LE end 3 end 5 end 3 end Space-filling modelPartial chemical structure Hydrogen bond Key features of DNA structure 0.34 nm 3.4 nm 1 nm The mechanism of DNA Replication When during the cell cycle is DNA synthesized? Draw

The Basic Principle: Base Pairing Each strand acts as a template for building a new strand in replication Parent dsDNA molecule unwinds & base pairs are broken - two new daughter strands built based on base-pairing rules Draw

LE 16-9_1 The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C.

LE 16-9_4 The nucleotides are connected to form the sugar-phosphate back- bones of the new strands. The first step is separation of the two parental DNA strands. Synthesis of complementary strands Predicted by Watson and Crick Semiconservative model of DNA replication A simple model of DNA replication

LE Conservative model. The two parental strands reassociate after acting as templates for new strands, thus restoring the parental double helix. Semiconservative model. The two strands of the parental molecule separate, and each functions as a template for synthesis of a new, comple- mentary strand. Dispersive model. Each strand of both daughter molecules contains a mixture of old and newly synthesized DNA. Parent cell First replication Second replication Various proposed models of DNA replication

Meselson and Stahl experimentally supported one of the replication models How & which one?

LE Bacteria cultured in medium containing 15 N DNA sample centrifuged after 20 min (after first replication) DNA sample centrifuged after 40 min (after second replication) Bacteria transferred to medium containing 14 N Less dense More dense Conservative model First replication Semiconservative model Second replication Dispersive model Heavy radioisotope Light radioisotope Why label nitrogen? Supported by data

Replication begins – at origin of replication (ori) Creation of replication bubble with replication forks at each end (Draw) Hundreds to thousands of oris on eukaryotic chromosome Usually one on bacterial chromosome Proceeds in both directions from each origin, until the entire molecule is copied

LE In eukaryotes, DNA replication begins at many sites along the giant DNA molecule of each chromosome. Two daughter DNA molecules Parental (template) strand Daughter (new) strand 0.25 µm Replication fork Origin of replication Bubble In this micrograph, three replication bubbles are visible along the DNA of a cultured Chinese hamster cell (TEM). Arrowheads mark replication forks.

Elongating a New DNA Strand Basic components Template DNA DNA polymerase DNA precursors deoxynucleotide triphosphates (dATP, dCTP, dGTP,dTTP)

LE New strand 5 end Phosphate Base Sugar Template strand 3 end 5 end 3 end 5 end 3 end 5 end 3 end Nucleoside triphosphate DNA polymerase Pyrophosphate 5’

Specificity of DNA polymerase only adds nucleotides to the free 3  hydroxyl end of dsDNA New DNA strand made only in 5’-3’direction Draw

LE Parental DNA 5 3 Leading strand Okazaki fragments Lagging strand DNA pol III Template strand Leading strand Lagging strand DNA ligase Template strand Overall direction of replication primer

LE Parental DNA 3 5 Overall direction of replication DNA pol III Replication fork Leading strand DNA ligase Primase OVERVIEW Primer DNA pol III DNA pol I Lagging strand Lagging strand Leading strand Leading strand Lagging strand Origin of replication

Other components of the DNA replication machinery? DNA helicase- to unwind DNA Single strand binding proteins- to stabilize ssDNA DNA ligase- to seal gap in sugar-phosphate backbone (make phosphodiester bond) between Okazaki fragments

LE 16-15_1 5 3 Primase joins RNA nucleotides into a primer. Template strand 5 3  Overall direction of replication A Closer Look at Lagging Strand Synthesis

LE 16-15_2 5 3 Primase joins RNA nucleotides into a primer. Template strand 5 3  Overall direction of replication RNA primer DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment.

LE 16-15_3 5 3 Primase joins RNA nucleotides into a primer. Template strand 5 3  Overall direction of replication RNA primer DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. Okazaki fragment After reaching the next RNA primer (not shown), DNA pol III falls off.

LE 16-15_4 5 3 Primase joins RNA nucleotides into a primer. Template strand 5 3  Overall direction of replication RNA primer DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. Okazaki fragment After reaching the next RNA primer (not shown), DNA pol III falls off After the second fragment is primed, DNA pol III adds DNA nucleotides until it reaches the first primer and falls off.

LE 16-15_5 5 3 Primase joins RNA nucleotides into a primer. Template strand 5 3  Overall direction of replication RNA primer DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. Okazaki fragment After reaching the next RNA primer (not shown), DNA pol III falls off After the second fragment is primed, DNA pol III adds DNA nucleotides until it reaches the first primer and falls off DNA pol I replaces the RNA with DNA, adding to the 3 end of fragment 2.

LE 16-15_6 5 3 Primase joins RNA nucleotides into a primer. Template strand 5 3  Overall direction of replication RNA primer DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. Okazaki fragment After reaching the next RNA primer (not shown), DNA pol III falls off After the second fragment is primed, DNA pol III adds DNA nucleotides until it reaches the first primer and falls off DNA pol I replaces the RNA with DNA, adding to the 3 end of fragment DNA ligase forms a bond between the newest DNA and the adjacent DNA of fragment 1. The lagging strand in the region is now complete.

Animation: Lagging Strand Animation: Lagging Strand

Animation: DNA Replication Review Animation: DNA Replication Review

Proofreading and Repairing DNA DNA polymerases proofread Replace mismatched nt in new DNA Also 1.Mismatch repair: repair enzymes correct errors in base pairing 2. Nucleotide excision repair: enzymes cut out and replace damaged stretches of DNA

Example DNA exposure to ultraviolet (UV) light induces chemical crosslinks between adjacent thymines (thymine dimers) How to repair?

LE DNA ligase DNA polymerase DNA ligase seals the free end of the new DNA to the old DNA, making the strand complete. Repair synthesis by a DNA polymerase fills in the missing nucleotides. A nuclease enzyme cuts the damaged DNA strand at two points and the damaged section is removed. Nuclease A thymine dimer distorts the DNA molecule.

Is DNA replication of linear chromosomes ever complete? Consider the tips (ends) of the leading and lagging strands.

LE End of parental DNA strands 5 3 Lagging strand 5 3 Last fragment RNA primer Leading strand Lagging strand Previous fragment Primer removed but cannot be replaced with DNA because no 3 end available for DNA polymerase 5 3 Removal of primers and replacement with DNA where a 3 end is available Second round of replication Further rounds of replication New leading strand Shorter and shorter daughter molecules

Ends of eukaryotic chromosomes –Tipped with many copies of a short DNA repeat called telomeres (e.g. human telomere sequence TTAGGG x 100-1,000) Added by telomerase, a ribozyme (made of RNA and proteins) Function:Telomeres postpone loss of important genes near ends after each cell division. Is telomerase found in all eukaryotic cells? NO, mostly in germ cells but NOT in somatic cells.

What will happen to DNA in cells that continually divide such as epithelial cells (skin, gut)? Make a prediction about the length of chromosomes in skin cells from a 80 year old versus a 4 year old. Cancer cells are characterized in part by their continuous cell division. Shouldn’t they ultimately die from loss of genes due to shortening of chromosomes? Hypothesize why they continue to divide without injury? Cancer cells express telomerase, which prevents chromosome shortening

LE µm Labelled telomeres Questions?