DNA: Structure and Replication Chapter 7

Replication Fork Also predicted by Watson and Crick Location the DNA double helix is unwound and produces two single strands that serve as templates for copying John Cairns tested this prediction using thymidine with 3H (tritiated) to label newly synthesized daughter molecule

Replication Fork in E. coli Autoradiograph is an image on an x-ray film or nuclear emulsion produced by the pattern of decay emissions (e.g., beta particles or gamma rays) from a distribution of a radioactive substance. Autoradiograph is an image on an x-ray film or nuclear emulsion produced by the pattern of decay emissions from a distribution of a radioactive substance.

DNA polymerases (pol) First DNA polymerase isolated and activity was demonstrated in vitro in 1959 by Arthur Kornberg To show activity: Add DNA polymerase, deoxyribonucleotides (dATP, dGTP, dCTP and cTTP), a single strand of DNA template There are 5 DNA pol in E. coli

DNA pol I Catalyze genes (DNA SYNTHESIS) in the 5’ to 3’ direction Have 3’ to 5’ exonuclease activity Have 5’ to 3’ exonuclease activity which degrades single strands of DNA or RNA Synthesizes at ~1000nt/second Dissociates with DNA after incorporating 20 to 50 nt THINK QUESTIONS: How long would it take to replicate DNA of a human cell (3 x 10^9) using just DNA pol 1? This pol is NOT responsible for catalyzing DNA synthesis at the replication fork 3,000,000,000 *2 = 6x10^9 /400 = 1.5 * 10^7= 6250 min = 104 hrs =4.34 days

Review question Which of the following is/are NOT a key structural property displayed by the heritable (transforming) material? A) The structure must contain complex information (genetic code). B) The structure must allow or tolerate small changes that facilitate nonlethal variation. C) The structure must be enzymatically active, able to mediate cellular metabolism. D) The structure must be composed of rare/unusual elements and must be enzymatically active, able to mediate cellular metabolism.

DNA pol III Catalyzes DNA synthesis at the replication fork Replication fork is the zone where DNA is unwinding DNA is always synthesized in the 5’ to 3’ direction MUST have free 3’ hydroxyl for DNA synthesis to start

Synthesizing the lagging strand

Synthesizing the lagging strand

Exceptions to the base pairs matching Tautomerization Keto - normal Imino – C and A Enol – T and G

DNA pol I and III proofread 3’ – 5’ direction

Quick Question The spontaneous (though infrequent) change of a nitrogenous base to a slightly distinct form is called: A) ketomerization. B) tautomerization. C) methylation. D) mutagenesis.

The replisome

DNA gyrase removes extra twists

Prokaryotic initiation of replication Orderly process Starts at Origin (precise location) E.coli – oriC Proceeds in both directions Take place on at certain times DnaA proteins bind to DnaA Box DNA unwinds and helicases bind Then DNA pol III and primase are recruited

What structural feature of DNA suggests a possible method of its replication? The nucleotides between the nucleotide strands are complementarily paired. The hydrogen bonds between A-T and G-C are the same on all DNA molecules. The DNA strands run anti-parallel. The phosphate bonds in the backbone of all DNA molecules.  

Replication in Eukaryotes

Coolest DNA replication video ever! https://www.youtube.com/watch?v=4jtmOZaIvS0

Cell Cycle DNA synthesis take place in S Cell cycle progress enables initiation of replication of the genome by providing Cdc6 and Cdt1

Eukaryotic initiation of replication In yeast, 3 proteins needed to assemble the replisome ORC, Cdt1 and Cdc6 All three recruit helicase MCM complex mini-chromosome maintenance

The replication problem at the end of chromosomes

Telomeres in Tetrahymena Elizabeth Blackburn and Joe Gall, 1978 Single-celled ciliate Micronucleus Macronucleus Fragmented chromosomes 40,000 telomeres TTGGGG repeated sequence Telomerase, adds the short repeats to the 3’ ends of DNA

Telomere lengthening Telomerase = Protein + RNA Lengthening of the 3’ overhang Telomerase = Protein + RNA Telomerase anneals to the 3’ overhang Replication of complementary strand Telomerase Primer is synthesized Primase Elongation Polymerase fills in the gap DNA polymerase Translocation The primer is removed and ligase seals the gap Elongation

Telomeric cap structure Telomeres preserve chromosomal integrity by associating with proteins that form a protective cap Without cap, the double stranded ends would be mistaken as a ds break Can lead to apoptosis telomeric repeat binding factor WRN - helicase

Telomeres and Aging Somatic cells produce very little telomerase Chromosomes get shorter with each cell division Many Cancers have increased levels of telomerase Telomeres the Secret of Life!!! Dysfunctional telomeres and mutation in WRN gene Werner Syndrome Picture below same person at 15 and 48

Concept question The number of proteins in the prokaryotic replisome is 13, while the number in a eukaryotic cell is 27. What are the important protein components of the replisome in general? Why might a eukaryotic cell have more components?