Lecture 4 DNA Replication, Repair, and Recombination

Lecture 4 DNA Replication, Repair, and Recombination -...

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Chapter 28 DNA Replication, Repair, and  Recombination  
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DNA replication, damage, and repair DNA structure; DNA modifying enzymes
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DNA replication Reaction direction complementary Semi-conservative required 5’ 3’
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Steps of DNA replication
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Klenow fragment  DNA polymerase I lacking 5’ end  First DNA polymerase identified DNA polymerase I 5’-3’exonuclease 3’-5’exonuclease Core polymerase Kleno w
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DNA polymerases   Two  metal ions (typically Mg 2+ ) participate  in the polymerase reaction (interact with  primer  and  dNTP )  Specificity of replication is dictated by  complementarity of shape between bases
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E. coli chromosomal replication  Replication structures can be seen by electron microscopy Theta structures Replication forks  Replication  starts at a unique site  (~245 bp) on the  E. coli  chromosome –  Ori C  Two replication forks progress around the chromosome in opposite directions  The two replication forks meet at a unique location (tre) and replication ends
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DNA replication : 5~6 nucleotides OH RNA (primer)removed (exonuclease)/DNA filled by DNA  polymerase I Ligated by DNA ligase = RNA polymerase  (DnaG) (RNA pol does not need primer) III (New DNA)
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Leading strand and lagging strand (Okazaki fragments) DNA polymerases catalyze synthesis of DNA strand in 5’ to 3’ direction  – but replication forks  appear to be synthesizing one strand in 5’ to 3’ direction and the other strand in 3’ to 5’ direction. •  Problem was solved by Okazaki who showed that one strand is synthesized continuously  (leading strand) and the other strand is synthesized  discontinuously  (lagging strand) . •  Discontinuous synthesis leads to production of DNA fragments of 500-1000bp known as  Okazaki fragments . •   DNA Polymerase III  synthesizes both  the leading strand and lagging strand. It is a highly  processive enzyme and incorporates many thousands of nucleotides before letting go of DNA. 5’ 5’ 3’ 3’ 3’ 5’ Theta structure Replication fork Replication fork
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• Lagging strand synthesis involves synthesis of short RNA primers by the RNA polymerase called  primase. DNA polymerase III then elongates these RNA primers to generate Okazaki fragments. • RNA is removed ( 5’ to 3’ exonuclease  activity) and gaps between Okazaki fragments are filled by  DNA polymerase I. • Finally, the DNA fragments on the lagging strand are joined by DNA ligase
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Lecture 4 DNA Replication, Repair, and Recombination -...

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