Biochem 2 review (test 3)

Biochem 2 review (test 3) - Component and functions in in...

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Component and functions in in vitro synthesis o ssDNA template strand – determine order that nt will be added o dNTPs – building blocks of new DNA; Provide energy o DNA Pol – catalyzes addition o Oligonucleotides – provides free 3’-OH that allows DNA synthesis to begine, determines start E. Coli DNA Pol I and Pol III holoenzyme o Both Exonuclease activity allows removal of mispaired bases – proofreading Adds nt to free 3’ hydroxyl of a primer Capable of adding more than 1 w/o dissociating Synthesizes in 5’ ->3’ direction; Reads template from 3’->5’ Catalytic mechanism relies on function of aspartate residues to coord. Mg2+ Conserved aspartate residues in active site and require Mg2+ as cofactor o Pol I Has 5’->3’ exonuclease activity Functions in cell as monomer Removal of RNA primer from lagging strand Relatively low processivity (<200) and doesn’t associate with other proteins Cleaved by mild protease treatment to produce frag with polymerase and proofreading activities (Klenow fragment) Has polymerase and nuclease activity on same polypeptide Nucleotide removed as dNMP by adding water o Pol III Has >= 10 subunits Adds mostly during replication High processivity (>500,000 nt) Requires b subunits for max processivity o Neither – Capable of RNA-dependent DNA synthesis Subunits of Pol III o a – polymerase activity o E – 3’ -> 5’ proofreading o Each core – aEO – synthesize each strand o y + d’ – clamp loader o d – clamp loader o B – required for optimal processivity o T - structual E. Coli initiation of replication o 1) DAM fully methylates GATC sites in oriC o 2) DnaA binds to 9 bp repeats at oriC o 3) HU aids DnaA in melting duplex at 13 bp A-T rich repeats o 4) DnaC loads DnaB onto DNA o 5) DnaB begins to unwind DNA duplex o 6) DNA gyrase relieves (+) supercoiling
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Enzymes o Primase – initiates Okazaki formation, synthesis of RNA using DNA o Telomerase – reverse transcriptase; synthesis of DNA using RNA, prevents replicative loss DNA o Topoisomerase – changes linking number in DNA by breaking and resealing phosphodiester bd Removes (+) supercoiling in front of advancing replication fork Active site in type I uses a Tyr residue to catalyze a transesterfication o DNA Pol – Synthesizes DNA using DNA at fork, proofreads new DNA o Helicase – Causes (+) supercoiling in front of fork, hexamer that encircles lagging at fork Unwinds DNA o DNA Ligase – Joins Okazaki, seals ss nicks in dsDNA, AMP-linked enzyme intermediate DNA Repair o Mismatch Requires cleavage of the newly synthesize strand at DAM site Involves MutH endonuclease cleavage of DNA Requires hemimethylation of DNA Involves exonuclease removal of >1000 bases o Direct One type involves AlkB One type involves breaking C-C between adjacent pyrimidines “ “ “ oxidative demethylation of a damaged base
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This note was uploaded on 03/18/2008 for the course BMB 463 taught by Professor Stolstice during the Spring '08 term at Michigan State University.

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Biochem 2 review (test 3) - Component and functions in in...

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