Replication of Nucleic Acids

2

Because sometimes this... 3

...leads to this! (Yikes!) 4

Cell Numbers must increase to get growth of an organism  Cells duplicate their contents  One parental cell gives rise to two daughter cells (blue)  Duplication of DNA must occur  Mutation rate must be low – replication with high fidelity  Mutation rate is 1 nucleotide/10 9 nucleotides (6.4x10 9 bp in a human diploïd cell) 5

DNA replication in cell cycle  The cell cycle is the ordered process of cellular duplication The replication of DNA only occurs in the S phase of the cell cycle M phase: mitosis G1 and G2: gaps, the cell gets ready for the next phase 6

7

Distribution of DNA strands – the possibilities  There are different possibilities: 1 parental:1 daughter strand (semi- conservative) (Watson-Crick) Parental and daughter (conservative) (Bloch) Breaks in DNA (dispersive) (Delbrück) 8

The Meselson-Stahl experiment – the setup  14 N (light) is the most abundant form of nitrogen  15 N (heavy) is not radioactive, but heavier  Centrifuge 1:1 mixture of DNA in CsCl gradient, then take the UV absorption reading 9 Meselson M, Stahl FW The replication of DNA in Escherichia coli. PNAS Vol 44:

The Meselson-Stahl experiment – the results  Generations are measured from time added 14 N  At generation 0: one band  At generation 1: 1 band  At generation 1.9: 2 bands 10 Why are there two bands at generation 1.9? Why are there three bands when you mix 0 and 1.9?

Distribution of DNA strands 11 This one is proven!

12

Basic needs for DNA replication In order to start, the following are required:  All of the 4 deoxyribonucleotide triphosphates (dNTPs) dATP, dTTP, dCTP, dGTP  DNA template  DNA/RNA primer to start the replication  DNA polymerase 13

DNA replication  Base pairing occurs according to Watson- Crick rules  Hydrolysis gives rise to a phosphodiester bond  Releases pyrophosphate (favourable energy release) 14

Phosphodiester bond formation 15 Nature :  The incoming nucleotide has 2 metal ions (Mg 2+ ) attached to the phosphates  The Mg 2+ also binds to Asp residues (conserved)  Lowers the affinity of O for H on 3’ OH, and allows for the reaction to occur  The exposed –OH group reacts with the triphosphate group of the incoming base 3’5’ 3’

DNA synthesis - polymerase  The incoming nucleotide must pair with the template strand to be recognized by the polymerase  DNA polymerase is the catalyst to the formation of the phosphodiester  Processivity is the number of phosphodiester bonds formed by the polymerase before it falls off the DNA 16

17

DNA Replication 18 Figure 5-6 Molecular Biology of the Cell (© Garland Science 2008)  On replicating bacterial DNA, two Y- shaped structures are occurring at the same time  Each arm of the Y: both replicated strands  The start point of replication is called the replication origin  Large DNA molecules can have many origins of replication

DNA replication fork  DNA is denatured (unwound)  Replication always occurs 5’→3’ in living organisms  An experiment with 3 H in bacteria showed the presence of DNA strands nt long: the Okazaki fragments  Okazaki in eukaryotes: nt long 19

Primers for the lagging strand  Primers are needed every nucleotides on lagging strand  Primers are made by the DNA primase  Made with RNA  About 10nt length 20

Replicating the lagging strand  RNA primers are extended by DNA polymerase III  Polymerase falls off when encounters double-strand structure  DNA polymerase I system removes RNA and replaces DNA  DNA ligase forms diester bonds 21

22

Types of DNA polymerases 23

Keeping the polymerase on  Polymerase will not stay on very long on its own  The sliding clamp keeps the polymerase on the leading strand  Clamp is loaded at the primer/template junction by the clamp loader 24 ATP- dependant!

Polymerase – more than a protein... 25

The sum of all parts 26 Each shape is a protein. Together, they form a complex that makes up the polymerase

Proofreading  DNA polymerase III has 3’→5’ exonuclease activity  A mismatched pairing causes the transfer of DNA strand from the polymerizing subunit to the editing subunit  The mismatched nucleotide is removed 27

Proofreading... 28

DNA Ligase 29 Phosphodiester bond is missing on the DNA strand In bacteria: ligase is NAD + dependent In eukaryotes: is ATP dependant DNA ligase residue

Unwinding DNA  To be replicated, DNA must be opened in front of the fork  Double helix very stable  Helicase uses ATP to propel it along the strand  Opens DNA helix at up to 1000bp/s 30

Keeping it straight  Hairpins in ssDNA will destabilize the polymerase  Single-strand DNA- binding protein (SSB) bind tightly and destabilize the helix  SSB do not cover the bases 31

32

The origin In E. coli:  Major initiator protein is DnaA The region is near an AT rich region  Helicase is DnaB  Primase is DnaG 33

The active replication fork 34

Putting it all together 35

36

DNA sequencing – Sanger Method 37 Each ddNTP is labelled with a different fluorescent dye

PCR  Standard technique in medical and research labs  After 25 cycles, the target sequence is amplified in the order of 10 6 (2 n =n is number of cycles)  Used to identify pathogens in infections, cancer types, genetic disorders