuclease activity DNA polymerase I: 5’ → 3’ Polymerase Activity 5’ → 3’ Exonuclease Activity - primer removal Leading and lagging strand DNA replication - Leading strand = continuous - Lagging strand = discontinuous - Both proceed in 5’ → 3’ direction - DNA ligase accomplishes the closing of nicks in DNA in lagging strand - Lagging strand DNA synthesis requires DNA ligase to maintain strand continuity - AMP= adenosine monophosphate A. starting DNA replication in E.coli - Priming of oriC B. RNA primers are used to initiate DNA synthesis - Provide 3’ -OH end C. unwinding the DNA helix to permit replication - During DNA replication, helicases unwinds on 5’-3’ stand D. keeping the unwound DNA “straight” - Single-strand DNA binding (SSB) protein keeps unwound strands in an extended form for replication Without a way to relieve the stress built up by advancing replication fork, the build-up of positive supercoils would stop replication: the DNA would be “overwound”: topoisomerases nick the DNA and release the positive supercoils A replication “fork” Replication mechanisms requires: - Topoisomerase - Helicase - Single-strand DNA binding protein (SSB) - Primase - DNA polymerase III - DNA polymerase I - DNA ligase -DNA strands have a defined 5’ to 3’ polarity Okazaki fragments - short lagging strand DNA fragments 3. Unique aspects of eukaryotic chromosome replication
● Shorter RNA primers and Okazaki fragments ● DNA replication only during S phase ● Multiple Polymerases (at least 15)[Pol α (alpha), ● Pol δ (delta) and Pol ε (epsilon) replicate the chromosomes] ● Multiple origins of replication ● Nucleosomes (two each of histones H2A, H2B, H3, H4 plus H1) ● Telomeres Disassembly and Assembly of Nucleosomes is Tightly coupled and rapid during DNA synthesis Telomere structure -Shelterin complex Protects the telomeres of mammalian chromosomes -protein complexes also protect yeast telomeres Telomere problem - chromosome end will be degraded causing chromosome shortening Telomere length and aging ● Most human somatic cells have low telomerase activity ● Shorter telomeres are associated with cellular senescence and death ● Diseases causing premature aging are associated with short telomeres - Progeria -
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- Fall '13