DNA REPLICATION. Anna Onofri Liceo «I.Versari»

Transcription

1 DNA REPLICATION Anna Onofri Liceo «I.Versari»

2 Learning objectives 1. Understand the basic rules governing DNA replication 2. Understand the function of key proteins involved in a generalised replication model

3 It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material Watson & Crick Nature (1953) Original drawing by Francis Crick

4 Four requirements for DNA to be genetic material Must carry information Cracking the genetic code Must replicate DNA replication Must allow for information to change Mutation Must govern the expression of the phenotype Gene function

5 DNA stores information in the sequence of its bases Much of DNA s sequence-specific information is accessible only when the double helix is unwound Proteins read the DNA sequence of nucleotides as the DNA helix unwinds. Proteins can either bind to a DNA sequence, or initiate the copying of it. Some proteins recognize the base sequence of DNA without unwinding it (e.g. a restriction enzyme). restriction enzyme EcoR V

6 DNA replication occurs with great fidelity Somatic cell DNA stability and reproductive-cell DNA stability are essential. Why? Identity Genetic diseases

7 DNA Replication Process of duplication of the entire genome prior to cell division Biological significance extreme accuracy of DNA replication is necessary in order to preserve the integrity of the genome in successive generations In eukaryotes, replication only occurs during the S phase of the cell cycle. Replication rate in eukaryotes is slower resulting in a higher fidelity/accuracy of replication in eukaryotes

8 The mechanism of DNA replication Arthur Kornberg et al (Nobel 1959) He was the first to discover how molecules of deoxyribonucleic acid (DNA) duplicate within bacterial cells and also the first to devise a method for synthesizing this process in a laboratory setting. For these achievements he shared the 1959 Nobel Prize in physiology or medicine with Severo Ochoa. REPLICATION STEPS Initiation Proteins bind to DNA and open up double helix Prepare DNA for complementary base pairing Elongation Proteins connect the correct sequences of nucleotides into a continuous new strand of DNA Termination Proteins release the replication complex

9 Basic rules of replication A. Semi-conservative B. Starts at the origin C. Can be uni or bidirectional D. Semi-discontinuous E. Synthesis always in the 5-3 direction F. RNA primers required

10 DNA replication Of the 3 possible models, replication is A) Semiconservative

11 Meselson and Stahl experiment Meselson and Stahl concluded that DNA employs semi-conservative replication. They placed the bacteria in an environment which contained a selected Nitrogen isotope. They first used N 14. The bacteria then integrated this isotope into their DNA. Later they used an environment that contained N 15. They then looked at which of the isotopes the bacterial DNA contained. They found that it contained both isotopes of nitrogen, which implies that conservative replication is not the correct conclusion

12

13 Semiconservative replication Half the original DNA molecule is saved, or conserved in the daughter molecules. This is why the process is called semi-conservative.

14 B) Starts at origin Initiator proteins identify specific base sequences on DNA called sites of origin Prokaryotes single origin site E.g E.coli - oric Eukaryotes multiple sites of origin (replicator) E.g. yeast - ARS (autonomously replicating sequences) Prokaryotes Eukaryotes

15 C) Uni or bidirectional Replication forks move in one or opposite directions

16 D) Semi-discontinuous replication Anti parallel strands replicated simultaneously Leading strand synthesis continuously in 5 3 Lagging strand synthesis in fragments in 5-3

17 Semi-discontinuous replication New strand synthesis always in the 5-3 direction

18 E) Synthesis is ALWAYS in the 5-3 direction Nucleotide recognition Enzyme catalysed polymerisation (DNA polymerase) Complementary base pair copied Substrate used is dntp

19 What happens if a base mismatch occurs? Where does energy for addition of nucleotide come from? DNA polymerase has 3 5 exonuclease activity in order to correct errors From cleavage of high energy phosphate of incoming triphosphate

20 PROOFREADING The term proofreading is used in genetics to refer to the error-correcting processes,. When an incorrect base pair is recognized, DNA polymerase reverses its direction by one base pair of DNA and excises the mismatched base. Following base excision, the polymerase can re-insert the correct base and replication can continue.

21 F) RNA primers required DNA polymerase can only join an incoming nucleotide to one that is base-paired RNA primase provides a base paired 3 end as a starting point for DNA pol by synthesising ~10 nucleotide primers

22 Core proteins at the replication fork Topoisomerase Helicases Primase DNA polymerase DNA ligase - regulate the overwinding or underwinding of DNA - separates 2 strands - RNA primer synthesis - synthesis of new strand - seals nick via phosphodiester linkage