Today you will… Explain the process of DNA replication. Identify the specific roles of the enzymes and proteins involved in DNA replication.

OUR DNA How many Chromosomes? 6 billion base pairs 12 pt single spaced font-> 1200 books How long to copy all DNA in 1 cell?

Enter question text... In which phase of the cell cycle is dna replicated? 1.G1 2.S 3.G2 4.M Phase

Meselson & Stahl

DNA Replication Overview Overview: 1.DNA strands separate. 2.Complimentary base pairing (A-T and G-C). 3.THREE STEPS 1.Initiation 2.Elongation 3.termination 4.Tons of enzymes involoved. An entire “team” of enzymes and proteins are responsible for each stage of replication.

Step 1: Initiation Origins of Replication (ori) – specific sequence of nucleotides along DNA where replication begins –Replication proceeds in BOTH directions of the replication bubble –Replication fork: Y-shaped area of bubble where DNA elongates

Enzymes Helicase SSB’s Topiosomerase TOPOISMOERASE

DNA directionality Review

Identify the 3’ carbon on the nucleotide. D CB A E 1.A 2.B 3.C 4.D 5.E

What would the complementary DNA strand be for the following sequence? 5’-CGTATG-3’ 1.5’GCATAC-3’ 2.5’GCAUAC-3’ 3.3’GCATAC-5’ 4.3’GCAUAC-5’

Stage 2: Elongation ADD new NT’s DNA Polymerase III – adds new NT’s to the growing end of the new DNA strand –Only added to 3’ side

Priming of DNA for synthesis Primase enzyme: attaches to parent strand and makes/lays down primers…Primase enzyme: attaches to parent strand and makes/lays down primers… PRIMERSPRIMERS = short segment of RNA nucleotides used to begin the replication Primer formation MUST PRECEDE DNA replication

It takes energy… Nucleotide added as Triphosphate…to provide energy Each equipped with energy! Polymerase

Addition of nucleotides

Question This cleaving is… Hydrolysis or Dehydration Synthesis Endergonic vs Exergonic

Replication Video

Leading vs lagging strand

New strands are synthesized differently Leading StrandLeading Strand: 3’-5’ template/parent –5’-3’ new strand/daughter –Continuous DNA synthesis towards Y of replication fork Lagging StrandLagging Strand: –5’-3’ template/parent –5’-3’ in fragments –Discontinuous synthesis away from opening replication fork

Replication of the LEADING strand 1.Primase 1.Primase adds an RNA primer DNAPolymerase III 2. DNA Polymerase III adds NT’s in the daughter 5’ to 3’ direction continuous 3.Elongation is continuous towards the opening replication fork

Replication of the LAGGING strand Same as leading except…Same as leading except… discontinuous Okazaki fragmentsReplication is discontinuous, through multiple segments (Okazaki fragments) Proceeds away from the direction of opening of the replication fork DNA ligaseDNA ligase bonds Okazaki fragments

All according to FORK

Stage 3: Termination When Replication Fork encounters neighboring ORI Real Time Video Real Time Video

DNA polymerases DNA polymerase III –1000 bases/second! –main DNA builder DNA polymerase I –20 bases/second –editing, repair & primer removal Arthur Kornberg 1959 Roger Kornberg 2006

Editing & proofreading DNA 1000 bases/second = lots of typos! DNA polymerase I (Nuclease Activity) –proofreads & corrects typos –repairs mismatched bases –removes abnormal bases –reduces error rate from 1 in 10,000 to 1 in 100 million bases

What does it really look like?

telomeres Repeated base pairs TTAGGG repeated x to make strands equal Non-coding Postpones DNA erosion at end

The scientist(s) whose experiment confirmed semiconservative replication. 1.Hershey & Chase 2.Meselson & Stahl 3.Avery, McCarty, & MacLeod 4.Watson & Crick 5.Griffith

The enzyme whose function is to separate the two strands of DNA prior to replication. 1.DNA ligase 2.Primase 3.Topoisomerase 4.Helicase 5.DNA Polymerase

The enzyme whose function copy the template DNA strand one nucleotide at a time. 1.DNA ligase 2.Primase 3.Topoisomerase 4.Helicase 5.DNA Polymerase

The enzyme whose function is to form phosphodiester linkages between okazaki fragments. 1.DNA ligase 2.Primase 3.Topoisomerase 4.Helicase 5.DNA Polymerase

The enzyme whose function is to correct the “overwinding” that occurs ahead of the replication fork. 1.DNA ligase 2.Primase 3.Topoisomerase 4.Helicase 5.DNA Polymerase