哈佛大学：《分子生物学》课程PPT教学课件（英文版）Replication1B

Collaboration of proteins at the Replication Fork Leadi ng strand DNA polymeraseⅢl on leadi ng strand Leadi ng strand template Helicase Laggi ng strand RNA primase 00000 template RNA primer Parent dna 5 5 Single-stranded DNA Kazak DNA polymerase Ill bindi ng proteins fragment on laggi ng strand

DNA polymerase iII dimer Lagging strand Looping the lagging strand to make both polymerases move in the 5′3′ same direction Lagging strand copy

Looping the lagging strand to make both polymerases move in the same direction

The discovery of dna polymerase. Arthur Kornberg and bob lehman pursued an enzyme in bacterial extracts that would elongate a chain of deoxyribonucleic acid just like glycogen synthase elongates a chain of glycogen The enzymatic activity was unusual: 1)Needed a template which dictates what nucleotide was added: substrate was directing enzymatic activity 2)Needed a primer annealed to the template. E. coli DNA pol I(pola) eas ily cleaved into two fragments b proteinase a large fragment(Klenow fragment) contains polymerase and 37-5 exonuclease (proofreading domain). USed in vitro for synthesis reactions (DNA sequencing) Klenow fragment(68 kD small fragment (35 kD) exonuclease polymerase 5-3 activity

The discovery of DNA polymerase. Arthur Kornberg and Bob Lehman pursued an enzyme in bacterial extracts that would elongate a chain of deoxyribonucleic acid just like glycogen synthase elongates a chain of glycogen. The enzymatic activity was unusual: 1) Needed a template which dictates what nucleotide was added: substrate was directing enzymatic activity 2) Needed a primer annealed to the template

(A)5→3 DNA synthesis Primer New DNA 5′ 国国■■■■■■口圆圆■■ Templat DNA polymerase (B)3′→5 exonuclease activity ncorrect nucleotide IIi DNA polymerase (C)5-3 exonuclease activity Displaced nucleotides

Nick translation by intact DNA pol I Fig.15.3 add DNase to nick dna 3-OH5°-P excised DNA fragments P add 32P-dNTP, cold dNTPs and DNA pol I note: nick moves 5 to 3>

in vitro DNA synthesis using the Klenow fragment DNA pol I Experimental Uses a. Fill-in reaction to label ends of dna b DNA sequencing

Wait a minute! 无法显示该图片 John Cairns mutated the gene for DNA polymerase, polA, and the bacteria grew just finel Either the polymerase hypothesis was all wrong,… or there were other dna polymerases in E. coli that carried out DnA synthesis in the polA strains

Wait a minute! John Cairns mutated the gene for DNA polymerase, polA, and the bacteria grew just fine! Either the polymerase hypothesis was all wrong,…… or there were other DNA polymerases in E. coli that carried out DNA synthesis in the polA strains

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Which polymerase is processive? 无法显示该图片

Which polymerase is processive?