DNA Replication C483 Spring 2013

1. Double stranded DNA that has been labeled with radioactive 14C is used as the template for replication. Replication is carried out in a medium containing only unlabeled nucleotides. After two rounds of replication, what percent of double stranded DNA molecules are radioactive? A) 25% B) 50% C) 75% D) 100% 2. Which of these statements is false? The primosome is part of the replisome DNA polymerase I is part of the replisome DNA polymerase III is part of the primosome The Klenow Fragment is part of DNA Polymerase I 3. Which of these statements is false? DNA Polymerase III contains 5’3’ exonuclease activity for proofreading 5’3’ polymerase activity of elongation of the DNA strand. Subunits to aid in processivity. Subunits that act as “sliding clamps.”

4. Because the replisome synthesizes both strands of DNA simultaneously Okazaki fragments are formed in the leading strand. The leading strand is synthesized 5’3’, but the lagging strand is synthesized 3’5’ The replisome needs to have primase activity to make RNA primers for the lagging strand Proofreading activity is required for the leading strand, but not the lagging strand. 5. Which of these activit(ies) is the last one in the process of lagging strand synthesis? Ligase Polymerase 5’3’ exonuclease primase

History of Nucleic Acid Biochemistry Miescher (1869) Kossel (1901) Discovers nucleic acid He called “nuclein” Discovers five nucleobases A, G, C, T, U Franklin and Wilkins Watson and Crick (1953) Avery, MacLeod, McCarty (1944)

Semiconservative Replication Template Protein machinery DNA polymerase Three stages Initiation Elongation Termination

E. Coli chromosome Circular, double stranded Origin Replication forks Bidirectional Replisome DNA polymerase is essential part of the replisome

Bacterial DNA Polymerase II DNA Polymerase I and II involved in repair and polymerization of part of one strand DNA Polymerase III is major component of replisome Multi-subunit, asymmetric dimer Many activities

Polymerase Activity dNTP is substrate Added to 3’ end of growing chain PRIMER is required 5’  3’ growth Selectivity based on H-bonding TEMPLATE is required Pyrophosphate generated

Processivity Replisome stays attached to replication fork Beta subunit is a sliding clamp Higher efficiency Few replisomes needed

Polymerase III Proofreads Incorrect base pair every 105 bp Epsilon subunit is proofreader Unmatched base pairs have different conformation 3’5’ exonuclease activity 99% of errors corrected here 99% of remaining errors are repaired

Polymerase Synthesizes Both Strands Simultaneouly! Synthesis is 5’ 3’ Leading Strand built on the 3’5’ template Lagging Strand built on the 5’3’ template Discontinuous Okazaki fragments

Primase RNA primer is needed for each polymerization RNA primer is made by primase enzyme Part of the primosome complex Primosome, along with DNA Polymerase III, is part of replisome

Closing the Nick Three actions needed Removal of RNA primer DNA polymerase I Filling in the nick with DNA Forming bond between adjacent fragments DNA ligase

DNA Polymerase I Two activities similar to DNA polymerase III Extend chain 3’5’ exonuclease Proofreading Unique activity 5’3’ exonuclease Removes RNA primer

Klenow Fragment DNA polymerase I was first discovered Kornberg Nobel prize Can be cut into two pieces that retain activity Klenow fragment Bigger piece with 5’3’ exonuclease removed “Usable” form of DNA polymerase III

Three Step Process

Step 1

Step 2

Step 3

Mechanism of Ligase

Answers B C A